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Über induktive Erregung zweier elektrischer Schwingungskreise mit Anwendung auf Perioden‐ und Dämpfungsmessung, Teslatransformatoren und drahtlose Telegraphie
|
Annalen der Physik
| 1,904
|
public-domain
| 13,549
|
512
5 . Uher induhtive Erregung xweier elehtrischer
Schwimgungskreise mit Anwemdung auf PerJodernu n d Ddmpfungsmessung, Teslatramsformatoren
und drahtlose Telegraphie;
vom P. Drzcde.
I n h a l t: Einleitung. I. Aufskllung und Integration der Differentialgleichungen p. 513. 11. Die niagnetisehe Koppelung ist sehr klein
p. 521. 111. Messung der Periode und der Dampfung p. 525. 1. Die
Maximalamplitude p. 528. 2. Der Integraleffekt p. 530. IV. Die magnetische Koppelung ist nicht sehr klein p. 534. V. Die Wirksamkeit der
Teslatransformatoren p. 540. VI. Abhangigkeit der Teslawirkung von
Diimpfung nnd Koppelung p. 544. VII. Anwendung auf die drahtlose
Telegraphie p. 650. a) Einfacher oder lose gekoppelter Empfanger p. 551
b) Eng gekoppelter Empfiinger p. 554. Hauptresultate p. 560.
J. v. G e i t l e r l ) , B. Galitzin2), A. Oberbecks) und
D o m a l i p und Kol&6ek4)haben nachgewiesen, daD, wenn zwei
elektrische Schwingungskreise geniigend intensiv aufeinander
einwirken, jede derselben nicht mehr eine, sondern zwei Eigenperioden hat, auch wenn die beiden Systeme aufeinander abgestimmt sind, d. h. wenn sie ohne gegenseitige oder bei
sehr schwacher gegenseitiger Einwirkung eine gleiche Eigenperiode besitzen. Das Problem ist spater in allgemeinerer
und vollstandigerer Weise von M. Wien K, behandelt worden.
Wien 6, wendet seine Resultate auch auf die drahtlose Telegraphie nach dem Braunschen System. V. Bjerknes’) hat
den Fall sehr schwacher Koppelung ausfiihrlich behandelt in
Riicksicht auf Perioden und Dampfungsmessung durch Diskussion der sogenannten Resonanzkurve.
1) J. v. G e i t l e r , Sitaungsber. d. k. Akad. d. Wissensch. au Wien,
Februar u. Oktober 1895.
2) Furst B. G a l i t s i n , Petersb. Ber., Mai u. Juni 1895.
3) A. O b e r b e c k , Wied.Ann. 66. p. 623. 1895.
4) R. D o m a l i p u. F. KolSfiek, Wied. Ann. 67. p. 731. 1896.
5) M. W i e n , Wied. Ann. 61. p. 151. 1897.
6) M. W i e n , Ann. d. Phys. 8. p. 686. 1902.
7) V. B j e r k n e s , Wied. Ann. 66. p. 120. 1895.
Induktive Erreguitg.
513
Die hier folgende Behandlung des Problems unterscheidet
sich von den genannten Bearbeitungen in folgenden Punkten :
1. Die Losung der Differentialgleichungen, speziell die
Berechnung der Amplituden aus den Anfangsbedingungen,
wird in einer mathematisch iibersichtlichen, Rechenfehler leicht
ausschlieBenden Form gegeben, die auch fiir kompliziertere
Verh'altnisse, z. B. eng gekoppelte Sender und Empfanger bei
drahtloser Telegraphie, anwendbar bleibt. l)
2. Der induzierte Kreis wird nicht, wie bei den genannten
Autoren, lediglich als ein langs des Leiters konstanter Strom
behandelt, an dessen Enden eine bestimmte Kapazitat liegt,
sonderri den wirklichen Verhaltnissen entsprechend.a)
3. Gegen die Wiensche Bearbeitung tritt ein Unterschied
auf fur das Resultat hinsichtlich der Dampfung beider Eigenschwingungen in eng gekoppelten Systemen.
4. B j e r k n e s diskutiert nur die Resonanzkurve des
Integraleffektes ausfuhrlich. Hier wird auch die der Maximalamplitude mit hinzugezogen und dadurch eine experimentell
einfache Methode zur Bestimmung der einzelnen Dampfungen
beider Schwingungskreise gewonnen.
5. Die Frage nach der besten Konstruktion eines Teslatransformators wird weiter gefiihrt. Ihre Losung hangt noch
von einer auszufiihrenden Experimentaluntersuchung ab.
I. Aufstellung und Integration der Differentialgleichungen.
Wir setzen zunachst voraus, daB die Sekundarspule (z. B.
Teslaspule) zentriert und symmetrisch zum Primarkreis liege.
Die allgemeinen Besultate gelten aber auch fur beliebige Lagen
der Sekundarspule. Der Primarstromkrois enthalte die (elektromagnetisch gemessene) Kapazitat C,. Die Potentialdifferenz
zwischen beiden Metallflachen der Kapazitat C, zu einer beliebigen Zeit t sei TI, die Stromstarke, die wir im ganzen
1) Nachtrflglich sehe ich, ds5 schon D o m a l i p und Kolhdek dime
Behandlung sehr 5ihihnlich gewghlt haben. Ich glaube nur, daS meine
Formeln etwas iibersichtlicher geblieben sind durch Einfuhrung einer
anderen Integralform bei starker Koppelung beider Schwingungskreise.
2) Daraus ergibt sich fur die in den Differentialgleichungen auftretenden Konstanteu der auf p. 518 gezogene SchluS L,, < LpI, wghrend
sonst L,, = L,, angenommen wurde.
514
P. Drude.
Primarkreis konstant annehmen konnen (da C, sehr groB gewahlt wird) , sei i l . Die Anzahl magnetischer Kraftlinien,
welche zu irgend einer Zeit den Primarkreis umschlingen, sei
N,. Dann gilt:
w1 ist ein Koeffizient, von dem die Dampfung der Primarschwingung abhiingt (Widerstand von Leitung und Funken,
sowie der [geringe] Strahlungsverlust des Kondensators, eventuell
auch elektrische Absorption in seinem Dielektrikum). w 1 wird
als Ton der Zeit unabhangig angenommen. Sollte diese Annahme nicht zutreffen, so ist unter w1 ein wahrend der Schwingungen stattfinder Mittelwert zu verstehen. Nl .hangt von il
und der Stromstarke iz in der Sekundarspule ab. ia ist langs
der Spule nicht konstant.
Wenn wir die Achse der (als Zylinder gewickelten) Spule
zur z-Richtung wahlen, 2 = 0 in die Mitte der Spule legen,
wahrend z = f h / , die beiden Spulenenden bedeutet, d. h. h
die Spulenhohe, so konnen wir als Anfangsglied einer F o u r i o r schen Reihc (Grundschwingung) setzen :
(3)
ii ist die Stromstarke in der Mittelwindung. Dabei ist
vorausgesetzt, daB die Spule frei, d. h. ohne angelegte Kapazitat, endigt, so daB f = 0 sein muB fur z = f hI2.
Die Kraftlinien ATl zerfallen nun in zwei Teile:
+
(4)
q = 3 1 1 q,,
Ton denen der erste Teil die Kraftlinien bezeichnen soll, welche
nur den Primarkreis umschlingen, wiihrend N,, die Zahl der
Kraftlinien sein soll, welche sowohl den Primarkreis umschlingen,
als auch Windungen der Spule. (Vgl. Fig. 1.)
F u r siimtliche Kraftlinien N,, ist die magnetomotorische
Kraft die gleiche, namlich 4ni1, wenn namlich der Stromkreis 1
als ein h e a r e r angesehen werden kann, was wir voraussetzen
mollen, und in praxi auch hinreichend erfullt ist. 1st also
lnduktive Ewepng.
515
Wll der magnetische Widerstand der (parallel geschalteten)
Kraftnjhren Nll, so ist:l)
(5)
Nll
4 n il
= Wyl-'
Fur die Kraftlinien ATl2 ist aber die magnetomotorische
Kraft nicht fur alle die gleiche, da die Spule nicht als linea-
2
2
Fig. 1.
rer Strom angesehen werden kann. Wenn eine dieser Kraftlinien den Spulenmantel an den Stellen z = & a schneidet,
so ist fur diese die magnetomotorische Kraft:
a
+ 8 n i ; sinn--3,
h
-a
falls n die Gesamtzahl der Spulenwindungen bedeutet, so dab
auf der Strecke d z die Anzahl (n/h)d z Windungen liegt. Wir
kijnnen aber fur die verschiedenen Kraftlinien N12 eine gleiche
mittlere elektromotorische Kraft ansetzen:
4 n i 1 + 8ni,Usin&,h
wobei a, jedenfalls kleiner als h / 2 und grofier als a'ist, falls
der Spulenmantel von der kurzesten der Kraftlinien N,,, die
gerade auch noch il umschlingt, an der Stelle a' geschnitten wird.
1) Vgl. z. B. P. Drude, Physik d. Athers p. 72. Stuttgart 1894.
2) Das +-Zeichen vor dem eweiten Gliede gilt, falls positives it
und i, gleichsinnig gerechnet wird.
P. Drude.
516
Es ist daher nach dem Gesetz vom magnetischen Kreislauf zu setzen:
4 n i l + 8 m ia sin (n a,/h)
N 12 =--- 1
(6)
Wl,
wobei F1,den magnetischen Widerstand siimtlicher Kraftrohren N,, in Parallelschaltung bedeutet.
Nach [2) und (4)entsteht daher:
Die Differentialgleichung fur die Stromstarke i, in der
Spule enthalt t und z als unabhangige Variabele.
Wenn e, die auf der Langeneinheit d z = 1 der Spule
liegende elektrische Ladung (nach elektromagnetischem MaBe)
bedeutet, Q, die Kapazitat dieser Langeneinheit, pZ das Potential auf der Spule an der Stelle z , so gilt:
Sind ferner N, die Zahl der magnetischen Kraftlinien,
welche den Spulenquerschnitt in der Hahe z der Spule durchsetzen, so gilt zwischen zwei Stellen z und z + d z , zwischen
denen ( n / h )d z Windungen liegen, falls ii tu, d z gleich dem
Energieverlust pro Zeiteinheit gesetxt wird (Widerstand und
Strahlung):
. ? a ax,
av,.
i2t u 2
--
h--at
- 8%
AUS (8) und (9) folgt:
a 2 A’,
h ata
a i2 - .1_Pi,
__
-_0 .
?a
-~
at
+m2
Ei da2
Diese partielle Differentialgleichung fur ia kijnnen wir in
totale fur ii umwandeln, wenn wir anch N, nach einer
F o u r i e r when Reihe entwickeln, beim Anfangsglied
~
nx.
N, = N; cos- h
stehen bleiben, und wenn wir unter tu, und B,, die eigentlich
auch von z abhangig sind, Mittelwerte fur die game Spulc
verstehen, d. h. sie als Konstanten behandeln. Dann ergibt
njimlich (3), (10) und (11):
m d’N8
di-! I n2 .o ._
(12)
h dta
3 d t
h2 Ea ‘2 +
~
~
Induktive Erreguny.
51 7
iV; bedeutet nach (11) die Anzahl Kraftlinien, welche den
Spulenquerschnitt an der Spulenmitte z = 0 durchsetzen. Auch
diese Zahl zerfallt in zwei Teile
+
N, = a,, Nl2,
wobei die Kraftlinien N,, nur die Spule durchsetzen, aber
nicht den Kreis 1 umschlingen, wahrend Nlz sowohl Spulenwindungen, als auch den Primarkreis 1 umschlingen. F u r die
A’,, ist nun wiederum die magnetomotorische Kraft nicht konstant, wir kijnnen aber fur sie eine gleiche mittlere magnetomotorische Kraft
8ni:sin(n.~)
(13)
in Ansatz bringen, wobei a, <a’, d. h. auch a, < al ist. Daher gilt
N,,
8 9;2.
=--.--,
1)
sin n -I:
(14)
We,
falls W,, der magnetische Widerstand der Kraftrohren N,,
ist. Dagegen ergibt (12):
2) K =
8nz(Ty
sin n3h
(15)
+
+
sinm-
1z d s i q
4nn deil
W),
dt2 +
m* h d-d-i ot2 -2.;6ns
=0.
,h
+
Diese Gleichung in Verbindung mit Gleichung (7) bildet
die Grundgleichungen unseres Problemes. Schreibt man jetzt
aur Vereinfachung i, fur ii, d. h. bedeutet von nun an i2 die
Stromstarke in der Bittelwindung der Teslaspule, so haben unsere
Grundgleichungen die bekannte Form :
2’. Brude.
515
AUS (17) folgt :
RCC
Ala: L,, = sin 1,
(18)
h
d. h. es ist n i c h t mehr, wie bei zwei linearen Stromkreisen, zu
setzen Lla = Azl, sondern es ist A,, < L2,, und zwar um so
mehr, je kleiner 4 ist, d. h. je naher der Primarkreis der
Mittelwindung der Teslaspule liegt und je hijher letztere im
Vergleich zu diesem Abstand vom Primarkreis ist. B e i verschiedenen Lagen der Teslaspule zum Primarkreis wechselt also
das Perhiiltnis LIZ: X a l , und zwar wird es urn so kleiner, j e
starker die gegenseitige Induktion (magnetische Koppelung) zwischen
Teslaspule und Primarkreis gemacht wird. Dieses Resultat, zu
dessen Ableitung die Interpretation der Koeffizienten der bekannten Gleichungen (16) unternommen wurde, gilt auch f u r beliebige und unspmetrische Lagen der Teslaspule turn Primarkreis.
Von den Koeffizienten 1; sind LIl und Lal verhaltnismabig einfach theoretisch zu berechnen, wahrend Laa und Lla
wohl nur durch sehr langwierige Rechnungen zu erhalten sind.
Lal riihrt von den Kraftlinien her, welche der Strom il durch den
Querschnitt 4 der Teslaspule hindurchsendet. Nennen wir diese
Anzahl, falls il = 1 ist, an einer beliebigen Stelle z der Spule N,,,
so ist das Anfangsglied N,4 der Fourierschen Entwickelung
72%
N,, = N,o, cos h
~
gegeben durcb
$. hI2
N , , = +JXzl cos
(19)
Daher ist
R %
d z.
-h/2
f h/2
A,, = n4 h%~ N a l c o s T7 2d%. z .
- 7k/2
Wenn die Primarwindungen Kreise sind, so ist die durch
i, an einer beliebigen Raumstelle hervorgebrachte magnetische
Kraft durch Kugelfunktionen darstellbar, daher ist Atal und
deshalb auch La1 zu berechnen. F’iir die Selhstinduktion All
sind schon bekannte Formeln entwickelt.’) Diese Formeln darf
1) Fur Spulen bedurfen die Stefanschen Formeln bei oszillatorischen Rondensatorentladungen einer Korrektion, vgl. P. Dru d e , Ann.
d. Phys. 9. p. 604. 1902.
Jnduktive 3rreguny.
519
man aber fur L,, nicht auch benutzen, da i2 langs der Spule 2
nicht konstant ist.
Setzt man an SteIle der Gleichungen (16) Gleichungen fur
die Potentialdifferenz 7, an der Kapazitiit C, im Stromkreis 1,
und fur das Potential Y2 (eigentlich 7,"
zu schreiben) an einem
freien Ende z = h / 2 der Spule, so ergibt sich vermoge (1)
und (8) aus (16):
Diese Gleichungen wollen wir entweder in der Form be-
wobei d s o gesetzt ist:
oder in der Form:
1) Diese Gleichungen nehmen die Gestalt von den Gleichungen bei
31. W i e n (Ann. d. Phys. 8. p. 694. 1902) an, wenn man beriicksichtigt,
daB 2 V, die Potentialdifferenz zwischen beiden Spulenenden ist. Daher
besteht auch die hier giiltige Gleichung
Die nachfolgenden Entwickelungen gelten daher auch fur den Fall daB
der Sekundarkreis aus keiner Spule, sondern aus einem linearen Strom
rnit angehangter groBer Kapazitiit C2 besteht. 2 V . ist daun die sekundare Potentialdifferenz.
P. Brude.
520
wobei gesetzt ist
Als Integral der Form (I) wahlen wir
(24)
Y,=Aemt, F2=Bezt, x=--6+iv,
als Integral von (11) wahlen wir:
Y1 = A e t i v ,
(25)
Yz = be ti^, y = - 9 + iz.
Dabei bedeutet i = 1
/17.
Es wird sich erweisen, daB in einem Falle die Form (I)
zweckmaI3ig ist, im anderen Falle die Form (11).
Aus (I) und (24) folgt:
+ 2 cYl 2 + V: + 6:)= p , , 'X
B,
B ( Z ~ + ~ - ~ , +Z- +
6 , 2V) ~
=p,,~~A.
A(za
(26)
Uagegen folgt aus (11) und (25):
+ 28,y + ~ ' ) = p , , ( r ; + 8 : ) B ,
{ qz;+ST
+ 9;+ 2 9,y + y3 p,, (r; + 9;)
A(.:
(27)
=
A.
Durch Elimination yon A : B folgen fur x und y die
biquadratischen Gleichungen :
(28) (x,
+ 2 Sl z + v; + 8;)(x2+ 2 cYs x + v; + 8;)= p , , p,,
{ !y 3- 2
.;
+ 8;)(yz+
+.: +
x4,
2 8,y
9.;)
= PizPai (r?+ ??:)(ti $3.
Wenn p , , = 0 ist, d. h. der Primarkreis allein vorhanden
ist, so ergibt (26):
(30)
x = - 8, & iv,.
(29)
aly+
+
v1 bezeichnet also die Frequenz, S, die Dampfung der Eigenschwingung des Primarkreises. Nennt man T, die Schwingungsdauer, 7, das logarithmische Dekrement, so ist
(30')
Analog bezeichnet v, und -6, Prequenz bez. Dampfung der
Rigenschwingung der Teslaspule allein. a,, r,, a,,t2 haben
52 1
Induktive Err egung.
nicht direkt so anschauliche physikalische Bedeutungen, jedenfalls ist aber fur die Eigenschwingungen zu setzen:
131)
y = - 8, f it, bez. y = - 8,f i t , .
Wenn 8 ; gegen r t , 8: gegen t: zu vernachlassigen ist,
so gilt
t
-~
=e
e-%+iTi
-
@ I t
21’
. e- - 1
t21.
Setzt man also
t
e
~-
-Ox+iz1
=e
-y.---
t
2’1
2 n
. t:i--tTI
7
so ist
(32)
tl =
TI
27c
’
9 - _rl ?!L.
1 -
4n2
Dabei ist Tl die Periode, y1 das logarithmische Dekrement
des Kreises 1.
Die Gleichung (I),(24) und (28) ist nun praktisch, wenn
die magnetische Koppelung k 2
(33)
ha = Pl, PZl =
4, L,,
so klein ist, daB sie gegen 1 vernachlassigt werden kann.
Verfolgen wir zunachst diesen ersten Fall.
11. Die magnetische Koppelung ist sehr klein.
Wenn p,,p,, gegen 1 vernachlassigt wird, so ergibt (28)
die vier Wurzeln fur x:
(34) XI = -8, +iV,, xi= - d; - i V 1 , x3 = - J2+iv,, x4= -8, --iV,.
Demnach ist das allgemeine Integral von (I):
{ TI
Y,
=A,ex~t+~ze2~t+A3e~~t+d4e2~t,
(W
= Bl e x i t
+ B, exat + B3 ex3t + B4 exat,
Aus (1) folgt:
(36)
-il - x A
c,
1
ez1t
+ z2A, e x : t + z3A ,e2zt + z4 n4
e4t,
aus (3) und (8):
(37)
n
i
-,$i- = xl B, ex1 t + xBB,
ex2
t
+ z3B3
ex3
t
+ x4B4
Als Anfangsbediiigung fur t = 0 gilt nun:
v1 = If’, Yz = 0 , il = 0, zz = 0.
(38)
Annalen der Physik. IV. Folge. 13.
31
ezd
t,
P.Brude.
522
Daher ergibt (35), (36), (37) und (38):
CAnxn=O,
C B , = O , CBnxn=O,
(39) C A n = F ,
n = 1, 2, 3, 4.
Nun ist nach der zweiten der Gleichungen (26):
B, (xn+ 2 8,
(40)
+
Durch Addition dieser vier fur n = 1, 2, 3, 4 gebildeten
Gleiuhungen entsteht wegen der Gleichungen (39):
Ferner folgt aus der zweiten der Gleichungen (26):
Durch Addition dieser vier fur n = 1, 2, 3, 4 gebildeten
Gleichungen folgt wegen (39) und (41):
7
1,
1,
%,
a17
(44)
I s:,
(45) i 1,
I
$1
% j
x4
%7
X87
$4
4 01,
a;:
XB,
4
4
xi,
= P 2 1 ~ + 3 ~ ~ ~ x1,8 ~1 ,4 * 1
1
1, 1, 1 = + ( X ~ - ~ ~ ) ( ~ - ~ ~ ) ( I C ~ - ~ ~ C ~ ) ( Z ~ - X ~ C ~ ' ) ( ~ G ~ - X
, z-2
' 2:,
I
1
e, 4,
z:,
F
1,
7
x3
1
22,,xi,
a:, m%,
x4
xi
4, x:
Induhtive Erregung.
523
Von besonderem Interesse ist das Potential P, am Spulenende. Durch (35) und (46) bis (49) ist dasselbe durch die
Anfangspotentialdiffereiiz im Primarkreis ausgedriickt.
Es ergibt sich B, zu B, konjugiert komplex, ebenso B4 zu B3,
gerade wie auch x2 zu xl, x4 zu x3.
Daher ergibt sich P, nach (35) gleich einer reellen GroBe.
Um das Resultat ubersichtlich zu gestalten, kann man
die Qleichung (36), wenn man fur die 5 die Werte (34) einsetzt, in der Form schreiben:
Setzt man also
v1
- vr
(50') B l e - a i t e t i T
+B3e-aale
- iPY:3
t
2
=
- ~ B ~ Z L ,
wobei B und x reelle GroBen sind, so wird
V,
(51)
= 2 B sin
(v ~ it + x)
"9
B bezeichnet also die Amplitude des Potentiales am Bnde der
Teslaspule. Es entstehen Schwebungen fur Y,, d a B von der
Zeit abhangt.
Aus (50) folgt nun, da B, konjugiert komplex zu B, ist,
und B4 zu B,:
(52)
{ B 2+= Bl
Zi2e-281t+ B 3 B 4 e - 2 8 s t
e-(S1+S"t[[B1B4ei(vi-VYa)t
+ B, B, e - i -
( ~ i 1'2)
1.
1
Nun ist nach (46)
(53)
B,
=
- p,,
- s, + i v,
P(Cy + v;).
;
2 2 Y1 [ - (hi - 3,) + i ( Y I
~
_
_
[ - (4 - 4) + i
_
_
~
- v~J ]
+ v2)I
Da nun Sl - S, stets klein gegen Y, + v,, und cYl klein
gegen v, ist, so kann man setzen bis auf erste Ordnung in
8, bez. 6,:
34 *
P.Brude.
524
Analog ergibt sich
.
Y[
- vt
~1
~
- 2 (5, - 8,) ’
-
Folglich ergibt (52):
wobei
Man kann auch B in der E’orm schreiben:
Fur den Fall der Resonanz vl = v, w i d
(58)
B nimmt einen maximalen Wert an, wenn ist
(59)
e(4
-
82)
f
= Ji :J,
d. h, fur t
= 1%
~~
4 I JB .
9, - 8,
Dieser maximale Wert (bei Resonanz) ist nach (58) uncl (59):
Dies stimmt mit der von B j e r k n e s l ) erhaltenen Formel
iiberein.
Fur Y, = v,, d1 = nimmt (58) die unbestimmte Form O/O
an. Wenn man aber in (57) nach Potenzen von v1 - v2 und
Jl - J, entwickelt, so entsteht, falls man fir sp in (56) Bur
schreibt sp=(vl-v,)t,eine bestimmteForm, d a ( v , - ~ , ) ~ + ) 8 ~ - J ~ ) ~
i m Ziihler nnd Nenner auftritt. Nan erhalt dadurch:
$ 1 3 t e - 6, t
(61)
= P,,
4
I) V. B j e r k n e s , Wied. Ann. 65. p. 134, Formel @a). 1895.
-
hiduhtive Erregung.
525
welche Formel ebenfalls von Bjerknes’) abgeleitet ist. Ob
es bei y gestattet ist, in seinem zweiten Gliede den Greazubergang v1 = v2 friiher auszufiihren, als in seinem ersten
Gliede (w,- v,) t, mag hier nicht untersucht werden.2)
111. Messung der Periode und der Diimpfung.
Wir nehmen den Sekundarkreis als unveranderlich an.
Von praktischem Interesse ist sowohl die Frage, ob die Wirkung
im Sekundarkreis bei stetiger Vednderung der Frequenz v1
des Primarkreises ein Maximum annimmt, wenn genau w1 = v2
ist, als auch die Frage, oh man aus quantitativer Susmessung
der Resonanzkurve, d. h. aus quantitativer Messung der Wirkung
im Sekundarkreis bei verschiedener Frequenz v1 des Primarkreises die Dampfungen 8, und S, der beiden Schwingungskreise bestimmen kann. Beide Fragen hat B j e r k n e s 1. c.
behandelt, sber nur fur den Integraleffekt erschopfend. der bei
elektrometrischen, bolometrischen, thermoelektrischeu (Joulewarme) MeBmethoden md3gebend ist. Indes ist sein Verfahren
insofern noch zu verallgemeinern, als es einen Unterschied
nusmacht, ob man zur Veranderung von vl die Kapazitat C,
verandert, wahrend Lll festbleibt3) (Fall a), oder ob man urngekehrt C, festhalt, aber Lll variiert (Fall b). Im Fall a) ist
namlich nach (22) der fur die Wirkung im Sekundarlrreise
maBgebende Koeffizient p,, nicht konstant; er ist, falls d2
gegen v2, d. h. ya gegen 4 7c2 vernachlassigt wird, in die Gestalt zu bringen :
1
(62)
~-
Ln
4.
Pa1 = a lJ,, v ;
7
~
1) 1. c., Formel @a). Aus ihr folgt:
%lax. = P 9 1
F3
4 e d ,-
2) Der Fall v1 = vz, dl = 8, erfordert streng genommen eine besonderc Behandlung schon von Gleichung (28) an, d a diese dann Iautet:
xa + 2 dl z + v: + 8 ; = & x* Ic, iind Ic nicht mehr gcgen 1 vernachlassigt
werden darf, d a nach (40) die B proportional zu Ic sind. Aus diesem
Grunde ist es auch unmtjglich, daE die Wurzeln z paarweise genau gleich
werden, was aus (34) fur 9, = d2, v1 = vp scheinbar hervorgeht.
3) Dieser Fall lie@ meinen Beobachtungen [Ann. d. Phys. 9. p. 293.
1902) zugrunde.
P. Brude.
526
im Fa11 b) dagegen ist pal konstant, vorausgesetzt, da6 bei
Anderung von All der Koeffizient der wechselseitigen Induktion AB1fest bleibt, was man experimentell leicht realisieren
kann. Den Bjerknesschen Betrachtungen liegt nur der Fall b)
zugrunde. Aber auch in anderer Hinsicht ist eine Erganzung
zu dieser grundlegenden Arbeit von Bj e r k n e s wunschenswert,
namlich um die Maximalamplitude ebenfalls erschopfend zu
behandeln, weil sich herausstellt, daB durch Kombination der
Beobachtungen der Resonanzkurve des Integraleffektes und
der Maximalamplitude die Dampfungen Jl und 8, beider
Schwingungskreise einzeln zu berechnen sind, was meines Erachtens ein einfacheres Beobachtungsverfahren bedeutet , als
die von Bj e r kn e s vorgeschlagenen. Die Maximalamplitude
(groBter Wert des Potentiales 7,) kann aus Funkenschlagweiten
oder besser aus der elektrischen Ablenkung der Kathodenstrahlen in einer Braunrohre experimentell quantitativ erhalten
werden.
Die Betrachtungen sowohl fur den Integral- als fur den
Maximaleffekt haben an die Formel (55) anzuknupfen. Setzt man
so schreibt sich ganz allgemein in jedem Falle der im Sekundarkreis eintretende Effekt E in der Form:
E =P(l
(64)
+
- bc'),
C Z ~
wobei a positiv und negativ sein kann, b aber stets positiv ist.
2, ist sehr grog gegen 1. Das Maximum Em von E tritt nach (64)
ein hei
(65)
und hat den Wert
zm= P (1 +
g).
Setzt man
(67)
5=Cm+q,
v1=va(1+Cm+q),
d. h. bezeichnet 4 die prozentische Differenz der Frequenz vl
von der Resonanzfrequenz v, (1
worunter die Frequenz
+ c,,),
Induktive Erreyung.
527
verstanden sein soll, welche die groi3te Wirkung im Sekundiirkreis ergibt, so folgt aus (64):
3 = P 1+
(68)
d. h. nach (66):
(
&-E
-~ -
(69)
Em
T+,e),
a*
= b97'.
q ist experimentell bestimmbar , wenn man den Primarkreis
nach Schwingungsdauer oder Wellenlange geeicht hat. Daher
gibt (69) an, in welcher Weise man quantitativ aus der Resonanzkurve den Koeffizienten b berechnen kann. Die Resonanzkurve ist um so steiler, je groBer b ist. Hat man so b gefunden,
so ist die Schwingungsdauer v2 aus (65) und (67) zu berechnen,
menn man a kennt.
Es wird nun die spezialisiertere Betrachtung lehren, daB
man wegen der Abhangigkeit l) der Dampfung 8, von der
Frequenz v, den Koeffizienten a nicht rechnerisch angeben
kann, aber wohl ist b aus (55) theoretisch ableitbar. 6 hangt
nur von den logarithmischen Dekrementen yl und yB (bez. den
Dtimpfungen 8, und 8.J der beiden Schwingungskreise ab.
N a n erhalt doher aus den Resonanzkurven Aufschlup uber diese
von beiden Dampfungen abhangende Qr6j3e b, dugegegen nicht
iiber den yenauen Wert der Frepuenz v2. Kombiniert man
aber die Resonanzbeobachtungen an den zwei verschiedenen
Effekten (Maximal- und Integraleffekt) miteinander, so kann
man auch v2 genau bestimmen, sowie die Dampfungen beider
Schwingungskreise einzeln. Dies wird nun gezeigt werden.
Wir behandeln jetzt die einzelnen Falle gesondert.
1) Eine solche kann bestehen sowohl direkt wegen veranderter
Frequenz, ais auch wegen veranderter Stromstarke il (bei variiertem Cl).
Auch B j e r k n e s zieht dies in Rucksicht.
2) Das scheinbar abweichende Resultat von B j e r k n e s wird nur
dadurch veranldt, daS er die Resonanzkurve auf gro6ere Werte ausdehnt, 81s wir es hier tun wolIen. Tatsiichlich bedeutet die B j e r k n e s sche Bestimmung der Differenz des Isochronitiititspunktes und des Resonanzpunktes (es handelt sich dabei urn die von B j e r k n e s mit S bezeichnete
GriiBe) auch nur eiue Taxierung der Gr66enordnung, vgl. V. B j e r k u e s ,
1. c. p. 150.
P.Brude.
528
1. Die Maximalamplitude. a) C, wird verandert. Formel
(57) und (62) liefert fur den nach (59) folgenden Wertl) von t
fur die im Laufe der Zeit erreichte groBte Amplitude des
Potentiales 7, :
(70) Bnfax.
=P L z l
L,1
wobei bedeutet
&w&-&
q
=
v
T
B'uhrt mail (63) ein, so entsteht:
wobei ist
(7 3)
Diese GriiBe b ist bestandig positiv und sehr grog gegen 1.
In der Tat liefert eine Reihenentwickelung :
(74)
b =-
V2
L2(1
11
103
+ x + =13x 2 + --x3
+ --x4
+ . ..) ,
15
140
gesetzt, gultig fur Sl > Sa, oder
128, \
x = 1 - cYa/S1
(75) 6 = - 2 , (V12
12 8,
,
1
S, < S,.
+ x + - - x13
~ + $ - 1 x 3 + - - - x 103
4+...
15
15
140
x = 1 - Sl/S, gesetzt, giiltig fur
Entwiclcelt man (72) nach 5 und behalt nur erste Potenzen von bei, abgesehen vom Gliede bCa (wegen der GrbBe
des Faktors 6) und setzt
<
(7 6)
$-= sg,
so wird (721:
1) Dieser Wert von t gibt streng genommen nur fur v1 = v, die
grijEte Amplitude BMax.Da aber v1 nahe benachbart zu vz angenommen
w i d , ktinnen wir diesen Wert von t benutzen. Die Beriicksichtigung
dieser Veranderung von 2 wiirde nur auf den Koeffizienten a in Formel (64j
EinfluB gewinnen, auf den es nicht genau ankommt nach oben im Text
nngestell ten Uberlegungen.
Induktive Errepng.
529
Nennt man Y z das Quadrat der maximalen Potentialdifferenz 2 BMax.
zwischen den Enden des Sekundarkreises, so
nimmt nach (77) B2 die Gestalt der Formel (64) an, wobei ist
wahrend 2, den aus (73), (74) oder (75) folgenden Wert hat.
Nach (69) gewinnt man also aus der quantitativ in der Niihe
der Resonanz ausgernessenen Resonanzkurve :
AufschluE uber den Wert von b, wahrend nach (65) die Resonanzfrequenz liegt bei
(79) v1 = v2 (1
+ 5,)
i
= u2 1 -
1
~
+
~-2
d (1
b, - fi2
- -c:Tlog+)).
d
2b
Ob der Wert von gm positiv oder negativ ist, d. h. ob
die Resonanxfrequenz groBer oder kleiner als die Frequenz vg
des Sekundairkreises ist, kann man ohne Kenntnis des Wertes 8,
d. h. der Abhangigkejt des
von ul, nicht sagen. Jedenfalls
ist 6 von der GroBenordnung des c?~ oder a,, so daB nach (79)
und (73) 5, von der GroBenordnung
ist, wobei y l , y2 die logarithmischen Dekremente der beiden
Schwingungskreise bedeuten (vgl. Formel (30') p. 520). Selbst
wenn yl = 1 sein sollte, was einer sehr gro6en Dtimpfung
entspricht, die nur eine schwach ausgepragte Resonanz geben
wurde, und y2 = 0, so wurde daher die Resonanzfrequenz des
Primarkreises nur urn etwa 1-2 Proz. von der Frequenz v2
des Sekundarkreises abweichen. l)
b) All im Primarkreis wird verandert. Nach (22) p. 519
ist p,, konstant. (57) liefert fur den nach (59) folgenden Wert
von t und nach (63):
-~
1) Bei meinen Versuchen (Ann. d. Phys. 9. p. 293. 1902), bei denen
ich die Eigenschwingungsdauern von Spulen durch Resonanz ermittelt
babe, war dieselbe sehr scharf ausgebildet, so daS der Febler sicher
unter 1/4 Proz. lag.
P. Brude.
530
Dagegen
wobei 6 denselben Wert (73) wie im Falle a) hat.
ist hier
3d 3= a g
(81)
dl;
im allgemeinen verschieden anzunehmen von dem Werte d 6,ld 5
im Falle a) (vgl. die dortige Formel (76)). Entwickelt man
(80) nach Potenzen von 5, so entsteht die Form (64), wobei
aber jetzt ist
Aus der ResonanzkurTe erhalt man also wiederum AufschluB uber die GroBe 6 , die Resonanzfrequenz liegt aber
nach (65) jetzt bei
(83) v1 = v,(l
+ 5,)
i+
= .u2 1
28’
4-4
3-
i l - - -. L
3
d,
fi2
lo$;:-))
26
Vergleicht man diese E’ormel mit der entsprechendeii
Formel (79) des Falles a), so erkennt man, daB, wenn nicht
eine starke Verschiedenheit der Anderungskoeffizienten d und 8
das Resultat beeinfluBt, die Resonanzfrequenz in diesem Fslle b)
groBer ist als im Fnlle a).
2. Der Integraleffekt.’) Beobachtet man die durch den
Sekundarstrom i2 in der Mitte des Sekundarkreises (i, = i i )
entwickelte J o ule-Warme, was durch ein dort eingeschaltetes
Thermoelement bequem geschehen kann, so kommt es auf den
Integraleffekt an
Da aus (3), (8) und (17) folgt
d TVh
ii = 2 C ----a
(vgl. Anm- 1 auf p. 519),
a
dt
1) Man kann auch aus dem Integraleffekt auf einen metallisch geschlossenen Sekundtirkreis (der also keine Eigenschwingungen besitzt) dic
Dtimpfung im Primfirkreis bestimmen (vgl. R. L i n d e m a n n , Ann. d. Phys.
12. p. 1012. 1903). Diese Methode ist besonders bequem, wenn man die
Anderung der Dtlmpfung yl durch Variation von C, oder F studieren
will. Ich gedenke bei anderer Gelegenheit ausfuhrlicher darauf einzugehen und die Messungen eu publizieren.
InduRtive Er regung .
53 1
so ergibt (51) auf p. 523:
m
=
2 C i (v, + vJ2l B 2(1 + cos [(v, + v2)t
+ 2 y]) d t.
0
Dei: Effekt des zweiten mit dem cos multiplizierten Gliedes
unter dem Integralzeichen ist zu vernachlassigen l) gegen den
Effekt des ersten Gliedes, so daB entsteht:
W
J=
ac;(v, + v , ) y B z d t .
0
Setzt man hierin den Wert von B nach (55) ein, so folgt,
da ist
1.-
+
cos (u t
+ @) d t
= e - (6, + 8,)
t
a sin@ t
+ B) - (d, + B2)cos(a t + @)
az
+ (a, + a2y
Y
Da'bei ist (vorubergehend) gesetzt nach (56):
Da man nun J2 gegen v 2 vernachlassigen kann, so folgt:
Nun ist in der Nahe der Resonanz (vl - u,)~ zu vernachliissigen neben (vl+ 9), vl v, (8, + 8,) : 8, vz 8, v1 , dagegen
oder neben (8, -8J2.
nicht zu vernachlassigen neben (8, +
Daher ist die letzte Formel zu schreiben:
+
l j Vgl. V. Rjerknes, 1. c. p. 137.
P. Drude.
532
oder:
Hieraus ist ersichtlich, d a b man aus Beobachhing der
Resonanrhurve des Integraleffektes Aufschlup iiber S1 8, erhalt,
+
ein Resultat, das schon B j e r k n e s l ) abgeleitet hat. Zur genaueren Diskussion, in welcher Weise
Sz aus der Resonanzkurve gewonnen wird, sind aber wieder die zwei Falle zu
unterscheiden, ob bei Variation von vl nur C, verandert wird,
oder nur Jll.
a) Die Kaparitat Cl im Primiirkreis zoird verandert. Nach
(62) p. 525 ist in diesem Falle zu setzen:
+
Daher wird (84) in Riicksicht auf v,”= 1 : LZ2C, [nach (22)]:
Setzt man nun nach (63)
ill
= w2(l
+ 5) und
nach (76)
d S , : d < = S < , so wird
d. h. man gewinnt wiederum die Form (64) mit der Bedeutung
der Koeffizienten
4, s
b=
”2
(Pny(85)
=-
( q+4 )’=
42-
rl + r2
Aus der Steilheit der Resonanzkurve nach (69) ergibt
sich also y1 + y 2 , wahrend die Resonanzfrequenz aus (65)
folgt zu
(8,- 64) .
v1 = v, (1 5,) = t,, 1 - 8, 6-
(4
+
i
~
2 4,
;)
Y2
Vergleicht man die Werte 6 nach (74) oder (75) mit dem
hier ermittelten Werte b nach (85), so erkennt man, dab
letzterer immer etwas grijBer ist als ersterer, am meisten fur
1) V. Rjerknes, 1. c. p. 148.
Indvhtiue Erregung.
533
den Fall S, =az. Da nun nach p. 527 groBeres b eine steilere
(ausgepragtere) Resonanzkurve ergibt , so ergibt die Beobachtung des Inteyraleffehtes scharfere Resonanz, als die des Maximaleffektes, ein Resultat, welches auch B j e r k n e s 1. c. p. 165 schon
gezogen hat. 1)
Durch Kombination der Beobachtunyen der Resonanzkurve
des Integraleffektes und des Maximaleffektes kann man die logarithmischen Dekremente y1, ya beider Schwingungskreise einzeln
berechnen, dn man nach (73) und (85) dadurch zwei verschieden
aus y1 und yz zusammengesetzte Ausdrucke berechnet. Dies
crgibt eine experimentell leicht ausfuhrbare Bestimmungsinethode von y1 und yz.
Wenn man in dieser Weise y1 und yz, oder Sl und S,
berechnet hat, so ergibt dann das Qerhaltnis der aus beiden
Bffekten erhaltenen Resonanzfrepuenzen (79) und (86) auch eine
Formel zur Berechnung des hderungskoeffitienten 8, d. h. auch
des yenauen Wertes der Frequent va des Sekundarkreises.
b) Ll1 im Primarkreis wird veruindert. Nach (22) p. 519
ist p z l konstant. Nach (84) wird
wobei 6' den nach (81) definierten Anderungskoeffizienten von Sl
bedeutet. Aus der Resonanzkurve ergibt sich also wiederum
v, ;Sl + 8, , die Resonanzfrequenz liegt bei:
Diese Resonanzfrequenz wird im allgemeinen grol3er sein, als
die Resonanzfrequenz im Falle a) nach (86). Ein Vergleich
mit (82) lehrt, dal3 man auch irn Falle b) durch Kombination
der Beobachtungen des Integral- und des Maximaleffektes S,,
a,, d', vZ experimentell bestimrnen kann.
1) Auch fur v1 = v2 hiingt der Integraleffekt vie1 mehr von der
Dlmpfung ab, da er nach (84) der vierten Potenz von J1,* umgekehrt
jmoportional ist, als die Maximalamplitude, da uach Anm. 1 p. 525 VGax,
proportional l/J: ist.
P. Drude.
534
1V. Die magnetiRche Koppelung iet nicht eehr klein.
Wenn die magnetische Koppelung ka (Formel (33)) nicht
gegen 1 zu vernachlassigen ist, so benutzen wir die Gleichungsform (II),(25) und (29). Fur
(89)
{
I
-
P(t;+ 793(t,”+ 9.;)=s0.
Die vier Wurzeln z dieser Gleichung miissen die Form
haben
(90) z , = / ? + i t , z 2 = @ - i t , z 3 = - / ? + i t ’ , z , - - P - i d .
Da nun (89) identisch ist mit
[z - 2,) (2 - Za) (z - z3)(2 - zg) = 0,
so ergibt die Koeffizientenvergleichung dieser letzten Gleichung
mit den Koeffizienten der Gleichung (89) und Benutzung der
Form (90):
+ t;ti - k2 (t;+ 9.:)(ti+ 8:).
Quadriert man die erste dieser Gleichungen und zieht davon
die mit 4 multiplizierte letzte Gleichung ab, so entsteht:
(92)
{
- 8 p a ( t 2 + t ’ 2 )= ( t : - ~ -, 2”()t ;~+ ~ i ) ( 9 ~ - 8 ~ ) ’
+ 4 P (ti+ q ( t ;+ 8;)= P.
(ta-t”)!’
Nun macht es einen wesentlichen Unterschied, ob diese
GroBe P positiv oder negativ ist. Bei nicht zu kleiner magnetischer Koppelung R2 ist P jedenfalls positiv. Wenn wir
in der Nahe der Resonanz tl= t, beobachten, so folgt aus
der zweiten der Gleichungen (91), da6 jedenfalls eine der beiden
GrGBen @ oder t 2 z ’ ~sehr klein sein mu6. Falls also P
-
535
Induktive Erregung.
positiv jst, muB dies nach (92) die GroBe
wir erhalten fur r1 = ra:
(93)
B
selhst sein, und
- t ’ 2 = 1/P fur tl = t2.
Die vier Wurzeln y haben also gemeinsame reelle An= 0,
22
teile, aber die imaginaren Anteile sind verschieden. Es entstehen also bei nicht zu kleiner magnetisc’ler Koppelung im Resonanzfall t, = r2 zwei Schwingungen mit uerschiedenen Perioden
und verschiedener I) Bampfiing, denn es ist dann, falls i
P gegen t 2
vernachkssigt wird, d. h. 2, das Quadrat des logarithmischen
Dekrementes gegen 4 z2, was stets zuliissig ist, wenn Resonanz
uberhaupt deutlich zutage tritt :
-
Die langsamere Schwingun,g (Periode
t) hat also kleinere
absolute Bampfung und kbineres loyarithmisches Bekrement als
die sclinellere Schwi?yung (Periode
t’,t’< r).
Wenn umgekehrt P negativ ist, was bei sehr kleiner
Koppelung k 2 in der Nahe der Resonanz eintreten kann, so
wurde t 2- t f 2klein,
grog sein. Mit Vernachlassigung von
k Z wiirde dann (91) die Wurzeln liefern: t = t,, z‘= t2,
@ = - +(sl
- tY2), d. h. nach (88) und (90) ware
y2 = - 6,- it,,
y1 = - i?, + i t , ,
y3 = - 7 9 2 + i t 2 , y4 = - 79, - 2 t2.
-
Dies ergibt also den unter 11. schon hehandelten und dort
erledigten Fall.
1) hf. W i e n (Wied. Ann. 61. p. 177. 1897; vgl. auch Ann. d.
Phys. 8. p. 695. 1902) hatte abgeleitet, daB der Fail der magnetischen
Koppelung sich nicht wesentlich untervcheidet von dem der elektrischen
Koppelung, und da8 daher die Dampfungen beider Wellen gleich seien.
Dies Resultat ist aber nur giiltig, wenn die Koppelung k2 (nach W i e n s
Bezeichnting q1 p2) klein gegen 1 ist, weil sonst die Wienechen Vernachlassigungen unstatthaft sind. Mit A. O b e r b e c k (Wied. Ann. 66.
p. 631. 1895) steht das hier abgeleitete Resultat in Einklang.
2) Vgl. oben p. 521, Formel (32).
P. Drude.
536
Wir haben also hier P als positiv anzunehmen. Die allgemeine Losung von (91), (92) gestaltet sich in der Nahe der
Resonanz t1 = t, einfach, weil dort Q klein ist, d. h. auch
pa klein ist gegen t a In (92) kann daher in dem Faktor
yon 8 pafur t 2+ i 2
derjenige Naherungswert t 2+ z ' =
~ r;"+ t i A
eingesetzt werden, der sich aus cler ersten der Gleichungen (91)
ergibt fur = 0 und mit Vernachlassigung von +(al
Daher
ergibt
dann
(92):
gegen t f ti.
+
(94)
- 8 p2(T; + ti)= P.
(r2- t")
Die zweite der Gleichungen (91) und (94) kann man nun
und p2 auflosen.
leicht nach den beiden Unbekannten t 2Man erhalt:
t I 2
Da in der Nahe der Resonanz 8 Q 2 ( t ; + ti) klein gegen
Y 2 ist, so kann man fur (95) setzen:
wahrend die erste der Gleichungen (91) liefert:
(9 7 )
22
+
7'2
=
.;+ t,"+ --.
2P
Q2
Es gilt nun weiter (fur beliebige Werte der Koppelung P )
nach (251, (88) und (90):
< = Al eti*l + A,
V2 = Bl
eth
+ B2
etlyz
&a
+ A, et/*g + A, et/ ,
+ B3 &s + B, efhr .
34
Induktive Xrregung.
537
Aus (1) folgt:
-A1 e t / y , + 2
A et/Yz + 3 et/Ys +
21
. --
(100)
Ys
Yz
Yl
et/Y, ,
Y4
aus (3) und (8):
n i a =
$h
(101)
4
et/Yi
+ Bz et/yz + B.et/Ys + 3 et/Yi .
~
Ya
Y1
Ys
Y4
Die Anfangsbedingungen (38) ergeben daher:
(102) z d n = P ,
z’-=O,
ZBn=O,
Yn
z”-=o,
Yn
n = 1, 2, 3, 4.
Nun ist nach der zweitenl) der Gleichungen (27):
Durch Addition dieser aus R = 1 , 2 , 3 , 4 gebildeten
vier Gleichungen entsteht wegen der Gleichungen (102):
C ’nyn = 0 .
(103)
Ferner folgt aus (27):
+ +
+
+
B n ( r ; 9.; 2 9,yn yn2)= P,, ”,(.
I F ; ) An.
Durch Addition dieser vier Gleichungen folgt wegen (102)
und (103):
C B n y 2 = p,, (xi + 9.i)F.
(104)
Aus (lO2), (103) und (104) lassen sich die B sofort als
Determinanten-Quotienten ausdriicken, z. B. ist
~.
B1
i
1,
1,
1,
1
Y1
Yz 9
YB?
Y;,
YS)
v4
I
Y?3, Y?
1) Aus ihr folgt nuch fur den Fall z1 = z
, und
= a2, daS
A l : B l = z : -r2:palzi ist, alsonegativ,dar>z,, unddaBA,:B,=r~-z’2:p,lz%
ist, d. h. positiv, da z’< ra ist. In Riicksicht auf (100) und (101) ergibt
gleich gerichtet ist, die Schwindies, daB die Schwingung z in il und iz
gung r’ aber in il und ia entgegengesetzt verliiuft. Daher kann man die
Periode T durch eine in der Nahe von il aufgestellte Resonanzleitung
(vgl. P. D r u d e , Ann. d. Phys. 9. p. 611. 1902) eventuell leichter messen
als die Periode z’.
Annalen der Physik. IV. I’olge.
13.
55
26
1
_.
2@
+
+ i l r + r’)
&T
i(r
+
fz’
T’)
l
und es ist, wenn man 19.; gegen r : sowie pz gegen 7 2 vernachlassigt, was gestattet ist, falls es sich iiberhnupt urn Resonanz handelt, und die Abkiirzung benutzt:
21
(106)
2
2 (T
+ z’) (4(la + ( T - T’)‘]
’
B,
=
+ pal r ;
7l$
r-r‘-27s+i
2 p + ,
L ( r t’) 14 pa (T T ’ ) “ ] - ~
+
+ -
1
daher wird nacli (98):
wobei (I) bedeutet:
Wenn wir zuiikchst fragen, fib noelcltes Vidiiiltnis tl:t,
h. & ein Maximum annehmen, so ist, gerade wie oben p. 526, zu unteischeiden, ob
bei der Variation von t, die Kapazitiit C, variiert wird, oder
die Selbstinduktiori Lll. Abgesehen hiervon kann man aher
ziir Taxierung der Verhkltnisse f‘olgendes iius (107) ablesen :
Bei Variation von tl (wfihrend t, fest bleibt) andert sich 7,
hauptsiichlich wegen des Nenners 4 p2 + (t- x ’ ) ~untl wegen
des Zahlers (b, wiihrencl p,, sicli iiur unwesentlich iindert.
Da nuii B in der Nahe der Resonariz selir klein ist, und da
aucli S ltleiri ist, so tiat @ einen Wertl), der bestandig kleiner
als 2 (t.- t’)ist; wir konnen also setzen:
7uird starkste Indiiktion eintreten, d.
109)
(b = 2 (1 (t - t’), (1
< 1.
Der Wert des echten Bruches (1 variiert iiiclit sehr stark,
wenn tl: t2 sich iindert. Mit der bei starker Koppelung zuliissigen Veiiiachlassigung von 4p2 gcgen (t- T ’ ) ~wird also
(107):
Maxirnitle Incluktion tritt also ein, wenn die Perioden der
beiden iriduzierteri Schwingungeii sich mijglichst nahe komnien,
1) Line Zerlegung des @ in eiii Produkt
hat nur dsnn physikalischen Sinn (Schwebungen), wenn z lmhezu gleich
z’ ist. Das korinen w i r hier bei starkerer Koppelung nicht voraussetzen.
35 *
P. Drude.
540
d. h. wenn t 2- t ' 2 ein Minimum ist.
naherungsweise zu setzen:
I(.:
Nach (92) ist nun
+
a. - t ' 2 =
- .:)a
4 k2 T: Z:
(111)
woraus her'vorgeht, daB f i r tl = z2 rlas BIinimurn fur t? - T '
eintritt. Starkste Induktion tritt also jedenfalls in der Nalie der
Isochrunitiit z1 = z, ein. Zugleich gelit aucb aus (1 10) und
(111) hervor, dak die Resonanzkurve urn so flacher wird, j e
stiirke~die Koppeluty istl), denn wenn ka groB ist, so andert
sich r2 bei Veranderung von tt - T: prozentisch vie1
weniger, als wenn k2 klein ist.
Zur Messung vun Schwingungsdauern durch Resonanzversuclte muF man also mit miylicltst kleiner Koppelung arbeiten.
V. Die Wirksamkeit der Teslatransformatoren.
Nach den zuletzt crhaltenen Resultaten konnen wir den
Resonanzfall z1 = T~ voraussetzcn, und erhalten dann aus (92)
und (110), da nach (91) /Istreng gleich Null zu setzen ist, fur
das Potential a m Ende der Teslaspulc den Wert:
Fur pa, ist der Wert nach (22) eingesetzt.
Da n u n nach (33) ist
so folgt
oder da i m Resonanzfall JllC, = LX2
C, ist, und unter Rucksicht auf (32):
__
1) Dies geht sowohl aus den Messungen von F. R i e b i t e , Ann. d.
Phys. 6. p. 895. 1901 hervor, der die flachere Resonanzkurve interpretierte
als eine Zunahmc der Diirnpfungen, als auch aus zahlreichen Beobachtungen von mir, nach denen ich stets die Rcsonanz um so scharfer erhielt, je schwitcher ich die Koppelung wiihlte.
~
54 1
Itiduktice Errepn,q.
Naoh (105) ist S = 9, + 8 , / 2 , und man kann setzen
t
il. t
1-s
cos - -- sin - = cos ____.
T
d a (S:
+
T
z
T
gegen 1 zu vernachlassigen ist.
Futirt man eine neue Zeit tl
=
Folglich wird
t - S ein, so wird
Da die Maximalwerte von Q zu Zeiten tl erreicht werden,
welche etwa gleich TI oder gr68er als TIsind, so kann man S
neben t, im Exponentialgliecl vernachlassigen und erhalt, wenn
man wiecleruni t fur tl schreibt:
Urn das absolute Maximum von & zu finden, ist diejenige
Zeit t aufzusuchen, zu welcher !I ein absolutes Maximum wird.
Bevor wir dies tun, mogeri einige allgemeine Bernerkungen
an (112) angelrniipft werden.
Wenn wir yon der Dampfung y1 und yz absehen kiinnteri,
so wurde QhIax. den Wert 1 haben und es wurde nach (112) K2
von der Starke der Koppelung ganz unabhangig sein, falls
wie bei linearen Stronilrreisen L,, = L,, zu setzen ware. Nach
1) Diese Formel stimmt mit der von A. O b e r b e c k (Wied. Anc.
65. p. 629. 1895) abgeleiteten Formel (20) bis auf den Faktor
p. 5 1 S 1r.iiclist aber L,, : L,, mit wacliscnder Koppcluiig, d d e r
iii@
ails diesenr G w n d e u n d we.qen vorIiande7acr Ilampf i ~ n g(lie
c
Koppelic ng zu iiehiri en.
Erreguiig dei. l’eslasptile n i it ~ c uf~sender
Nich t induktiv iuirkeidc (tote) 6 e Zbs tiizduhtion des l’i*iniU P
k r ~ i s e sist uus zwei Grunden scfiudlicli fur die beste Wirltsamkcit
des Teslatraiisformatols. Ersteiis wird, da bei vorliegender
Tcslnspule die Scli\\.ii,gungsperioda ~orgcschricben ist, die
Kapazitkt Cl ilcs 1’i.iniiirlireises lier;tbgesetzt, zweitens wird
(lie Koppelung ltleiiier, \vie ails (33) hcrrorgeht.
D a nacli (112) yZ proportioiial zu VC, : C, ist, so mup aZ.w
;UT besteiz lionstruktioii eines l’es1atransforniatoi.s dcr Primarkreis
uus eiricr 1Yindung miiyliclist clicken Brahies bestekoi (dn dadurcli L,,
nioglichst lilcin wird uiid Cl bci gcgcbenem 1: moglichst groW),
wiihreiid die 8ekundarspulc iecht kleine Kupazitat im Yergleicli
zu C, haben mup. Eiiie einzige offeiic Ki~ciswindung alz
Yekund.iirlwcis ist dalier wegcii der daiiiit, verbundeiien Kleinlieit dcs C, relativ gunstig, jedcnfalls giinstigcr als eine aub
wenigcn Windungen (2-10) besteheniie Sekundiirspule, da die
diclit beieinander liegenden Eiidwindungen derselben wclclie
j u eiitgcgengcsetzte elektrische Ladungen tragen , riel grijBere
Kapazitat C, ergebcn. Dieses ltesultat wird durch Vcrsuche,
tlic ich angestcllt habe (Potcntialmessung durch Beobaclitung
yon Funkenlii,ugcn oder qualitativ durch Aufleuchten von
Vakuunirohren) durcliaus bestatigt.
Man muB also Spulen von viol mehr Windungeii anwendeii,
uiii wieder auf giinstige kleine Werte dcr Kapnzitiit C, z u
konimen.
Es sei ?I die Hohc, r dcr Radius, g die Galigliohe, d: die
Drahtdicke, 11 die absolute Wiiidunpzahl der Tcslaspule.
D a m ist die M’ellcnliingc 3. ihrer Eigeiiscliwinguiig bestirnuit
durc11
l / 2 h = l . f ‘ ( f 1 / r914,
,
wobei 1 = 2 r TC n die Drahtliinge der Teslaspule bedeutet,
wahrend f ein voii h / r 1q / S uiid voii der Natur des Spuleiilieriles abhfngeiider Paktor ist, fur den ich fruher l) Tabelleii
iingegeben habe.
Neiiiit man r1 den Radius des Kreises, welcheii derPrimiirh e i s bildet (wir wolleii ihn entsprecheiid unseren obigeii Uber~~~~
1) P. D r u d e , Ann. d. Phys. It. p. 322. 1902.
~
lnduuktive Ewegung.
543
legungen als einen Kreis von einer Windung annehmen), 0, den
Radius seines Drahtes (halbe Drahtdicke), so gilt1) fur die
Selbstinduktion des Primarlrreises:
All = 4 7z r1 [(lognat 8 r1/q,) - 21 ,
daher mu8 fur C, gelten, da ist
2 7z
=
f
a,,ia
falls C, nach elektrostatischem MaBe gemessen wird:
rama f 9
c =---A: 4 zaL,,
z r,
(log 8 r1/el - 2) *
Die Kapazitat C, der Teslaspule (nach elektrostatischem MaBe)
konnen wir jedenfalls in der Form schreiben
C, = r y ( h / r ,g / S ) .
Da nun nach (17)und (1%)p. 517 Azl : Al, und ebenso k 2 nach
(33) nicht von n abhangen, sondern nur von hlr, yl/rl und da
der Maximwlwert, den Q im Laufe der Zeit annimmt, nur von
k2, y1 und 7, abhangt, so gewinnen wir aus (112) den Satz:
Bei konstanten Yeyhaltnissen h l r , r1/ r , r1/el , und bei
konstanten Bekrementen y, und y2 ist das Maximumpotential der
l'eslaspule pivportional mit ihrer Wiiidungszahl.
Es wiirde nun vor allem die fur die Praxis wichtige Frage
zu untersncheii sein: Fur welches Yerhaltiiis h l r der Teslaspule
ist ihre Wirhung bei gegebener Windungszahl n um grGPten? Die
Beantwortung dieser Frage werde ich spater auf experimentellem
Wege geben. Ungefahr liegt das heste Verhaltnis h/r zwischen
1,5 und 3.
Wenn man , was bei Teslatransformatoren meiet erlaubt
ist, (yl - y J 2 : 4 nc9neben k2 veriiachlassigt, so schreibt sich
(I 12) unter Benutzung der Umgestaltung
C, = rI . r / r , . y (I+, g/b) = L,, .y, (h/r, r / r , , r l / p l , g/J)
Nun ist nach (22) und (30')
Daher wird
1) ill. W i e n , Wied. Ann. 53. p. 931. 1894.
P. Drude.
544
w1 besteht nun fast nur aus dem Funkenwiderstande.
Derselbe ist von Jl1unabhangig, dagegen abhangig von F und
von GI. Mit wachsendem C, nimmt er a b . 3 Q nilnmt mit
wachsendem y1 ab. Bei schwacheii Koppelungen kann man
annahernda) p y1 als konstant betrachten (vgl. Abschnitt VI),
wobei unter Q der im Laufe der Zeit erreichte Maximalwert
verstanden sein SOL Daher folgt aus (114'):Die Wirksamkeit
eines nicht sehr stark gekoppelten Teslatransformators hangt bei
konstanten Yerhaltnissen h/r, r / r l , r l / ~ l ,g / S nuv von der Kapazitat Cl des Primarkreises ab, einerlei, ob die Spule kleine Dimensionen iind vie1 Windungen oder gr6pere Dimensionen und wenipr
Windungen besitzt. Bei sehr starken Koppelungeu wachst dagegen p yl mit yl. Baher ist dann gropes n und kleines r
etwas giinstiyer als kleines n und gropes r.
I n jedem Falle kommt es darauf an, dap das zur Resonanz
notQe C, miiglichst grop ist.
Die Ab hangigkeit der Teslawirkung vom Pr ini arf unkenpotential P ist innerhalb gewisser Grenzen geriny; das haben
mir sowohl Versuche gezeigt, als auch geht es aus (114') hervor?
da w1 mit der Funkenlange, daher auch mit P zunimmt.
Eine Einbettung der Spule in Petroleum ist wegen der
damit verbundenen Erhohung ihrer Kapazitat ungiinstig, wegen
der Moglichkeit starkerer Koppelung ka (r annahernd gleich rl)
eventuell giinstig. Der Vorteil wird bei starken Dampfungen den
Nach teil uberwiegen ; bei schwachen Dampfungen dagegen nicht.
Jetzt sol1 untersucht werden, in welcher Weise der Maximalwert des Faktors p in Formel (112) von Dampfung yl,y, und
Koppelung ha abhangt.
VI. Abhangigkeit der Teelawirkung von DEmpfung und
Koppelung.
Nach (92) ist fur den Resonanzfall
,f3 = 0 ist:
z2
- 512 =
tl= z,, da nach (91)
~ _ _ _ _
p=2 z'; l/ka)>-, ; (a,-*
2-
- 2 z: k ' ,
wobei k' eine neue Abkiirzung ist fur
1) Vgl. R. Lindemann, Ann. d. Phys. 12. p. 1012. 1903.
2) Dttbei ist y2 nls klein neben yl augenommen.
lnduhtive Xrreyuny.
545
Nach (91) ist, dn Q (al
- 6 J 2gegen 2 t;zu vernachlassigen ist:
t 2 + .’a=
2t;.
Hieraus folgt
t2= t: (1 + z)? t”= t;(1 - I{).
(116)
Nach (115) ist K 2 nur bei nicht zu kleiner Koppelung k2 direkt
a;ls Koppelungskoeffizient zu deuten. Ganz allgemein knnn
man k’ aus (116) definieren, namlich
Nach (113) urid (32) ist nun
t
2 9 = t‘- a - = .
wobei gesetzt ist
(1 18)
COS-
t
- e -dL cos,, t
7 ‘ .
Fur numerisches Rechnen ist bequemer, ( I 18) in der Form
zu schreiben
t
t
- t
-at
(120)
20=e
7 . ~ ~ ~ - - ea . cos ?,
wobei ist:
22
uJ’= a.-.
(121)
d
Die Abhiingigkeit des g von dem Verhaltnisse t :t‘ und
von dem mittleren logarithmischen Dekrement 9 (ul+ yz) kann
nur durch numerische Auswertung ubersichtlich erhalten werden.
I n den folgenden Tabellen sind die sukzessiven Maxima von Q
und die Werte t/Tl, bei denen sie stattfinden, fur verschiedene
Verhaltnisse 1’:T = t :t‘ und fur verschiedene logarithmische
Dekremente y1 + yz / 2, wie sie in praxi vorkommen k6nnen l),
angegeben. Die absoluten Maxima sind durch Druck hervorgehoben. 1; ist die (gemeinsame)Schwingungsdauer des Primaroder Sekundarkreises, wie sie ohne Koppelung jeder Kreis fur
sich allein besitzt. Die Maxima von 2 0, welche auf das letzte
in den Tabellen angegebene Maximum folgen, sind stets
kleiner, als clieses zuletzt angegebene.
1) Dns Dekrement y = 0,15 ist schon ziemlich klein. Es ist von mir
bei Vermeidung Hertzscher Strahlung beobachtet in Abhmdl. d. stichs.
Gesellsch. d. Wissensch. 23. p. 99. 1896. - Man kann aber noch kleinere
Dekremente herstellen (2. B. 7, =0,09), woruber ich spxter berichten werde.
P. Brude.
546
k' = 0,095,
1': (r' = 1,1,
Nr.
des Mar.
Nr.
les Max.
t : T,
~-
.~
~~
1
2
3
4
a
6
I
S
9
10
Id2 = 0,0090.
0,34
0,78
1,25
1,75
5,24
2,74
3,23
3,73
4,23
4,73
2
t : TI
~~
+ 0,17
0,42
+ 0,63
....
+ 0,97
....
. . . ..
27
2s
29
13,tiG
14,18
14,69
15,19
15,GS
11
....
-
- O,Y3
-
21 *
1,07
+ 1,16
- 1,lS
+ 1,18
90
- 1,15
31
...
+ 142
5,23
... ..
. . . ..
. . . ..
. . . ..
+ 0,lO
10,45
. . . ..
+ 0,36
-
0,37
+ 0,36
- 0,35
+ 0,34
.....
. . . ..
* Vgi. die Anmerkung zur n%Asten Tabcllc bci Max. Nr. 9*
T: T' = 1,25,
6
7
S
9'
10
1I
12
13
, 2,69
'
I
3,18
3,G8
4,41
5,13
5,62
G,11
G,G2
i l
* Xr.
-1,23
+0,93
-0,5G
+0,15
-0,45
+0,64
-0,73
+0,73
2,G9
S,l8
'
3,79
4,41
5,08
5,Gl
G,LO
G,62
-0,94
+0,67
-0,39
+O,IG
-0,29
+0,38
-0,40
+0,38
9 ist fur klcines
Ic' = 0,219,
2,70
' 3,19
3,75
4,41
5,07
5,58
G,O8
G,G2
2
-448
+0,31
-0,17
+0,11
-0,lO
+0,11
-0,lO
+0,09
Iif2 = 0,048.
2,7o
3,20
3,77
4,41
4,99
5,54
6.07
6,G2
-0,25
+0,15
-0,08
+0,05
-0,05
+0,03
-0,02
+0,02
,
2,70
3,22
3,79
4,41
-0,13
+0,07
-0 04
+0.02
-
-_
-
-
= 0,15 und 0,25
>laximum, sonderii cin Xinimum. Zwischeil detn achteu und zehlitcii
Jlusimurn iiimmt uiitnlich dam Q zwei positirc Maxima und cin positives
-
llinimuin au. Letztercs ist unter Kr. 9* rubriziert. Fur grofieres k:-y'
ist xber 9 * wirklich ein Maximum, da zwischc~i8 uud 10 daun nur dim
cine Maximum Nr. 9 licgt.
~-
~
t : TI
t'g
0,2G
0,70
o,(i(i 1,18
- 0,54 1,68
0,37 ?,IS
+0,33
- O,ti4
+
+
T : T' = 1,332,
k' = 0,279,
~ _ _ _ _ .
+0,4G
-1,03
+1,31
-1,25
'1
0,28
0,72
1,20
1,G9
I
T :2"= 1,412,
f0.54
1'
--1,18
+1,40
2': 2" = 1,5U,
~
+ 0,95
- o,s2
~
~
+
0,27
0,7 1
1,19
1,69
+0,41
- 0,85
k ' = 0,332,
~~
k'* = 0,078.
.- - -__
0,37
-0,71
+0,71
- 0,54
0,25
0,69
1.16
- O,b9
1,68
- O,%
4
5*
6
7
0,30
O,i2
1,17
1,64
2,35
3,05
3,53
l
0,27
0,70
1,17
+0,45
~
0,25
-0,Sl
0,68
+0,i7
1,15
1;'' = 0,148.
I;' = 0,384,
~
~~
~~
+O,titi
--1,44
0,29
+0,ti3
0,71
--1,3?
+l,63o 1,li +1,44
0,27
0,60
1,17
-1,22
1,G5 - 1 , O l 1 1,tiG
+0,21 2,35 +0,2G 2,35
-0 9 i 3,04 -0,71
3,Ol
3,5?
+1,10 13,52 +0,77
~
7 ' : 2" = 1,tiG7,
_____
~
1
a
3
4*
5
0,28
O,G9
1,13
1,82
2,50
~-~
-1,0'3
+1,04
-0,Gti
+0,28
-0,3ti
+0,34
~'
- 1,615
0,tiY
-1,49
- 0,24
1,13 +1,31
1,82 -0,31
2,49 +o,uo
1
1
1
~
+
0,26
o,ti9
0,67 -1.21
1,13
0,94
1,82 - 0,36
2,4G
0,50
+
+
0,2G
0,m
+0,51
1,lG
+0,77
-0,45
+0.22
--0,21
+O,l6
-0,91
1,67
2,35
2,97
S,j2
Is' = 0,450,
. __
+0,7G
i
+0,5i
_ _ _ ~
._ _ _ _ ~ .--
0,27
+1,17
+ 0;52
~_________
0,28 +0,49
0,72 -0,97
1,18 +1,03
~~
1
2
3
+u,34
k ' * = 0,110.
~~~~_
~
+0.30
- 0,64
+ 0,50
- 0,313
+ 0,22
.
~
~
I
i
1
I
0,25
0,G7
1,15
1,G8
2,35
2,96
3,51
-~
_
+0,48
- 0,77
f0.58
- 0,31
+0,16
- ,012
+0,OR
A,' L3 = 0,221.
- __
~
0,25
0,6G
1,13
1,82
"44
I
+O,ti2
-1901
+0,70
-0,32
+0,31
0,20
0,65
1,14
1,82
2,43
+0,58
-485
+0,53
-0,2G
+O,?O
Y.Drxde.
648
T:T' = 2,0,
k' = 0,60,
k ' s = 0,360.
--
T: T = 2,5,
--
k" = 0,524.
k' = 0,724,
-__.-1
1
2'
1
0,19
0,67
+1,04 0,17
0,18 +1,18 0,18
0,67 -0,65 ' 0,67
1,34 +0,65 " 1,34
2901 -0,57
2,68 +0,49
-0,68
+0,57
-0,45
+0,35
I/ 2,Ol
2,68
I
I
0,67
1,34
2,OI
2,68
+0,95
+0,65
+0,49
-0,31
+0,24
-__
S
I
I1
1
, 0,15 +l@'1 4 1 5
2
0,69 -1,39
0,69
3' , 1,37 +0,73 ,, 1,37
4 i 2,06 -0,93
2,013
5* 11 2,74 +0,77 11 2,74 I
1
I
1
2*
3'
4+
ti*
6'
,
0,12
0,69
1,39
2,08
2,77
3,47
+1,b(1
-0,69
+0,88
-0,83
+0,81
-0,77
0,11
0,69
1,39
2,08
2,77
3,47
+1942
-1,16
+0,82
-0,W
+0,69 I
+1,46 0 , l i
-0,SO
+0,82
-0,76
+0,69
10,
1. MHX. (zugleicb absolutes) bei
I
,
I
0,69
1,39 +0,70
2,08 -0,58
2,77 +0,49
-0,41
k'= 0,98,
2
T:T = 100,
1. Mux. (zugleich absolute) bei
+1 3 7
-0,83
2
- -_
0.10
+1,12
0,69 -0,76
1,39
0,58
2,08 -0,4B
2,77
0,35
3,47
0,26
+
+
-
+
+
__
0,10
1,OJ
0,69 -030
1,39
049
2,08 - 0,34
a,?? +0,24
3,47 -0,17
V' = 0,96.
= 0,15 f i r 1/
= 0,066: 2 Q =
+ 1,bL
Y = 0,9998.
= 0,15 fir t /
= 0,0017: 2 4 = + 1,OZ.
549
Induktive Erregung.
Die mit * bezeichneten Maxima sind fur kleine y1 +y2/2
iiicht Maxima, sondern Minima des absoluten Betrnges von 2 g
('61. Anm. z u p. 546). Z. B. ist fur
I':Y"=3,
2': T = 2,
T : T = 1,5,
-=0,15,
2
~~
~
2
7; +?''
2
t:5!:=0,45,
20=-1,22.
- 0,15, t : I, = 1,02, 2 p = + 0,82.
- 0,15,
-
t : T, = 2,09,
20=
+ 0,44.
Der Verlauf von 2 g ergibt sich auch aus den graphischen
Darstellungen (vgl. unten).
Zunachst interessieren die absoluten Maxima: die in den
'i'abellen durch Druck hervorgehoben sind. Man bemerk t,
dat3 dieselben mit wachsender Koppelung und abnehmender
Dampfung im allgemeinen zunehnien , jedoch ist bei nicht zu
gropen Dampfunyen (yl y2/2 2 0,5) die Koppelungl) k' = O,S,
k 2 = 0,36, T:T = 2 urn gunstigsten, d. h. auch besser, als
starkere Koppelungen. Die Abhangigkcit der absoluten Maxima2
y o n der Koppelung ist in der beistehcnden Fig. 2 dargestellt,
bei der der Koeffizient It' und das absolute Maximum 2 i j als
Ordinaten aufgetragen sind.
Die Kurven sind von kompliziertem Charakter. Bei der
Koppelung k = 0,6 ist fur y1+yz/2 = 0,15 % i j = 1,74, d. h.
es bleibt um 1 3 Proz. zuriick hinter dew Werte 2 F = 2 , der
fur verschwindende Dampfung eintreten wiirde.
F u r y1+yz/2 = 1 ist bei k'= 0,6 2 F = 0,89, d. h. es ist
etwa halb so grog als 2 F bei y1+y1/2 = 0,15. Bei so starker
Koppeluug hangt also die Naximalamplitude nur roenig von der
+
2lampfuri.q ab, d a g y e n sehr bedeutend bei schwacher Koppelung.
Aus Fig. 2 hat man die beste Anschauung davon, in welchen
Grenzen die Wirksamkeit eines mit guter Resonanz gebauten
'i'eslatransformators durch wechselnde Dampfung schwanken
kann. Dabei ist noch zu beriicksichtigen, daB nach (112)
ivegen des Falrtors
1) Ich gebe hier immer nur den Koeffizienten k' oder K e an. Der
cigentliche Koppelungskoeffizieut k9 ist nach Formel (115) tlus k'¶ zu bercehneti, wenn man 7, - 72 kennt. Fur V 2 = 0,36 ist der Unterschied
zwischen Id4 und kZ schon zu vernachllssigen.
550
P. Dmtle.
das Maximum von T z bei kleinein K 2 rioch wesentlich groWer
wird als
namlich wenn ( y , - y z / 2 n)2 einen merklichen Betrag hat
gegen k 2 ,
Fig. 2.
VII. Anwendung auf die drahtloee Telegraphie.
Verwendet man einen Sendeappnrat mit induktiver Erregung (magnetischer Koppelung), so bleibt Formel (112) im
wesentlicheii bestehen , obwohl der Sekundarkreis (Teslaspule)
zur Vermehrung der Strahlung mit Antennen versehen wird.
Verzichtet man nun irn Empfmgsapparat m f Resonanz, so
kommt es nur auf Erzielung eines moglichst g i d e n ahsoluten
Maximums von P z an. Man wird also die Kuppelung ilt’=O,G
wahlen (nur bei sehr starker Diimpfung y1 + y s / 2 z 0 , 7 5 miiglichst
uoch eine starkere). Verwendet mail aber das Resonawprinzip,
In rlu ktiva Br repiny.
55 1
urn den Kmpfanger nuf den Sender abstimmen zu konnen, so ist der
zeitliche Verlauf des Faktors g i n FormeI (112) wichtig. EY sind
nun zwei Fiille zu unterscheiden, j e nachdem man einen einfacben
ungekoppelten, oder nur sehr lose gekoppelten (zweifachen) Empfangsapparat benutzt, der nur eine Eigenschwingungsperiode besitzt, oder
Kurce in der Umgebung der wit ''
versehenen Maximia) aus der graphischen Darstellung der Fig. 3, welche den zeitlichenverlauf von <)
bei verschiedenemk' nur fur die kleinste Dampfung y1 + y 2 / 2= 0,15
darstcllt, da das Charakteristische dabei am meisten zutage tritt.
Der allgemeine Charakter dieser Kurven besonders bei
kleiiier Koppelung ( T :T = 1,l und 27: T = 1,25) ist d e r der
sogenannten Scfmelrungen, Die erste Schwebung ist bei dem in
den Tabellen mit * bezeichneten Maximum beendet (in den Kurven
durch einen Silttel erkenntlich). Innerhalb einer Schwebung ist
Fig. 3.
P. Drude.
552
der Verlauf von p anniihernd periodisch mit der Periode 'la
TI,
jedoch ist die zweite Schwebung um n in Phase versetzt gegen
die erste Schwebung, so daB auf einen Empfangsapparat, der
als einfacher Resonator mit der Periode
gebaut ist, die
zweite Schwetung schadlich f i r die Wirkung der ersten Schwetung
ist. Wenn man daher bei der drahtlosen Telegraphie Wert
auf Abstimmung legt, so muB man die Koppelung beim Sendeapparat derartig wahlen, dalS die zweite Schwebung eine Maximalamplitude enthalt, die relativ klein ist im Vergleich zur
Maximalamplitude in der ersten Schwebung. Dies ist um so
mehr der Fall, je kleiner die Koppelung im Sendeapparat ist.
Denn nach den Tabellen sind I, R. fur y1+ya/2 = 0,15 die
Maximalamplituden Q1 und
der beiden ersten Schwebungen :
Bei T:T'= 1,1,
,,
= 1,25,
19
=
1,50,
k'=
0,095,
= 0,219,
= 0,384,
2 & = 1,18,
= 1,44,
= 1,63,
2 & = 0,37,
= 0,73,
= l,lO,
&:& = 3,19.
= 1,97.
= 1,48.
Daher muB im Sender die Koppelung sehr klein sein,
wenn in einein einfachen Empfanger scharfe Resonanz erzielt
werden s01l.l) Wenn man nun aher neben scharfer Abstimmung zugleich auch noch Wert legt auf moglichste Empfindlichkeit des Kmpfangsapparates, so ist zu berucksichtigen, daB
die Maximalamplitude
der ersten Schwebung mit abnehmender Koppelung abnimmt. Aus diesem Grunde wird man die
Koppelung nicht zu klein wahlen. Es hangt die beste Wahl
der Koppelung im Sender sowohl von der Dampfung y1+y2/2
im Sender, als auch von der Eigendampfung des Empfangers
ab. Denn es ist klar, daB fur einen stark gedampften Empfanger, dessen Eigenschwingung nach etwa 20 halben Eigenperioden nahezu abgeklungen ist (7=0,4, Amplitude nur 2 Proz.
der Anfangsamplitude), die zweite Schwebung der Koppelung
1':T = 1,1, R'= 0,095, welche uberhaupt erst nach 21 halben
Eigenperioden TI einsetzt, vollkommen unschadlich ist. Daher
el
1) Dies Resultat deckt sich mit dem schon von W. W i e n (Ann. d.
Phys. S. p. 711. 1902) abgeleiteten. Fur kleine Koppelungen ist ja auch
kein Unterschied zwischen den W i e n schen und meinen Entwickelungen
hier vorhanden, sondern nur fur starke Koppelungen, da nach meinen
Entwickelungen fur sehr starke Koppelung, Yon der allerdings zweifelhaft
ist, ob sie sich realisieren Iaflt, wiederum gute Resonanz moglich ware
(vgl. weiter unten im Text).
553
Induhtice Erreguiig.
wiirde fur einen solchen Empfanger jedenfalls nicht eiqe noch
kleinere Koppelung im Sender zu wahlen sein. Aber auch
von der Dampfung y1 y,/2 im Sender hangt die richtige Wa,hl
der Koppelung ab, denn das Verhaltnis Q, : $i2 der Maximalamplituden der beiden ersten Schwebungen wird urn so groUer,
je groBer y1 + y2/2
y2/2 ist, wie folgende Tabelle lehrt:
+
T:!!"= 1,25,
k'=
0,%19.
0,219.
~~
Yl
+ Y,l2
2 I1
(2 e z
91 : Bz
0,15
1,44
0,73
1,97
T:T'=
rt
+ rzl2
2 Pl
2 Be
G1
:a 2
0,15
1,63
1,lO
1,48
0,5
0,25
1,20
0,40
3,o
olll
1,50,
k'= 0,384.
0,25
1,44
0,77
1,87
0,5
1,09
0,36
390
0,82
735
0,75
0,61
0,05
12,2
1,o
0148
0,02
24,O
0,75
0191
0,22
471
1,o
0,77
0,16
4,s
Wenn also z. B. die zweite Schwebung der zehnte Teil
der Amplitude der ersten Schwebung sein darf, ohne daB ihre
schadliche Wirkung bemerkbar wird, und es ist im Sender
yl + yz/2 = 0,75, so braucht man keine schwachere Koppelung
als h'=0,219 zu wahlen, weil schon fur diese $il : &=12,2 ist.
Wenn wir nun die unteren Kurven der Fig. 3 betrachten.
welche fur sehr starke Koppelungen gelten, so treten wieder
andere Verhaltnisse ein, weil d a m Q einen periodischen Verlauf ohne Schwebungen annimmt. Dies tritt fur Verhaltnisse
T : P, welche groBer als 3 sind, d. h. fur Koppelungen k', die
groBer als k = 0,8 sind, aus. den Kurven der Fig. 3 deutlich
zutage. Es liegt dies daran, daB fur sehr starke Koppelungen
die schnellere Schwingung der beiden Perioden T , 1" des
Senders vie1 starker gedampft ist, als die langsamere, so da8
dann nur diese allein merklich bleibt. Wenn also Koppelungen,
die 9roper als h'= 0,8 sind, experimetitell realisiert werden honnen,
YO
kann rnati zur Brzielung guter Resonaiiz und zugleich krafiiger
Wirkun.7 auch mit sehr starker Koppelung im Sender arbeiten.
Die Eigenperiode des Empfangers mup dann aber @= 1,41 ma1
so grop sein, als die Eigenperiode 7; der beidea (ungekoppelten)
Schwingunphreise des Senders.
Annslen der Physik. IV. Folge. 13.
36
P. Brude.
554
Nach (60) p. 524 hangt die Maximalamplitude eines einfachen Empfangers stark uon der Bampfung des Senders und
Empfangsrs ah, der Integraleffekt nach (84) noch mehr.
b) En,qgekoppelter Empfangsapparat. Besitzt derselbe (wegen
der engen Koppelung seiner beiden Teile) zwei Eigenperioden
11 und I”, und nennt man P, das Potential am freien Ende
die Potentialdifferenz zwischen
der Antennen des Empfangers,
den Belegungen des Kondensators in dem Stromkreise 1 des
Empfangers, der mit dem Stromkreise 2 des Empfangers,
welcher die Antennen enth<, eng gekoppelt ist, so gelten entsprechend den Gleichungen (11)der p. 519 oben die Formeln:
Dabei beziehen sich jetzt alle GraBen, abgesehen ron P i ,
auf die beiden Stromkreise 1 und 2 des Empfangsapparates,
nur V,’ bezieht sich auf den Sender und bedeutet die in dem
bisherigen Teil dieser Arbeit mit Ya bezeichnete GroBe, d. h.
das Potential am Antennenende des Senders, fiir welche ja in
Formel (112) und (113) genauere Ausdriicke gegeben sind.
Der Term a 7,’bezeichnet die Erregung des Empfangers
(strenger ausgedriickt der Antennen des Empfangers) durch
die vom Sender ausgestrahlten Wellen elektrischer Kraft. Der
Faktor a hangt also von der Entfernung des Senders vom
Empfanger und von der spezielleren Konstruktion, z. B. Antennenlange des Empfangers ab.
Nach (112) und (113) ist zu setzen
Wir konnen setzen:
a P,’ = S, e t k + Sa e t
(125)
wobei
(126)
{
z1 = -
und
(127)
gesetzt ist.
cT+it,
+ 8,e t h + S4e tiza
z,=-d-iiz,
z4 =
5, =
h
%,=--d+iT’,
- s - ir‘,
s, = s, s, = s, = - s
555
IrLduAtive Erregung.
Als Integral von (122), (123) schreiben wir analog wie
friiher F'ormel (25) p. 520:
JTl = A , et/Yi + 8,e t / Y s + A, eth8 + A, e t / ~ a
+ D,e t h + U , e t/zz + U , e t/zs + B4e t/z4 ,
5 = B, etlyl + B, ethp + B, et/y8 + B4 et1~4
+ 3,e t h + E, e tlzl + 3,e t h + E4 e t h .
(128)
Die mit A und B proportionalen Glieder sind die Eigenschwingungen des Empfangers, die mit B und E proportionalen
Glieder die durch den Sender erzwungenen Schwingungen. Daher
sind die y die vier Wurzeln der Gleichung (vgl. (29) p. 520):
I
1
(129)
1
(YZ
.;
+ 29.,Y + + 9.:)(y2 + 2 9 , y + .:+ 9.;)
- P l S P , , (4+ W(r; + 9,"
= (Y - Yl) (Y - Yz)(Y - Y3)(Y - Y4) = 0.
Als Anfangsbedingungen (fur t = 0) gilt:
6 = o , Y, = o , il = o , i z = o ,
daher (vgl. (102) p. 537) gilt:
Nun gilt nach (122):
+
+ +
+ +
+
+
(131)
A1 (r:
79;
2 9.1 ~1
3:) = pi, (r:
B1,
(132)
B,(tt 9.: 2 8
1 21
2;) = Pi,:.(
$:)El,
und noch sechs analoge Gleichungen fur A,, ya,B, und D,,z,, E, etc.
Dividiert man(l31) durchy,, (132)durchz,und addiert,so entsteht:
;.(
+
+9.:,(2
+ $) +
+ + A,Y, + D,
= p,, (
.: + 9.:): ( +) :
29.1(8,
01)
2,
*
Addiert man hierzu die drei analogen Gleiehungen, die
man erhllt, wenn man in A, B, y, z den Index 1 durch 2, 3, 4
ersetzt, so entsteht wegen der Relationen (130):
C a y C B Z = 0.
(133)
Addiert man (131) und (132), so entsteht:
+
+
(.?
9.;)(4
+
01)
+2
=PI,
9.1
(A, Y1
(.:
+ D, + 8, y; + D,
2:
21)
+ 1T)(B1+ 4-
36 *
P. Brude.
556
A=
1,
1,
Y I ~~
1,
1
i YS-
~4
=
( ~ -Y~)(YI
1
- YJ(YL
-~
4 )
(YZ-Y~)(Y~---~)(Y~-Y~),
Y;, Y;, Y:
YBl
Y:? Y:
!l;1
Y:,
4 i = 4 , =
2
1,
1,
1
Yet
Ysr
Y4
=-(Y~-Y*)(Y~-Y~)(Y~-Y~),
Y;, Y h Y:
=
4 s
=
11
’7
;il
=-
rY1-Y,)(Y*-YII(YJ-Y4)(Y2YsfY1YIfY3Y4)l
=-
(Y~-Y/~)(Y~-Y~)(Y~-Y~)(YB+Y~+Y~).
Y i l Y:
Y t , Y:, Y:
1,
1,
1
YZl
Y39
Y4
Y L Y l t Y:
’
A1
Y1
+ + +
(135)
\
-c
D
(Yl - YJ (.Yl - ?/3) (Y1 - Y4) Yz Y3 Ye z’y
- (Yz ?/3 + Yz Y4 + Y3 Y3
(Y,
Y3
Y4)CB z Dz2
=-
c7
D
(2 -
c
Ya)(2 - Y3)(2 - Yo).
Induktive Erregung.
B, [(z;
557
+ 2 8,z1 + .;+ a;)(%;+ 2 LY2 z1 + + 8;)
+ 9 . ; ) ( t : + $:)I =
+ 9.3
t;
pi2 (t:
~ i z ~ (T?
a 1
$1 2 ; i
oder wegen (1 29) :
(136) 4 (5- YJ (21 - YJ (21 - Y J (z1- Y4) = P,, (t:+ 9.3 =? 4.
Setzt man diesen Wert in (135) ein, so folgt:
ff
k?1 -g2)
(Y1 -y3)
(!./I
-y4)
= -p12
(f'
+':)z
*
Unter dem 2-Zeichen hat S und z sukzessive die vier Indizes
n = 1,2,Y, 4,wahrend yl seinen Index nicht andert. Die Formeln
fur A,, d3,8,ergeben sich aus (137)durch zyklische Vertauschung
der Indizes 1, 2, 3, 4 bei den GroBen A, 8, y, z der Formel (137).
Die Formeln (128), (136) und (137) geben die vollstandige Losung der Aufgabe fur F'. bei beliebigen Werten y und
z, d. h. bei beliebigen Diimpfungen und Eigenschwingungen
sowohl vom Sender wie vom Empfanger. EB ist klar, daB
letzterer im Resonanzfall am starksten angeregt wird ; man
wird es also so einrichten, daB die beiden Eigenperioden des
Senders identisch mit denen des Empfangers sind. Wenn z. B.
identisch (auch hinsichtlich der Koppelung) konstruierte Apparate als Sender und Empfanger benutzt werden, nur mit dem
Unterschied, daf3 beim Sender der Stromkreis 1 eine Funkenstrecke enthalt, beim Empf'anger dagegen an Stelle der Funkenstrecke einen Strom- oder Potentialindikator ( R u t h e r f o r d s
magnetischer Stromindikator oder Koharer), so sind die Eigenperioden von Sender und Empfanger gleich. In Hinsicht auf
(126) ist also dann zu setzen:
Die DampfungsgroBe cr der Eigenschwingungen des Senders ist im allgemeinen verschieden anzunehmen yon der
DampfiingsgroBe 8 des Senders, bei identisch konstruierten
Apparaten wird wohl 6 meist groBer als 6 sein, weil der Sender
eine Funkenstrecke enthalt. Da aber der Wellenindikator im
Empfanger auch elektrische Energie verzehrt , so kann es
eventuell auch eintreten, daB B = 6 wird.
Wir wollen der weiteren Rechnung den Fall 8 = c zugrunde legen, weil dadurch die Formeln sehr einfach werden, und
weil das Wesentliche dabei schon zur Geniige hervortreten wird.
P. Brude.
558
Unter der Annahme S = (r merden die y und z gleich.
d. h. es wird y1 = zl,yz = z 2 , y, = i 3 ,y4 = z4. Wir kiinnen
also nach (136) schreiben:
iWlc-+'
(139) ~ ( Y , - ~ ~ ~ ~ ) ( Y ~ - ~ ~ ) =P&
( Y ~ - Y+
Y~)
und nach (137):
(140) A, (Y1-
22
xi Y1
- Ys) (n- Y4) = - 2 3 2 it: + w19 x_
_
1 - Yl
?/,)(!I1
,
~
Von der 2 auf der rechten Seite von (137) ist niimlich
nur das mit 8l behaftete Glied beizubehalten, weil es den
Nenner z, - yl = 0 enthalt.
Aus (128) folgt nun weiter:
+ ( A , + B,)et/v?
[ Yl + (A, ++ D,)et/yl
D3)eL/~a
+ (A, + D 4 ) e h
=
(141)
(Al
Durch Addition von (139) und (140) ergibt sich:
(4+ 4)
(Yl - ?/,I
(Yl - Y3)(Yl - YJ = PI, (r; + 19;)4 !/I
*
oder nach (138):
(A,
Ol)(t2- t ' 2 ) = - + p l z (t; 8;)
8, (1 + 2 /. t)
und analog:
(A, D,)(t,- t',) = - + p l z ( t ; + 9.;)8,(1 - io/t),
(A,
D s ) ( t 2 - z ' ~=
) - + p l z ( t ; + t 9 ; ) S 3 ( 1+ j o l t ' ) ,
(A,
D 4 ) ( t z- t") = - +p,,(t," + 8:)S4(1 - i o l t ' ) .
U-nter Rucksicht auf (127) wird also nach (141), wenii man
17;gegen t: vernachlassigt, und cz gegen t2 resp. gegen T ' ~ :
+
+
+
+
+
Setzt man t - o = t', und sieht man von Anfangszeiten
ah, die von der GroBenordnung t = c sind, so kann man (142)
schreiben :
Nach den Entwicklungen des Abschnittes V p. 540 ist
dies nun umzugestalten in:
Induk tive Brregung
5
.
559
Die Potentialdifferenz
im Empfanger wird also durch
eine analoge Formel dargestellt, wie das Potential
am
Sender nach (112), nur daB Cl Lz1 und C2L,, ihre Rolle als
Zahler und Nenner vertauschen. Man mug daher, wenn Pi
mijglichst hoch werden soll, Cl klein gegen Yz wahlen, wenn
man aber einen moglichst starken Primarstrom erhalten will,
mug Cl grot3 gegen C, sein, weil
5
ist, nach (1) p. 514. Fur den Ruthezfordschen magnetischen
Wellenindikator ist jedenfalls diese Anordnung zu wahlen,
und selbst fur den Koharer scheint sie mir wegen seiner groBen
und unkonstanten Kapazitat zweckmagiger zu sein, a19 die
neuerdings meist gewahlte Transformierung auf hohes Potential Y, , wenigstens wenn man mit deutlicher Resonanz arbeiten
will. I)
Die Abhangigkeit des
von der Zeit ist dieselbe, wie
sie in V. beim Teslatransformator ausfuhrlich behandelt ist,
namlich sie ist die Differenz zweier verschieden gedampfter
Schwingungen verschiedener Periode. Wie dort dargelegt wurde,
muB also auch hier die Koppelung k' = 0,6 (t: t' = 2) beider
Teile d es Empfangers besonders gunstig sein. Veranderungen
von Dcimpfungen machen dann auch, wie aus Fig. 2 hervorgeht, f u r die Maximalamplitude wenig Unterschied aus, jedenfalls vie1 weniger, als f u r den lntegraleffekt", da dieser, wie
aus (143) leicht zu erhalten ist, proportional zu 1 : 6 ist. Jedenfalls sieht man, daB beim zweifach gebauten (gekoppelten) Empfanger der unperiodische Verlauf des Senderpoteritials fur die
Resonanz nichf mehr hinderlich ist.
Immerhin wird man die scharfste Resonanz (aber mit Verzicht auf moglichst groBe Intensitat) erhalten, wenn man nach
M. Wien einen sehr lose gekoppelten Sender und einen sehr
lose gekoppelten Empfanger benutzt, bei welchen die Dampfungen
<
1) Diesen Punkt werde ich bei spaterer Gelegenheit ausfubrlicher
behandeln.
2) Der Koharer reagiert auf die Maximalamplitude, R u t h e r f o r d s
magnetischer Indikator vermutlich auf den Integraleffekt. Daher wird
man bei letzterem mehr als bei ersterem auf kleine DLmpfung achten
mussen.
P. Ilrude.
560
der beiden Stromkreise 1, welche die groBe Kapazitat Cl enthalten, miiglichst klein zu machen sind. Ferner ist fur scharfe
Resonanz besser ein auf den Idegraleffekt reagierender Indikator zu benutzen, als :ein auf die Maximalamplitude reagierender. Denn ganz allgemein muB die Resonanz bei der
Anordnung am besten wahrzunehmen sein, bei der die Intensitat am starksten (selbst im Resonanzfall) von der Dampfung
beeinflufit wird.
Es ist eine Frage fur sich (die spater behandelt werden
soll), wie man die Antennen am zweckmal3igsten im Vergleich
zur Sekundarspule nnd wie diese nach Hohe, Radius, Wicklungszahl am besten zu dimensionieren ist fur den Zweck der
Ausstrahlung elektromagnetischer Energie.
Zusammenfassung der Hauptresultate.
1. Aus der kombinierten Beobachtung der Resonanzkurve
der Maximalamplitude und des Integraleffektes ergibt sich die
Bampfimy beidet. Schwinyungskreise einzeln, sowie die Freguenz
des Resonators.
2. Die Resonanzkurve wird um so ausgepragter, je
schwacher die Koppelung beider Schwingungskreise ist. Ferner
ist sie fur den Integraleffekt ausgepragter, r21s fur die Maximalamplitude.
3. Der wirksamste Teslatransformator besteht aus einer
Primarwindung und vielen Sekundarwindungen, die einen Spulenkorper von bestimmtem (noch nicht genau ermitteltem) Yerhiiltnis
von HolLe zu Durchmesser bildon. Die Anzahl der Sekundarwindungen ist begrenzt durch die Nebenforderungen, daB die
Isolation nicht durchschlagen wird, und die Drahtdicke nicht
zu klein wird. Ferner erfordert eine Steigerung der Zahl
der Sekundarwindungen einen Speise-Induktor von hoherer
Schlagweite. Tote (nicht induktiv wirkende) Selbstinduktion
des Primarkreises ist mijglichst zu vermeiden , seine Drahtdicke soll moglichst groB sein und die Koppelung zwischen
Primarkreis und Teslaspule soll den V e r t k2 = 0,36 (Verhaltnis
der beiden durch Koppelung entstandenen Frequenzen 1 : 2)
mzglichst erreichen. Die Primarkapazitat mu13 den Primarkreis
in Resonanz mit der Teslaspule bringen und soll (wegen Ver-
Induktive Erregung.
561
meidung von E n tladungen und Ruckstand) aus Metallplatten
in gut isolierendem, riickstandsfreiem Dielektrikum (Petroleum,
nicht a l a s oder Luft) gebildet sein.
Bei schwacher Koppelung kommt es nur darauf an, dap
diese Primarkapazitat C, moglichst grop ist, einerlei, ob dies
durch kleine Spulen von hoher Wickelungszahl n erreicht wird,
oder durch groBere Spulen von kleinerem n ; bei starker Koppelung jst hohes n etwas wirksamer. - Vom Funkenpotential
hangt die Teslawirkung innerhalb gewisser Grenzen wenig ab.
4. Bei schwacher Koppelung hat die Dampfung der Primar- und Sekundarschwingungen starken EinfluB auf die Wirksamkeit des Teslatransformators, bei starker Koppelung (schon
von k2 = 0,16 an) vie1 weniger.
5. Bei der drahtlosen Telegraphie erhalt man scharfste
Resonanz (aber mit Verzicht auf Intensitat) bei schwach gekoppeltem und wenig gedampften Sender und Empfanger.
Letzterer enthalt dann zweckmaBig einen auf den Integraleffekt
reagierenden Indikator.
ti. Verwendet man einen schwach gekoppelten (oder einfachen) Empfanger und einen stark gekoppelten Sender , so
tritt keine deutliche Resonanz auf. Erst bei sehr starker
Koppelung im Sender (lc2 > 0,6) kunn man den Empfan.qer auf
den Sender abstimmen. Die Empfangerfrepuenz mup dunn aber
im Perhaltnis 1 : f j kleiner sein, als die Irrequenz der beiden
(aufeinander abgestimmten) Senderschwi7igun.~sRrei.Feeinzeln.
7. Verwendet man zwei gleich gebaute und gleich gekoppelte Apparate als Sender und Empfanger, so kann man
groBte Intensitat erhalten und miiBige Deutlichkeit der Resonanz, letztere mehr beim Integraleffekt, als bei der Maximalamplitude. Zur Erzielung groBter Wirkung ist in beiden Apparaten die Koppelung K 2 = 0,36 zu wahlen. Bei Benutzung
eines auf Maximalamplitude reagierenden Indikators im Empfanger hangt dann die Wirkung wenig von den Diimpfungen
ab, dafiir wird die Resonanz aber auch undeutlicher, als wenn
man einen auf Integraleffekt reagierenden Indikator wahlt.
G i e s s e n , November 1903.
(Eingegangen 25. November 1903.)
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Expression of a Cu,Zn superoxide dismutase typical for familial amyotrophic lateral sclerosis increases the vulnerability of neuroblastoma cells to infectious injury
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BioMed Central BioMed Central Published: 12 November 2007 BMC Infectious Diseases 2007, 7:131
doi:10.1186/1471-2334-7-131 This article is available from: http://www.biomedcentral.com/1471-2334/7/131 This article is available from: http://www.biomedcentral.com/1 © 2007 Goos 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. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creative
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cit BMC Infectious Diseases Open Access amyotrophic lateral sclerosis increases the vulnerability of
neuroblastoma cells to infectious injury
Miriam Goos1, Wolf-Dieter Zech1, Manoj Kumar Jaiswal2,
Saju Balakrishnan2, Sandra Ebert1, Timothy Mitchell5, Maria Teresa Carrì3,4,
Bernhard U Keller2 and Roland Nau*1 Address: 1Department of Neurology, Georg-August-University of Göttingen, Göttingen, Germany, 2Department of Physiology, Georg-August-
University of Göttingen, Göttingen, Germany, 3Dept. of Biology, University of Rome "Tor Vergata", Rome, Italy, 4Laboratory of Neurochemistry,
Fondazione S. Lucia IRCCS, Rome, Italy and 5Glasgow Biomedical Research Centre, University of Glasgow, Glasgow, UK Email: Miriam Goos - mlotz@gwdg.de; Wolf-Dieter Zech - wolf.zech@web.de; Manoj Kumar Jaiswal - manoj.jaiswal@medizin.uni-
goettingen.de; Saju Balakrishnan - sajbal@gmail.com; Sandra Ebert - sebert1@gwdg.de; Timothy Mitchell - T.Mitchell@bio.gla.ac.uk;
Maria Teresa Carrì - carri@uniroma2.it; Bernhard U Keller - bkeller1@gwdg.de; Roland Nau* - rnau@gwdg.de * Corresponding author Received: 29 April 2007
Accepted: 12 November 2007 Open A
Research article
Expression of a Cu,Zn superoxide dismutase typical for familial
amyotrophic lateral sclerosis increases the vulnerability of
neuroblastoma cells to infectious injury
Miriam Goos1, Wolf-Dieter Zech1, Manoj Kumar Jaiswal2,
Saju Balakrishnan2, Sandra Ebert1, Timothy Mitchell5, Maria Teresa Carrì3,4
Bernhard U Keller2 and Roland Nau*1 Open
Research article
Expression of a Cu,Zn superoxide dismutase typical for familial
amyotrophic lateral sclerosis increases the vulnerability of
neuroblastoma cells to infectious injury Background vated microglia are more susceptible to stimulation by
infectious agents (e.g., via TLR agonists) and can therefore
cause stronger neuronal damage during the course of an
infection by the release of nitric oxide, reactive oxygen
species and cytokines. g
Infections may increase the risk or accelerate the progres-
sion of various neurodegenerative disorders including
Alzheimer's disease, Parkinson's disease, stroke and
amyotrophic lateral sclerosis (ALS) [1,2]. Moreover,
patients with neurodegenerative diseases are more suscep-
tible to systemic, in particular lung infections than healthy
persons subsequent to swallowing disturbances and a
decreased strength of their respiratory muscles. In support
of the link between infections and ALS, an epidemiologi-
cal study found evidence for infection with Mycoplasma
spp. in the blood of more than 80% of patients suffering
from ALS and in less than 10% of age-matched control
subjects [3]. Neuronal injury in infection can originate from systemic
inflammation, stimulation of local immunocompetent
cells and direct action of bacterial toxins on neurons
[1,18]. During CNS infections, Streptococcus pneumo-
niae primarily causes damage by the direct action of the
cholesterol-binding pore-forming hemolysin pneumo-
lysin and through microglia/monocyte activation by ago-
nists of receptors of the innate immune system,
particularly Toll-like receptor 2 (TLR2) [18,19]. Both
mechanisms may be also of importance in patients with
neurodegenerative diseases during extracerebral infec-
tions. Here we demonstrate the particular vulnerability of
G93A-SOD1 transgenic neuroblastoma cells to both
modes of infectious injury. ALS is an ultimately lethal disease with a high inter-sub-
ject variation of progression. It is characterized by the
degeneration of cortical and spinal motor neurons. ALS
appears to be a multifactorial disease, where motor neu-
ron degradation is initiated by mitochondrial dysfunction
or/and by enhanced motor neuron excitability. Mitochon-
drial function can be disturbed by mutations in the gene
encoding Cu,Zn superoxide dismutase (EC 1.15.1.1,
SOD1). Clinically, ALS occurs both sporadically and as a
familial form. In 5–10% ALS is a familial disease, and
approximately 20% of the familial ALS cases are caused by
a mutation in the gene encoding SOD1 [4,5]. The point
mutation G93A is one of those occurring in familial
amyotrophic lateral sclerosis (FALS). Here, in position 93
the amino acid glycine is replaced by alanine in the SOD1
enzyme. Families with the G93A-SOD1 mutation are
indistinguishable from sporadic ALS by clinical and path-
ologic criteria [5]. http://www.biomedcentral.com/1471-2334/7/131 http://www.biomedcentral.com/1471-2334/7/131 http://www.biomedcentral.com/1471-2334/7/131 http://www.biomedcentral.com/1471-2334/7/131 Background An in-vitro model to study the cellular
alterations associated with mutations of SOD1 was con-
structed by transfection of the human neuroblastoma cell
line SH-SY5Y with G93A-SOD1 [6]. This particular muta-
tion was chosen, because it does not affect the activity of
SOD1. Abstract Background: Infections can aggravate the course of neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). Mutations in the anti-oxidant enzyme Cu,Zn superoxide dismutase (EC 1.15.1.1, SOD1) are associated with familial ALS. Streptococcus pneumoniae, the most frequent respiratory pathogen, causes damage by the action of the cholesterol-binding
virulence factor pneumolysin and by stimulation of the innate immune system, particularly via Toll-like-receptor 2. Methods: SH-SY5Y neuroblastoma cells transfected with the G93A mutant of SOD1 typical for familial ALS (G93A-SOD1) and
SH-SY5Y neuroblastoma cells transfected with wildtype SOD1 were both exposed to pneumolysin and in co-cultures with
cultured human macrophages treated with the Toll like receptor 2 agonist N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-
[R]-cysteinyl-[S]-seryl-[S]-lysyl-[S]-lysyl-[S]-lysyl-[S]-lysyl-[S]-lysine × 3 HCl (Pam3CSK4). Cell viability and apoptotic cell death
were compared morphologically and by in-situ tailing. With the help of the WST-1 test, cell viability was quantified, and by
measurement of neuron-specific enolase in the culture supernatant neuronal damage in co-cultures was investigated. Intracellular calcium levels were measured by fluorescence analysis using fura-2 AM. Results: SH-SY5Y neuroblastoma cells transfected with the G93A mutant of SOD1 typical for familial ALS (G93A-SOD1) were
more vulnerable to the neurotoxic action of pneumolysin and to the attack of monocytes stimulated by Pam3CSK4 than SH-
SY5Y cells transfected with wild-type human SOD1. The enhanced pneumolysin toxicity in G93A-SOD1 neuronal cells
depended on the inability of these cells to cope with an increased calcium influx caused by pores formed by pneumolysin. This
inability was caused by an impaired capacity of the mitochondria to remove cytoplasmic calcium. Treatment of G93A-SOD1 SH-
SY5Y neuroblastoma cells with the antioxidant N-acetylcysteine reduced the toxicity of pneumolysin. Conclusion: The particular vulnerability of G93A-SOD1 neuronal cells to hemolysins and inflammation may be partly
responsible for the clinical deterioration of ALS patients during infections. These findings link infection and motor neuron disease
and suggest early treatment of respiratory infections in ALS patients. Page 1 of 12
(page number not for citation purposes) BMC Infectious Diseases 2007, 7:131 Preparation of human macrophages Human macrophages were derived from peripheral blood
mononuclear cells [21]. Shortly, after centrifugation over
a Ficoll-Hypaque density gradient, mononuclear cells
were plated in RPMI-1640 + 10% FCS and maintained at
37°C in an atmosphere containing 5% CO2. Monocytes
were allowed to adhere and were then cultivated until dif-
ferentiation into macrophages for 10 to 14 days as
assessed by morphologic criteria such as adherence of the
cells and the sprouting of ramifications and functional
properties. CD-68 staining showed 98–99% purity of the
macrophage cultures. Calcium changes in defined regions of interest (ROIs)
were monitored online using the TILL Vision Software
V3.3 (TILL Photonics, Martinsried, Germany). Back-
ground fluorescence was subtracted from the recorded
values. The measured fluorescence ratio [R] at wave-
lengths 360 and 390 nm was used to calculate the intrac-
ellular calcium concentration [Ca2+]i using the equation
of Grynkiewicz et al [25]. The Kd of fura-2 was experimen-
tally determined as 224 nM [23,26]. Excitation of fura-2
was alternatively done at 360 nm and 380 nm, emitted
light was directed to a dichroic mirror with mid-reflection
at 425 nm filtered by a band pass filter (505–530 nm). Fluorescence ratio F(360)/F(380) was taken as an esti-
mate of the cytosolic calcium concentration and, accord-
ingly, changes of [Ca2+]i in fura-2 AM loaded cells are
shown. Further analysis was performed off-line with the
IGOR software (Wavemetrics, Lake Oswego, OR, USA). Bathing solutions were either RPMI-1640 or (in mM)
NaCl 140, KCl 2, CaCl2 2.5, MgCl2 1, HEPES 10, glucose
40, and bovine serum albumin 0.05% at pH 7.3. For nom-
inally Ca2+-free solutions MgCl2 was substituted for CaCl2
without adding EGTA. Methods
SH-SY5Y G93A-SOD1 and Wt-SOD1 neuroblastoma cell
cultures and measurement of cell viability Transfected human neuroblastoma cell lines constitu-
tively expressing either wild-type (Wt) human SOD1 or
the G93A mutant of this enzyme associated with familial
amyotrophic lateral sclerosis (FALS) were previously
described [6]. They were routinely maintained in Dul-
becco's MEM-F12 (Gibco, Invitrogen, Karlsruhe, Ger-
many) containing 15% fetal calf serum (FCS), 100 U/ml
penicillin and 100 μg/ml streptomycin (Invitrogen, Karl-
sruhe, Germany) at 37°C at a humidified atmosphere
with 5% CO2. Cell lines were kept in selection by addition
of 200 μg/ml geneticin (G418 sulfate, Gibco, Invitrogen,
Karlsruhe, Germany); geneticin was removed two days
before performing the experiments. For investigation of the differences in vulnerability to
pneumolysin both cell lines were seeded into 96-well
plates at a density of 105 cells/cm2. Cultures were treated
with medium that contained pneumolysin at a concentra-
tion of 0.5 μg/ml. After three hours of exposure cell viabil-
ity was determined by use of the WST-1 cell proliferation
reagent (Roche Applied Science, Mannheim, Germany). The assay is based on the cleavage of the tetrazolium salt
WST-1 by active mitochondria, which produces a soluble
formazan. Cells were incubated with WST-1 for 2 hours. Then, the formazan dye formed was quantified by meas-
uring the optical density at 490 nm by use of a Genios
multiplate reader (Tecan, Crailsheim, Germany). The
absorbance directly correlates with the number of meta-
bolically active cells. A significant inflammatory component contributes to the
pathology of ALS [7,8]. This comprises elevated tissue lev-
els of cyclooxigenase-2 and various cytokines and chem-
okines in the CNS tissue of ALS patients and mouse
models [7,9,10] and the presence of activated microglial
cells as demonstrated in post mortem spinal cord tissue of
ALS patients [10,11] and by positron emission tomogra-
phy using [11C](R)-PK11195 in living ALS patients [12]. The presence of activated microglial cells in the vicinity of
neuronal death in ALS suggests that stimulants of micro-
glial activation are produced by stressed neurons. Con-
versely, activated microglia can injure neurons both in
vitro and in vivo [13-16]. The simultaneous action of
host-derived and exogenous stimulants can lead to an
additive or supra-additive microglial activation [17]. We
hypothesize that in neurodegenerative diseases preacti- Page 2 of 12
(page number not for citation purposes) BMC Infectious Diseases 2007, 7:131 http://www.biomedcentral.com/1471-2334/7/131 http://www.biomedcentral.com/1471-2334/7/131 Antioxidant N-acetylcysteine (NAC) To examine the effect of the antioxidant N-acetylcysteine
(NAC) (Sigma, Deisenhofen, Germany) on cell viability,
SH-SY5Y G93A-SOD1 and SH-SY5Y Wt-SOD1 human
neuroblastoma cells were kept in culture medium con-
taining 1 mM NAC for periods of 24 and 72 hours prior
to the experimental procedure. After exposure to pneumo-
lysin for a period of 3 hours cell viability was determined
by the WST-1 test. Measurement of intracellular calcium levels f
Changes in the cytosolic calcium ([Ca2+]i) were measured
in SH-SY5Y cells expressing either the Wt-SOD1 or the
G93A-SOD1 gene attached to glass coverslips after 2–5
days in culture. Cell layers were incubated with RPMI-
1640 + 10% FCS (Gibco, Invitrogen, Karlsruhe, Germany)
containing 10 μM fura-2 AM at 37°C for 30 min. The
RPMI-1640 medium used contains 0.846 mM Ca 2+ (sup-
plier's data). Cells were rinsed with RPMI and further
incubated for 20 min at 37°C to allow complete deesteri-
fication. Changes in [Ca2+]i were measured using a CCD
camera system (TILL Photonics, Martinsried, Germany)
[23,24]. A computer-controlled monochromator (Poly-
chrome II, TILL Photonics) was connected to an Axio-
scope microscope (Zeiss, Goettingen, Germany) via
quartz fiberoptics and a minimum number of optical
components for maximum fluorescence excitation (objec-
tive Achroplan W 63×, 0.9 W). The CCD camera displayed
12-bit dynamics and an A/D converter with 12.5 MHz
sampling rate. Pneumolysin For activation of macrophages co-cultures were exposed to
N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-[R]-
cysteinyl-[S]-seryl-[S]-lysyl-[S]-lysyl-[S]-lysyl-[S]-lysyl-[S]-
lysine × 3 HCl (Pam3Cys-SKKKK × 3 HCl, EMC Microcol-
lections, Tuebingen, Germany; Pam3CSK4) [22] in con-
centrations of 10 μg/ml. After 72 hours neuron-specific
enolase was determined in 250 μl of the culture superna-
tant with a luminescence enzyme immunoassay (LIA)
using the Liaison® Analyser from Byk Sangtec and reagents
from Diasorin (Dietzenbach, Germany). Cells were fix-
ated with 4% formaldehyde for staining procedures. Pneumolysin was purified after overexpression of the
recombinant toxin in Escherichia coli strain JM109 by
hydrophobic and ion exchange chromatography as
described before [20]. Toxin purity was assessed by SDS-
polyacrylamide gel electrophoresis followed by Coomas-
sie-blue staining which showed a single 52 kD band
accounting for 95% of the protein. Endotoxin content of
purified pneumolysin was determined using the Limulus
amebocyte lysate kinetic-QCL kit (Cambrex, Nottingham,
United Kingdom). The purified protein had less than 0.6
endotoxin units per μg of protein, i.e. a very low level
which is unlikely to have a biological effect. Increased toxicity of pneumolysin for neuroblastoma cells
transfected with G93A-SOD1 After incubation of Wt-SOD1 and G93A-SOD1 neurob-
lastoma cells with the pneumococcal virulence factor
pneumolysin for 3 hours, the G93A-SOD1 mutant cells
showed a significantly decreased cell viability as evi-
denced by the WST-1 test (24.3 ± 9.6% of the living
untreated cells) compared to the Wt-SOD1 cells (48.8 ±
19.5%; p < 0.0001, Fig. 1). This phenomenon was morphologically confirmed by
staining of the cell somata with hemalum. A significantly
higher density of living cells after pneumolysin treatment
was observed in cultures of Wt-SOD1 cells compared to
G93A-SOD1 neuroblastoma cultures (Fig. 2). By in-situ
tailing, morphology (arrows) (Fig. 3), and by immunocy-
tochemistry for activated caspase-3 (Fig. 4) of G93A-
SOD1 neuroblastoma cells it became apparent that a large
proportion of these cells died by apoptosis. The rate of
apoptotic neuroblastoma cells in the different treatment
groups is presented in Fig. 5. Results CD68 and light green staining was used to distinguish
between human macrophages and the SH-SY5Y cells in
co-culture and to visualize the morphology of the cells;
immunostaining for activated caspase-3 was used to
detect apoptosis in pneumolysin-treated G93A SOD1
cells: fixated cells were permeabilised with Triton X (0.1%
in PBS) for 30 minutes and then incubated with CD68
antibody (clone KP1, dilution 1:50, DAKO, Glostrup,
Denmark) or activated caspase-3 antibody [rabbit anti-
caspase-3 (cleaved), dilution 1:100, kindly donated by
Zytomed Systems, Berlin, Germany] for 90 minutes. Sec-
ondary anti-mouse biotinylated antibody or secondary
anti-rabbit biotinylated antibody (dilution 1:200, both
from Amersham Biosciences, Munich, Germany) were
added for 45 minutes. Thereafter, cells were treated with
avidin-biotin complex (ABC, Vector, Burlingame, CA) for
30 min, and diaminobenzidine was used for visualisation
(5 minutes) resulting in a brown staining of the somata of
macrophages (CD68)/apoptotic G93A SOD1 cells (Cas-
pase 3). SH-SY5Y cells were counterstained by light green
SF yellowish solution (Chroma-Gesellschaft Schmidt &
Co, Stuttgart, Germany) after CD68 immunocytochemis-
try and with hemalum after caspase-3 immunocytochem-
istry for 1–2 minutes, rinsed in water, dehydrated and
mounted with DePeX (Serva, Heidelberg, Germany). Impaired ability of neuroblastoma cells transfected with
G93A-SOD1 to cope with the pneumolysin-induced
calcium influx For a period of 20 minutes both cell lines were treated
with pneumolysin in concentrations of 0.5 μg/ml. During
this time cytoplasmic calcium levels were measured in
both cell lines. Fig. 6 shows intracellular calcium levels
[Ca2+]i as cytoplasmic calcium-versus-time curves in two
representative cells during pneumolysin treatment. The
ability of G93A-transfected SH-SY5Y cells to maintain low
cytoplasmic calcium levels was strongly reduced. Analysis
of the cytoplasmic calcium-versus-time curves of 25
G93A-SOD1 transfected and 25 Wt-SOD1 transfected
cells after exposure to pneumolysin for 20 minutes
revealed an approximately 4-fold increase of the area
under the curve (AUC) in G93A-SOD1 SH-SY5Y cells
(634.0 ± 309.0 in Wt-SOD SH-SY5Y versus 2658.0 ± SH-SY5Y G93A-SOD1 and Wt-SOD1 neuroblastoma and
human macrophage co-culture For co-culture experiments 5 × 104 neuroblastoma (G93A-
SOD1 or Wt-SOD1) cells/well were seeded on glass cover-
slips in 24 well plates and allowed to adhere for 24 hours. Trypsin was used initially to separate the neuroblastoma
cells, but was washed out twice by centrifugation and PBS
washing. The following day after PBS washing and change
of culture medium human macrophages were mechani-
cally displaced with a cell scraper and added in concentra-
tions of 105 cells/well. Co-cultures were maintained for 24
hours in RPMI 1640 medium (Biochrom, Berlin, Ger-
many) containing 10% FCS at 37°C with 5% CO2 prior to
stimulation experiments. The macrophages did not come
into contact with trypsin during this procedure to avoid
an activation prior to the experiments. Page 3 of 12
(page number not for citation purposes) Page 3 of 12
(page number not for citation purposes) http://www.biomedcentral.com/1471-2334/7/131 http://www.biomedcentral.com/1471-2334/7/131 BMC Infectious Diseases 2007, 7:131 developed with 4-nitroblue-tetrazolium (NBT) chloride/
5-bromine-4-chloride-3-indolyl-phosphate. The cover
slips were counterstained with nuclear fast red aluminum
hydroxide (reagents from Roche). The area under the concentration-versus-time curve
(AUC) was calculated with the baseline 0 by the equation:
In [Ca2+]it = t1∫t2 [Ca2+]i•dt with the help of Origin soft-
ware, version 7.5 (In = fluorescence intensity under the
concentration-versus-time curve, [Ca2+]i = cytosolic cal-
cium, t = time). The area under the concentration-versus-time curve
(AUC) was calculated with the baseline 0 by the equation:
In [Ca2+]it = t1∫t2 [Ca2+]i•dt with the help of Origin soft-
ware, version 7.5 (In = fluorescence intensity under the
concentration-versus-time curve, [Ca2+]i = cytosolic cal-
cium, t = time). Statistics Graph Pad Prism Software (GraphPad Software, San
Diego, California, USA) was used to perform statistical
analyses and graphical presentation. Experiments were
reproduced at least three times. Data were expressed as
means ± SDs. Groups were compared by two-tailed para-
metric one-way ANOVA, and p values were adjusted for
repeated testing by Bonferroni's multiple comparison test. P < 0.05 was considered to be statistically significant. Hemalum staining, CD68 and activated caspase-3
immunocytochemistry and light green staining Hemalum staining, CD68 and activated caspase-3
immunocytochemistry and light green staining Pneumolysin-stimulated SH-SY5Y cell mono-cultures
(Wt-SOD1 and G93A-SOD1) were plated on glass cover-
slips and were fixated with 4% formaldehyde, dehydrated
through graded steps of water/ethanol and histolene and
then stained with Meyer's hemalum solution (1: 1 dilu-
tion in water). Toxicity o
neuroblast
Figure 2 y
p
y
g
Toxicity of pneumolysin for Wt-SOD1 and G93A- Toxicity of pneumolysin for Wt-SOD1 and G93A-
SOD1 neuroblastoma cells. Hemalum staining showed a
substantially higher density of living Wt-SOD1 SH-SY5Y cells
than G93A-SOD1 SH-SY5Y cells after PLY treatment. Please
note the shrinkage and clustering of severely damaged/dead
G93A-SOD1 neuroblastoma cells. In-situ tailing (IST) In order to assess the quantity of cells which had died by
apoptosis, formaldehyde-fixated cells on cover slips were
treated with 50 μg/mL proteinase K (Sigma) for 15 min at
37°C in a reaction mixture that contained 10 μL of 5× tail-
ing buffer, 1 μL of digoxigenin DNA labeling mix, 2 μL of
cobalt chloride, 12.5 U of terminal transferase, and the
amount of distilled water necessary to give a volume of 50
μL. After washing, the cells were incubated with 10% FCS
for 15 min at room temperature and then washed again. A solution of alkaline phosphatase labeled anti-digoxi-
genin antibody in 10% FCS (1:250) was placed on the sec-
tions for 60 min at 37°C. The color reaction (black) was Page 4 of 12
(page number not for citation purposes) Page 4 of 12
(page number not for citation purposes) http://www.biomedcentral.com/1471-2334/7/131 BMC Infectious Diseases 2007, 7:131 Toxicity of pneumolysin for Wt-SOD1 and G93A-SOD1
neuroblastoma cells
Figure 2
Toxicity of pneumolysin for Wt-SOD1 and G93A-
SOD1 neuroblastoma cells. Hemalum staining showed a
substantially higher density of living Wt-SOD1 SH-SY5Y cells
than G93A-SOD1 SH-SY5Y cells after PLY treatment. Please
note the shrinkage and clustering of severely damaged/dead
G93A-SOD1 neuroblastoma cells. Toxicity of pneumolysin for Wt-SOD1 and G93A-SOD1
neuroblastoma cells
Figure 1
Toxicity of pneumolysin for Wt-SOD1 and G93A-
SOD1 neuroblastoma cells. Cell viability measured by the
WST-1 test after 3 h of incubation with pneumolysin (PLY) at
a concentration of 0.5 μg/ml. Values are given in % of mito-
chondrial metabolic activity of unstimulated control cells ±
standard deviation (SD). G93A-SOD1 mutant SH-SY5Y cells
were more vulnerable to the action of PLY than Wt-SOD1
SH-SY5Y cells (p < 0.0001). Toxicity of pneumolysin for Wt-SOD1 and G93A-SOD1
neuroblastoma cells
Figure 1
Toxicity of pneumolysin for Wt-SOD1 and G93A-
SOD1 neuroblastoma cells. Cell viability measured by the
WST-1 test after 3 h of incubation with pneumolysin (PLY) at
a concentration of 0.5 μg/ml. Values are given in % of mito-
chondrial metabolic activity of unstimulated control cells ±
standard deviation (SD). G93A-SOD1 mutant SH-SY5Y cells
were more vulnerable to the action of PLY than Wt-SOD1
SH-SY5Y cells (p < 0.0001). Page 5 of 12
(page number not for citation purposes) In-situ tailing (IST) Toxicity of pneumolysin for Wt-SOD1 and G93A-SOD1
neuroblastoma cells
Figure 2
Toxicity of pneumolysin for Wt-SOD1 and G93A-
SOD1
bl
t
ll
H
l
t i i
h
d Toxicity of
neuroblast
Figure 1 y
p
y
g
Toxicity of pneumolysin for Wt-SOD1 and G93A-
SOD1 neuroblastoma cells. Cell viability measured by the
WST-1 test after 3 h of incubation with pneumolysin (PLY) at
a concentration of 0.5 μg/ml. Values are given in % of mito-
chondrial metabolic activity of unstimulated control cells ±
standard deviation (SD). G93A-SOD1 mutant SH-SY5Y cells
were more vulnerable to the action of PLY than Wt-SOD1
SH-SY5Y cells (p < 0.0001). y
p
y
g
Toxicity of pneumolysin for Wt-SOD1 and G93A-
SOD1 neuroblastoma cells. Cell viability measured by the
WST-1 test after 3 h of incubation with pneumolysin (PLY) at
a concentration of 0.5 μg/ml. Values are given in % of mito-
chondrial metabolic activity of unstimulated control cells ±
standard deviation (SD). G93A-SOD1 mutant SH-SY5Y cells
were more vulnerable to the action of PLY than Wt-SOD1
SH-SY5Y cells (p < 0.0001). 502.3 in G93A-SOD1 SH-SY5Y cells, p < 0.0001) (Fig. 7). The pneumolysin-induced [Ca2+]i peak values in G93A-
SOD1 cells amounted to approximately 1.6-fold of the
corresponding [Ca2+]i peak values in Wt-SOD1 cells (3.1 ±
0.4 versus 1.9 ± 0.5, p < 0.0001) (Fig. 8). Toxicity of pneumolysin for Wt-SOD1 and G93A-SOD1
neuroblastoma cells
Figure 2
T
i i
f
l
i
f
W SOD1
d G93A Toxicity of
neuroblast
Figure 2 The pneumolysin-induced neuronal injury was attenuated
by the anti-oxidant N-acetyl-cysteine (NAC) Incubation with the antioxidant N-acetyl-cysteine (NAC)
in a concentration of 1 mM starting 72 and 24 hours in
the absence of pneumolysin suggested a slightly higher
mitochondrial metabolic activity of G93A-SOD1 cells
compared to G93A-SOD1 cells kept in medium without
NAC (difference not significant). After 72 hours of NAC
pre-treatment, pneumolysin exposure to G93A-SOD1
cells resulted in a metabolic activity of 27.2 ± 3.5% of
G93A-SOD1 control cells not exposed to pneumolysin
compared to a metabolic activity of 20.8 ± 3.1% of respec-
tive pneumolysin-challenged G93A-SOD1 cells in the
absence of NAC (p < 0.001) (Fig. 9). After pre-incubation
of G93A-SOD1 cells in NAC-containing medium for a
period of 24 hours similar results were observed: NAC
exerted a neuroprotective effect on G93A-SOD1 cells
treated with pneumolysin as determined by the WST-1 test after 3 hours of pneumolysin exposure (22.5 ± 4.1%
mitochondrial metabolic activity of control cells in G93A
cells with NAC treatment versus 15.1 ± 5.4% without NAC
treatment, p < 0.0001) (Fig. 10). Contrarily, there was no significant difference in the met-
abolic activity of NAC-treated and untreated Wt-SOD1
cells after pneumolysin incubation, i.e., NAC did not pro-
tect Wt-SOD1 neuroblastoma cells from the toxic action
of pneumolysin (Fig. 9 &10) (56.9 ± 4.3% mitochondrial
metabolic activity in pneumolysin-treated Wt-SOD1 cells
versus 54.5 ± 6.6% in pneumolysin-exposed Wt-SOD1
cells with 72 hours pre-incubation with NAC, p > 0.05). Page 5 of 12
(page number not for citation purposes) BMC Infectious Diseases 2007, 7:131 http://www.biomedcentral.com/1471-2334/7/131 Toxicity of pneumolysin for G93A-SOD1 neuroblastoma
cells
Figure 4
Toxicity of pneumolysin for G93A-SOD1 neuroblast-
oma cells. Apoptotic G93A-SOD1 SH-SY5Y cells after incu-
bation with 0.5 μg/ml PLY for three hours detected by
immunocytochemistry for activated caspase-3. Toxicity of pneumolysin for G93A-SOD1 neuroblastoma
cells
Figure 3
Toxicity of pneumolysin for G93A-SOD1 neuroblast-
oma cells. Apoptotic G93A-SOD1 SH-SY5Y cells after incu-
bation with 0.5 μg/ml PLY for three hours (in-situ tailing,
apoptotic cells marked by arrows). Toxicity o
cells
Figure 3 y
p
y
g
Toxicity of pneumolysin for G93A-SOD1 neuroblast-
oma cells. Apoptotic G93A-SOD1 SH-SY5Y cells after incu-
bation with 0.5 μg/ml PLY for three hours (in-situ tailing,
apoptotic cells marked by arrows). y
p
y
g
Toxicity of pneumolysin for G93A-SOD1 neuroblast-
oma cells. Apoptotic G93A-SOD1 SH-SY5Y cells after incu-
bation with 0.5 μg/ml PLY for three hours (in-situ tailing,
apoptotic cells marked by arrows). Increased vulnerability of neuroblastoma cells transfected
with G93A-SOD1 to the attack of monocytes stimulated
with the Toll-like receptor-2 agonist Pam3CSK4 p
g
3
4
After stimulation of human neuroblastoma and macro-
phage co-cultures with the TLR2 agonist Pam3CSK4 for a
period of 72 hours the release of neuron-specific enolase
(NSE) was measured in the culture supernatants and
expressed as per cent of the NSE release induced by cell
lysis. Co-cultures of macrophages with G93A-SOD1 SH-
SY5Y cells showed a significantly higher release of NSE
compared to co-cultures with Wt-SOD1 SH-SY5Y cells
after stimulation with Pam3CSK4 (27.8 ± 2.4% vs. 19.0 ±
3.3%; p < 0.001) (Fig. 11). After cell staining with light
green and macrophage staining with CD 68 significantly
less neuronal cell somata of G93A-SOD1 SH-SY5Ycells
per mm2 than of equally treated Wt-SOD1 cells were
counted by microscopy (Fig. 12). In-situ tailing detected
macrophages attacking the apoptotic neuroblastoma cells
(arrow) (Fig. 13). SH-SY5Y neuroblastoma cells transfected with the G93A
mutant of SOD1 typical for familial ALS (G93A-SOD1)
are more vulnerable to infectious stimuli than neuroblas-
toma cells overexpressing normal SOD1. The increased
vulnerability was observed after exposure to the bacterial
hemolysin pneumolysin and after co-incubation with
activated monocytes. SH-SY5Y neuroblastoma cells transfected with the G93A
mutant of SOD1 typical for familial ALS (G93A-SOD1)
are more vulnerable to infectious stimuli than neuroblas-
toma cells overexpressing normal SOD1. The increased
vulnerability was observed after exposure to the bacterial
hemolysin pneumolysin and after co-incubation with
activated monocytes. Hemolysins are important virulence factors of a variety of
bacteria. The cholesterol-binding hemolysin pneumo-
lysin binds to eukariotic lipid membranes. There it oli-
gomerizes into ring-shaped structures and forms non-
selective pores within the lipid bilayer with a diameter of
25–50 nm [27-32]. At sublytic concentrations, pneumo-
lysin rapidly activates Rho and Rac GTPases and leads to
the formation of actin stress fibers, filopodia, and lamel-
lipodia. At these low concentrations, pneumolysin does The pneumolysin-induced neuronal injury was attenuated
by the anti-oxidant N-acetyl-cysteine (NAC) Toxicity of pneumolysin for G93A-SOD1 neuroblastoma
cells
Figure 4
Toxicity of pneumolysin for G93A-SOD1 neuroblast-
oma cells. Apoptotic G93A-SOD1 SH-SY5Y cells after incu-
bation with 0.5 μg/ml PLY for three hours detected by
immunocytochemistry for activated caspase-3. Increased vulnerability of neuroblastoma cells transfected
with G93A-SOD1 to the attack of monocytes stimulated
with the Toll-like receptor-2 agonist Pam3CSK4 Page 6 of 12
(page number not for citation purposes) Discussion Patients with neurodegenerative diseases can experience
irreversible deterioration during infections. In an in-vitro
model of amyotrophic lateral sclerosis we showed that Page 6 of 12
(page number not for citation purposes) Page 6 of 12
(page number not for citation purposes) http://www.biomedcentral.com/1471-2334/7/131 BMC Infectious Diseases 2007, 7:131 Toxicity of pneumolysin for Wt-SOD1 and G93A-SOD1
neuroblastoma cells
Figure 5
Toxicity of pneumolysin for Wt-SOD1 and G93A-
SOD1 neuroblastoma cells. Rate of apoptotic cells in
Wt-SOD1 SH-SY5Y and G93A-SOD1 SH-SY5Y neuroblast-
oma cells after incubation with 0.5 μg/ml PLY for three hours
(all cells = 100%). Please note the strong difference of the
rate of apoptosis in wild-type and G93A-SOD1 transgenic
cells exposed to pneumolysin (p < 0.001). cellular Ca2+ concentration originates from the influx of
extracellular Ca2+ and not from mobilization of intracellu-
lar stores and is mediated by the pneumolysin pore itself
and not via voltage-gated Ca2+ channels [30]. The massive
Ca2+ influx causes activation of p38 MAPK, opening of the
mitochondrial permeability transition (MPT) pore and
consecutive caspase activation. At low concentrations,
pneumolysin primarily leads to apoptosis [30]. The
amount of pneumolysin released by S. pneumoniae can
be influenced by the onset and choice of antibiotic ther-
apy [33]. SH-SY5Y cells transfected with G93A-SOD1 suffer from a
decreased mitochondrial membrane potential and an ele-
vated intracellular Ca2+ concentration already at rest as
determined by fluo-3 AM staining [6]. We also found a
slight elevation of the intracellular Ca2+ already in unchal-
lenged G93A-SOD1-transfected cells. After exposure to
pneumolysin, the capacity of G93A-SOD1 cells to control
cytosolic Ca2+ by transport to the extracellular space or to
intracellular storage sites was much lower than the capac-
ity of neuroblastoma cells not transfected with mutant
SOD1. This accounted for the increased vulnerability of
SH-SY5Y cells possessing a mutant G93A-SOD1 gene to
low concentrations of pneumolysin. SH-SY5Y cells transfected with G93A-SOD1 suffer from a
decreased mitochondrial membrane potential and an ele-
vated intracellular Ca2+ concentration already at rest as
determined by fluo-3 AM staining [6]. We also found a
slight elevation of the intracellular Ca2+ already in unchal-
lenged G93A-SOD1-transfected cells. After exposure to
pneumolysin, the capacity of G93A-SOD1 cells to control
cytosolic Ca2+ by transport to the extracellular space or to
intracellular storage sites was much lower than the capac-
ity of neuroblastoma cells not transfected with mutant
SOD1. Discussion The pneumo-
lysin-induced neuronal injury in G93A-SOD1 neuroblastoma
cells, but not in Wt-SOD1 cells, was attenuated by pre-incu-
bation for 72 hours with the anti-oxidant N-acetylcysteine
(NAC) in a concentration of 1 mM (p < 0.001). Strongly elevated calcium influx into G 93A-SOD1 neurob-
lastoma cells in comparison to wild-type SOD1 cells
Figure 7
Strongly elevated calcium influx into G 93A-SOD1
neuroblastoma cells in comparison to wild-type
SOD1 cells. Comparison of the intracellular calcium con-
centration-versus-time curves (n = 25 cells each; means ±
SD; p < 0.0001). Radical scavenging by N-acetyl-cysteine
Figure 9
Radical scavenging by N-acetyl-cysteine. The pneumo-
lysin-induced neuronal injury in G93A-SOD1 neuroblastoma
cells, but not in Wt-SOD1 cells, was attenuated by pre-incu-
bation for 72 hours with the anti-oxidant N-acetylcysteine
(NAC) in a concentration of 1 mM (p < 0.001). Strongly el
lastoma ce
Figure 7 Strongly elevated calcium influx into G 93A-SOD1 neurob-
lastoma cells in comparison to wild-type SOD1 cells
Figure 7
Strongly elevated calcium influx into G 93A-SOD1
neuroblastoma cells in comparison to wild-type
SOD1 cells. Comparison of the intracellular calcium con-
centration-versus-time curves (n = 25 cells each; means ±
SD; p < 0.0001). Radical sca
Figure 9 G93A-SOD1 transgenic mice, a 1% solution of N-acetyl-
cysteine administered as drinking water from 4–5 weeks
of age delayed the onset of motor dysfunction and pro-
longed survival [35]. The in-vitro and in-vivo data availa-
ble suggest that N-acetylcysteine, a drug with low toxicity G93A-SOD1 transgenic mice, a 1% solution of N-acetyl-
cysteine administered as drinking water from 4–5 weeks
of age delayed the onset of motor dysfunction and pro-
longed survival [35]. The in-vitro and in-vivo data availa-
ble suggest that N-acetylcysteine, a drug with low toxicity Radical scavenging by N-acetyl-cysteine. The pneumo-
lysin-induced neuronal injury in G93A-SOD1 neuroblastoma
cells, but not in Wt-SOD1 cells, was attenuated by pre-incu-
bation for 72 hours with the anti-oxidant N-acetylcysteine
(NAC) in a concentration of 1 mM (p < 0.001). Strongly elevated calcium influx into G 93A-SOD1 neurob-
lastoma cells in comparison to wild-type SOD1 cells
Figure 8
Strongly elevated calcium influx into G 93A-SOD1
neuroblastoma cells in comparison to wild-type
SOD1 cells. Comparison of the pneumolysin-induced peak
calcium intracellular concentrations (n = 25 cells each, means
± SD, p < 0.0001). Discussion This accounted for the increased vulnerability of
SH-SY5Y cells possessing a mutant G93A-SOD1 gene to
low concentrations of pneumolysin. Toxicity of
neuroblast
Figure 5 y
p
y
g
Toxicity of pneumolysin for Wt-SOD1 and G93A-
SOD1 neuroblastoma cells. Rate of apoptotic cells in
Wt-SOD1 SH-SY5Y and G93A-SOD1 SH-SY5Y neuroblast-
oma cells after incubation with 0.5 μg/ml PLY for three hours
(all cells = 100%). Please note the strong difference of the
rate of apoptosis in wild-type and G93A-SOD1 transgenic
cells exposed to pneumolysin (p < 0.001). The anti-oxidant N-acetylcysteine has been shown to
restore mitochondrial function and to lower the produc-
tion of reactive oxygen species in neuroblastoma cells
expressing mutant SOD1 [34]. In this study, it inhibited
pneumolysin-induced neurotoxicity. Accordingly, in not appear to form macropores, but micropores with ion
channel properties [29]. Formation of pores leads to a
Ca2+ flux from the extracellular to the intracellular space
causing an increase of intracellular Ca2+ in the micromolar
range and affecting cell survival. The increase of the intra- Strongly elevated calcium influx into G 93A-SOD1 neuroblastoma cells in comparison to wild-type SOD1 cells
Figure 6
Strongly elevated calcium influx into G 93A-SOD1 neuroblastoma cells in comparison to wild-type SOD1 cells. Representative recordings of intracellular calcium concentrations in single cells as measured by the fura-2 AM method. Strongly elevated calcium influx into G 93A-SOD1 neuroblastoma cells in comparison to wild-type SOD1 cells
Figure 6
Strongly elevated calcium influx into G 93A-SOD1 neuroblastoma cells in comparison to wild-type SOD1 cells. Representative recordings of intracellular calcium concentrations in single cells as measured by the fura-2 AM method. Page 7 of 12
(page number not for citation purposes) BMC Infectious Diseases 2007, 7:131 http://www.biomedcentral.com/1471-2334/7/131 Strongly elevated calcium influx into G 93A-SOD1 neurob-
lastoma cells in comparison to wild-type SOD1 cells
Figure 7
Strongly elevated calcium influx into G 93A-SOD1
neuroblastoma cells in comparison to wild-type
SOD1 cells. Comparison of the intracellular calcium con-
centration-versus-time curves (n = 25 cells each; means ±
SD; p < 0.0001). G93A-SOD1 transgenic mice, a 1% solution of N-acetyl-
cysteine administered as drinking water from 4–5 weeks
of age delayed the onset of motor dysfunction and pro-
longed survival [35]. The in-vitro and in-vivo data availa-
ble suggest that N-acetylcysteine, a drug with low toxicity
Radical scavenging by N-acetyl-cysteine
Figure 9
Radical scavenging by N-acetyl-cysteine. Discussion After stimulation of monocytes with
the Toll-like receptor 2 agonist Pam3CSK4 G93A-SOD1 SH-
SY5Y cells were more severely injured by activated macro-
phages than Wt-SOD1 cells. Radical scavenging by N-acetyl-cysteine
Figure 10
Radical scavenging by N-acetyl-cysteine. 24 hours of
pre-incubation with the anti-oxidant N-acetylcysteine (NAC)
in a concentration of 1 mM also resulted in an attenuation of
the pneumolysin-induced neuronal injury in G93A-SOD1 (p
< 0.0001), but not in Wt-SOD1 neuroblastoma cells. Radical scavenging by N-acetyl-cysteine
Figure 10
Radical scavenging by N-acetyl-cysteine. 24 hours of
pre-incubation with the anti-oxidant N-acetylcysteine (NAC)
in a concentration of 1 mM also resulted in an attenuation of
the pneumolysin-induced neuronal injury in G93A-SOD1 (p
< 0.0001), but not in Wt-SOD1 neuroblastoma cells. Vulnerability of G93A-SOD1 and wild-type SOD1 neuroblas-
toma cells to the attack of monocytes stimulated with
Pam3CSK4
Figure 11
Vulnerability of G93A-SOD1 and wild-type SOD1
neuroblastoma cells to the attack of monocytes stim-
ulated with Pam3CSK4. Release of neuron-specific eno-
lase (values expressed in per cent ± SD of the NSE release
induced by cell lysis). After stimulation of monocytes with
the Toll-like receptor 2 agonist Pam3CSK4 G93A-SOD1 SH-
SY5Y cells were more severely injured by activated macro-
phages than Wt-SOD1 cells. Vulnerability of G93A-SOD1 and wild-type SOD1 neuroblas-
toma cells to the attack of monocytes stimulated with
Pam3CSK4
Figure 11
Vulnerability of G93A-SOD1 and wild-type SOD1
neuroblastoma cells to the attack of monocytes stim-
ulated with Pam3CSK4. Release of neuron-specific eno-
lase (values expressed in per cent ± SD of the NSE release
induced by cell lysis). After stimulation of monocytes with
the Toll-like receptor 2 agonist Pam3CSK4 G93A-SOD1 SH-
SY5Y cells were more severely injured by activated macro-
phages than Wt-SOD1 cells. Radical scave
Figure 10 g g y
y
y
g
Radical scavenging by N-acetyl-cysteine. 24 hours of
pre-incubation with the anti-oxidant N-acetylcysteine (NAC)
in a concentration of 1 mM also resulted in an attenuation of
the pneumolysin-induced neuronal injury in G93A-SOD1 (p
< 0.0001), but not in Wt-SOD1 neuroblastoma cells. g g y
y
y
g
Radical scavenging by N-acetyl-cysteine. 24 hours of
pre-incubation with the anti-oxidant N-acetylcysteine (NAC)
in a concentration of 1 mM also resulted in an attenuation of
the pneumolysin-induced neuronal injury in G93A-SOD1 (p
< 0.0001), but not in Wt-SOD1 neuroblastoma cells. centrations [48,49]. Discussion used to liquefy the pulmonary secretion in pneumonia
and to protect the liver after paracetamol poisoning,
should be explored in a randomized trial concerning its
ability to lower the speed of progression of amyotrophic
lateral sclerosis in humans. Within the CNS, microglial cells and meningeal and
perivascular macrophages participate in the resistance to
infection, removal of cell debris from sites of injury and
promotion of tissue repair [36-38]. Microglia and mono-
cytes/macrophages are derived from the same progenitor
cells, express TLRs and other receptors mediating innate
immunity [39-42]. Because primary human microglia are
not readily available and tumor-derived microglial cell
lines behave differently from primary microglia upon
stimulation [43], in this study we used primary cultures of
monocytes/macrophages from the systemic circulation of
blood donors. Monocytes and microglia activated by single TLR agonists
can kill neurons [[13-16,44,45], present data]. Mitochon-
drial damage contributes to neuronal death both in
inflammation and ALS [46,47]. Neurotoxicity of micro-
glia has been observed in vitro following stimulation with
the TLR4 agonist lipopolysaccharide (LPS) [44,45]. and Strongly elevated calcium influx into G 93A SOD1 neurob
lastoma cells in comparison to wild type SOD1 cells
Figure 8
Strongly elevated calcium influx into G 93A-SOD1
neuroblastoma cells in comparison to wild-type
SOD1 cells. Comparison of the pneumolysin-induced peak
calcium intracellular concentrations (n = 25 cells each, means
± SD, p < 0.0001). Page 8 of 12
(page number not for citation purposes) Page 8 of 12
(page number not for citation purposes) http://www.biomedcentral.com/1471-2334/7/131 BMC Infectious Diseases 2007, 7:131 Vulnerability of G93A-SOD1 and wild-type SOD1 neuroblas-
toma cells to the attack of monocytes stimulated with
Pam3CSK4
Figure 11
Vulnerability of G93A-SOD1 and wild-type SOD1
neuroblastoma cells to the attack of monocytes stim-
ulated with Pam3CSK4. Release of neuron-specific eno-
lase (values expressed in per cent ± SD of the NSE release
induced by cell lysis). After stimulation of monocytes with
the Toll-like receptor 2 agonist Pam3CSK4 G93A-SOD1 SH-
SY5Y cells were more severely injured by activated macro-
phages than Wt-SOD1 cells. Vulnerability of G93A-SOD1 and wild-type SOD1 neuroblas-
toma cells to the attack of monocytes stimulated with
Pam3CSK4
Figure 11
Vulnerability of G93A-SOD1 and wild-type SOD1
neuroblastoma cells to the attack of monocytes stim-
ulated with Pam3CSK4. Release of neuron-specific eno-
lase (values expressed in per cent ± SD of the NSE release
induced by cell lysis). Discussion Acti-
vated microglia are observed in various diseases including
autoimmune- and infection-mediated inflammation,
trauma, ischemia and neurodegeneration [37,38]. In
inherited ALS, after an initial phase of the disease predom-
inated by motor neuron damage caused by mutant SOD,
in a later phase of disease progression is linked to the
inflammatory response of microglia [8]. Discussion Co-stimulation of microglia with
host-derived
compounds
(β-amyloid,
fibronectin,
advanced glycation end products) and bacterial products
can lead to an additive or supra-additive microglial activa-
tion [17,50-52]. In addition to their greater vulnerability
to bacterial hemolysins, increased susceptibility of neu-
rons expressing the G93A mutant in their SOD1 to the
attack of activated immune cells may be the pathophysio-
logical basis of the vulnerability of the nervous system of
patients with motor neuron disease to systemic infections. Endogenous compounds deposited in the extracellular
space (e.g., β-amyloid) [17], entering the brain through
the leaky blood-brain barrier (e.g., fibronectin) [51] or
released by dying neurons and oligodendrocytes
(advanced glycation end products) [50,52] can transform
microglia from a dormant into an activated state, which
renders them more susceptible to the stimulation by bac-
terial products. analogues of bacterial DNA (TLR9 agonist) [13]. The neu-
rotoxic mechanisms of microglia and macrophages
involve generation of nitric oxide (NO) and other reactive
oxygen species [13,44]. Here we demonstrate that activa-
tion of macrophages by the TLR2 agonist Pam3CSK4 can
also cause neuronal death, and that neuroblastoma cells
expressing G93A-SOD1 are more susceptible to the attack
of activated immune cells than those expressing wild-type
SOD1. analogues of bacterial DNA (TLR9 agonist) [13]. The neu-
rotoxic mechanisms of microglia and macrophages
involve generation of nitric oxide (NO) and other reactive
oxygen species [13,44]. Here we demonstrate that activa-
tion of macrophages by the TLR2 agonist Pam3CSK4 can
also cause neuronal death, and that neuroblastoma cells
expressing G93A-SOD1 are more susceptible to the attack
of activated immune cells than those expressing wild-type
SOD1. In the brain of healthy individuals, microglia are in a rest-
ing state. Conversely, in several neurodegenerative dis-
eases, endogenous compounds present in the extracellular
space lead to a chronic activation of microglia [1]. Acti-
vated microglia are observed in various diseases including
autoimmune- and infection-mediated inflammation,
trauma, ischemia and neurodegeneration [37,38]. In
inherited ALS, after an initial phase of the disease predom-
inated by motor neuron damage caused by mutant SOD,
in a later phase of disease progression is linked to the
inflammatory response of microglia [8]. In the brain of healthy individuals, microglia are in a rest-
ing state. Conversely, in several neurodegenerative dis-
eases, endogenous compounds present in the extracellular
space lead to a chronic activation of microglia [1]. Page 9 of 12
(page number not for citation purposes) Competing interests Competing interests
The author(s) declare that they have no competing inter-
ests. immune cells than neuronal cells expressing wild-type
SOD1. In many neurodegenerative diseases, microglia are
chronically stimulated by host-derived compounds and
change their phenotype from a dormant to the activated
state. Activated microglia are more susceptible to stimula-
tion with bacterial products than resting microglia. Increased vulnerability of neurons and increased suscepti-
bility of immune cells to bacterial products probably are
two causes why patients with neurodegenerative diseases
frequently deteriorate during infections. Our in vitro find-
ings must be confirmed in animal experiments and
human studies, before conclusions concerning changes in Conclusion Human immune cells of the monocyte/macrophage/
microglia type activated by a TLR2 agonist can kill neu-
rons. Neuronal cells expressing a SOD1 mutant frequently
encountered in familial cases of amyotrophic lateral scle-
rosis are more vulnerable to the direct action of the bacte-
rial hemolysin pneumolysin and to the attack of activated Low concentrations of different TLR agonists can cause
additive or supra-additive stimulation rendering micro-
glial cells very susceptible to bacterial products at low con- Page 9 of 12
(page number not for citation purposes) Page 9 of 12
(page number not for citation purposes) BMC Infectious Diseases 2007, 7:131 http://www.biomedcentral.com/1471-2334/7/131 Vulnerability of G93A-SOD1 neuroblastoma cells to the
attack of monocytes stimulated with Pam3CSK4
Figure 13
Vulnerability of G93A-SOD1 neuroblastoma cells to
the attack of monocytes stimulated with Pam3CSK4. In-situ tailing shows a macrophage internalising an apoptotic
nucleus of a G93A-SOD1 neuroblastoma cell (arrow). Vulnerability of G93A-SOD1 neuroblastoma cells to the
attack of monocytes stimulated with Pam3CSK4
Figure 13
Vulnerability of G93A-SOD1 neuroblastoma cells to
the attack of monocytes stimulated with Pam3CSK4. In-situ tailing shows a macrophage internalising an apoptotic
nucleus of a G93A-SOD1 neuroblastoma cell (arrow). Vulnerability of G93A-SOD1 neuroblastoma cells to the
attack of monocytes stimulated with Pam3CSK4
Figure 13
Vulnerability of G93A-SOD1 neuroblastoma cells to
the attack of monocytes stimulated with Pam3CSK4. In-situ tailing shows a macrophage internalising an apoptotic
nucleus of a G93A-SOD1 neuroblastoma cell (arrow). Vulnerability of G93A-SOD1 neuroblastoma cells to the
attack of monocytes stimulated with Pam3CSK4
Figure 13
Vulnerability of G93A-SOD1 neuroblastoma cells to
the attack of monocytes stimulated with Pam3CSK4. In-situ tailing shows a macrophage internalising an apoptotic
nucleus of a G93A-SOD1 neuroblastoma cell (arrow). treatment can be drawn. A therapeutic strategy directed at
minimizing the release of proinflammatory/toxic bacte-
rial compounds like TLR-agonists and pneumolysin can
be achieved either by the early treatment of infections
with antibiotics and/or by the use of bactericidal drugs
which minimize the release of bacterial products through
inhibition of bacterial protein synthesis [53-55]. Both
strategies may be beneficial in patients suffering from
neurodegenerative diseases. Vulnerability
toma cells to
Pam3CSK4
Figure 12 y
yp
y
3
4
g
Vulnerability of G93A-SOD1 and wild-type SOD1
neuroblastoma cells to the attack of monocytes stim-
ulated with Pam3CSK4. Conclusion After staining of Pam3CSK4-stimu-
lated co-cultures with light green and macrophage staining
with CD 68 less neuronal cell somata of G93A-SOD1 SH-
SY5Y (lower panel) cells than of Wt-SOD1 cells in equally
treated co-cultures (upper panel) were visible. Please note
the clustering of severely damaged/dead G93A-SOD1 cells
and the area devoid of neuronal cells in the vicinity of groups
of macrophages. y
yp
y
3
4
g
Vulnerability of G93A-SOD1 and wild-type SOD1
neuroblastoma cells to the attack of monocytes stim-
ulated with Pam3CSK4. After staining of Pam3CSK4-stimu-
lated co-cultures with light green and macrophage staining
with CD 68 less neuronal cell somata of G93A-SOD1 SH-
SY5Y (lower panel) cells than of Wt-SOD1 cells in equally
treated co-cultures (upper panel) were visible. Please note
the clustering of severely damaged/dead G93A-SOD1 cells
and the area devoid of neuronal cells in the vicinity of groups
of macrophages. Page 10 of 12
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human macrophage preparation and the co-cultures of
SH-SH5Y and human macrophages. They developed
methods to quantify neuronal damage via WST-1 test,
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the cells. TM provided endotoxin-free pneumolysin used Page 10 of 12
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51. Goos M, Lange P, Hanisch UK, Prinz M, Scheffel J, Bergmann R, Ebert
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Energy Storage on a Distribution Network for Self-Consumption of Wind Energy and Market Value
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i ht General rights
The copyright and moral rights to the output are retained by the output author(s), unless otherwise stated by the document licence. General rights
The copyright and moral rights to the output are retained by the output author(s), unless otherwise stated by the docum Unless otherwise stated, users are permitted to download a copy of the output for personal study or non-commercial research and are
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The copyright and moral rights to the output are retained by the output author(s), unless otherwise stated by the document licence. Link to publication record in Ulster University Research Portal Document Version
Publisher's PDF, also known as Version of record Energy Storage on a Distribution Network for Self-Consumption of Wind Energy and
Market Value Ademulegun, O., Keatley, P., Bani Mustafa, M., & Hewitt, N. (2020). Energy Storage on a Distribution Network
for Self-Consumption of Wind Energy and Market Value. Energies, 13(11), 1-17. Article 2688. https://doi.org/10.3390/en13112688 Ademulegun, O., Keatley, P., Bani Mustafa, M., & Hewitt, N. (2020). Energy Storage on a Distribution Network
for Self-Consumption of Wind Energy and Market Value. Energies, 13(11), 1-17. Article 2688. https://doi.org/10.3390/en13112688 Link to publication record in Ulster University Research Portal Article Received: 4 April 2020; Accepted: 21 May 2020; Published: 26 May 2020 Abstract: Wind energy could be generated and captured with a storage device within the customer
premises for local utilization and for the provision of various services across the electricity supply
chain. To assess the benefits of adding a storage device to an electricity distribution network that has
two wind turbines with a base load of 500 kW and a typical peak load under 1500 kW, a 2 MW/4 MWh
storage is installed. To observe the effects of adding the storage device to the network, a technical
analysis is performed using the NEPLAN 360 modelling tool while an economic analysis is carried
out by estimating the likely payback period on investment. A storage potential benefit analysis
suggests how changes in integration policies could affect the utility of adding the storage device. With the addition of the storage device, self-consumption of wind energy increased by almost 10%. The profitability of the project increased when the device is also deployed to provide stacked services
across the electricity supply chain. Policies that permit the integration of devices into the grid could
increase the profitability of storage projects. Keywords: distributed energy resources; economics of storage; energy storage; self-consumption of
wind; storage services; wind energy energies energies Take down policy Take down policy
The Research Portal is Ulster University's institutional repository that provides access to Ulster's research outputs. Every effort has been
made to ensure that content in the Research Portal does not infringe any person's rights, or applicable UK laws. If you discover content in
the Research Portal that you believe breaches copyright or violates any law, please contact pure-support@ulster.ac.uk Download date: 24/10/2024 www.mdpi.com/journal/energies 1. Introduction The need for low-carbon energy systems in achieving energy sustainability has encouraged the
adoption of different techniques for increasing cleaner energy generation and utilization through
distributed energy resources (DER). For instance, in the UK where a net-zero emission target has been
set [1] and in Northern Ireland where an increasing level of system non-synchronous penetration (SNSP)
is to be achieved on the electricity grid [2], it is desirable to generate clean energy from renewables like
wind turbines. The variable nature of the renewables reduces their effectiveness where the stability
and reliability of the electricity grid is to be maintained. To address the challenges in the variability of
the renewables for a resilient grid, some solutions have been proposed, namely demand-side energy
management and the use of energy storage devices [3,4]. Integrating renewables and energy storage devices into the grid comes with challenges and
opportunities. The opportunities include optimal power management and economic benefits [5], better
utilization of relatively cheap renewable resources [6], increased consumption of the energy produced
from renewable sources [6], less pollution from energy production activities, and reduction of the
curtailments and constraints of renewables [7]. The storage could also be deployed for stacked services
in multi-use purposes [8,9]. The challenges in the integration include the complex nature of the real benefits of storage, the
locational nature of the values for renewables and storage [10], the dynamics of storage economics,
and certain inconsistencies in policies that could discourage innovation. The peculiarities in the
characteristics of the aggregate power system within a region (the structure of the grid, the fuel mix Energies 2020, 13, 2688; doi:10.3390/en13112688 www.mdpi.com/journal/energies www.mdpi.com/journal/energies 2 of 17 Energies 2020, 13, 2688 of the grid, the load profile of attached loads to the grid, the point on the grid where DER are to be
located, the availability of different energy sources, and the electricity market at the location) make the
value derivable from installing DER rather unique, typically varying from location to location [10]. In [11], the market designs for and the characteristics of different ancillary services are described
with emphasis on the increasing role of DER in providing the ancillary services that have historically
been provided by conventional synchronous generators. The procurement schemes and the emerging
ancillary services that may be offered by the distributed resources are also described. 1. Introduction The roles that
DER may play in decarbonization within the distribution network through the provision of ancillary
services have been described in [12]. In [13], a multi-source energy storage model that consists of a
conventional energy storage, multi-energy flow resources, and a demand response resource, at the
demand and the supply sides, has been described for achieving economic self-management of energy
through an intelligent control management method. The integrated distributed energy system was
deployed to deal with the variability in loads and renewable supply. In [14], an energy management
system that maximizes renewable energy utilization while providing certain ancillary services using
a heat pump and a thermal energy storage system has been reported to help achieve cost saving,
reduction of purchased energy imbalance from the grid, more reliable use of the heat pump, and a
more stable surrounding temperature. This work investigates the use of an energy storage device for increasing self-consumption of
wind energy and providing market services within a distribution network having features given
in [15,16]. It is well known that energy storage techniques could be used to capture renewable energy
for later use. However, there is a knowledge gap in ascertaining the real value of deploying the storage
at the specific locations having a unique network, market, and policy characteristics. Moreover, as
reported in [17], it is often uneconomical to deploy storage devices at high investment costs when the
other possible storage application revenues are not considered. The work explores the other value
streams that could make deploying the storage device more profitable at the distribution network. The
addition of the storage device is modelled and technically analyzed using the NEPLAN 360 software
while the economic feasibility of the storage project is assessed by estimating the likely payback period
on investment. The local network is a campus site where the base load is 500 kW while the typical peak load is
below 1500 kW. The distribution network has two behind-the-meter (BTM) wind turbines which are
connected to an alternating current electricity grid through an 11 kV substation. Currently, any excess
energy production from the turbines is fed to the grid at a price fixed by the utility. 1. Introduction Instead of feeding
the excess locally generated wind energy to the grid, the work examines installing a 2 MW/4 MWh
storage device to capture the excess energy—to increase self-consumption of wind energy while also
using some capacity of the storage device for providing certain ancillary services to the grid. As
reported in [18], wind turbines could be deployed to provide grid services; in this work, only the
storage device is deployed for the grid services. To see how changes in policies could impact the
profitability of the project, a potential benefit analysis for adding the device is undertaken using an
existing market structure. 2.1. Description of Distribution Network To investigate how the energy storage device could be used to increase local consumption of wind
energy and provide certain ancillary services, a model of the distribution network is created using
the NEPLAN 360 software. There are 10 substations that feed different loads on-site. There are two
grid-connected wind turbines running on-site. The site is connected to the electricity grid via an 11 kV feeder. From a typical one-year data of the
site, a total energy of 3,720,642 kWh was imported from the grid. A total energy of 3,042,075 kWh was
generated from the wind turbines; whereas 601,780 kWh—representing about 20% of the total energy 3 of 17
e base Energies 2020, 13, 2688
the same one-year
l
d
k
d generated from wind—was exported to the grid. The total annual energy consumption within the
same one-year period was 6,189,647 kWh. The load profile depicts a campus site where the base load
is 500 kW and the typical peak load is less than 1500 kW, Figure 1. the same one-year period was 6,189,647 kWh. The load profile depicts a campus site where the base
load is 500 kW and the typical peak load is less than 1500 kW, Figure 1. 1200
1400 0
200
400
600
800
1000
1200
1400
LOAD (kW)
TIME (Calendar Year)
Figure 1. One-year (365 days) load profile of site. Figure 1. One-year (365 days) load profile of site. A high voltage connection agreement puts the maximum energy that may be exported from
0
200
400
600
800
1000
LOAD (kW)
TIME (Calendar Year) Figure 1. One-year (365 days) load profile of site. tion agreement puts the maximum energy that Figure 1. One-year (365 days) load profile of site. tion agreement puts the maximum energy that may be exported from the Figure 1. One-year (365 days) load profile of site. A high voltage connection agreement puts the maximum energy that may be exported from the
site to the grid (the maximum export capacity) at 1242 kW; the maximum energy that may be
A high voltage connection agreement puts the maximum energy that may be exported from the
site to the grid (the maximum export capacity) at 1242 kW; the maximum energy that may be imported
from the grid to the site (the maximum import capacity) is 2500 kW. 2.1. Description of Distribution Network site to the grid (the maximum export capacity) at 1242 kW; the maximum energy that may be
imported from the grid to the site (the maximum import capacity) is 2500 kW. The line diagram of Figure 2 and Equation (1) both describe the initial configuration of the
distribution network site to the grid (the maximum export capacity) at 1242 kW; the maximum energy that may be
imported from the grid to the site (the maximum import capacity) is 2500 kW. Th
li
di
f Fi
2
d E
i
(1) b
h d
ib
h
i i i l
fi
i
f h
The line diagram of Figure 2 and Equation (1) both describe the initial configuration of the
distribution network. distribution network. ܶଶ± ܩௗ= ܶଵ+ ܼ + ܮ
(1) and Equation (1) both describe the initial configuration of the
T2 ± Ggrid = T1 + Z + L
(1)
ଶ
ௗ
ଵ
( )
nsumed in the aggregated system load Z represents the total f the
(1)
( )
otal ܶଶ± ܩௗ= ܶଵ+ ܼ + ܮ
(1)
where L denotes the total power consumed in the aggregated system load, Z represents the total
power expended in system impedances T1 represents the power supplied from the turbine number
where L denotes the total power consumed in the aggregated system load, Z represents the total power
expended in system impedances, T1 represents the power supplied from the turbine number one,
Ggrid represents the energy from the power grid, and T2 represents the power supplied from turbine
number two. where L denotes the total power consumed in the aggregated system load, Z represents the total
power expended in system impedances, T1 represents the power supplied from the turbine number
one, Ggrid represents the energy from the power grid, and T2 represents the power supplied from
turbine number two. y
p
p
p
pp
nts the energy from the power grid, and T2 represents the powe
wo. Figure 2. Line diagram of distribution network. Figure 2. Line diagram of distribution network. Figure 2. Line diagram of distribution network. Figure 2. Line diagram of distribution network. Figure 2 Line diagram of distribution network
The BTM energy storage device is installed to capture any excess energy generation from the
wind turbines T1 and T2. The network elements of the site are depicted in Figure 3. 2.1. Description of Distribution Network The BTM energy storage device is installed to capture any excess energy generation from the
wind turbines T1 and T2. The network elements of the site are depicted in Figure 3. Figure 2. Line diagram of distribution network. The BTM energy storage device is installed to capture any excess energy generation from the
wind turbines T1 and T2. The network elements of the site are depicted in Figure 3. i
u
i e
a
e
e
o
e e
e
o
e i e a e
epi e
i
igu e
Meanwhile, the loads in the local network are constantly linked to the grid for continuous power
supply irrespective of the power output of the wind turbines. Rather than feeding the excess wind
energy from the turbines to the grid, a storage device is installed on the network to take up the excess
wind energy for later consumption on-site. 4 of 17 4 of 17 Energies 2020, 13, 2688 Figure 3. Arrangement of network elements. Figure 3. Arrangement of network elements. Figure 3. Arrangement of network elements. Figure 3. Arrangement of network elements. Meanwhile, the loads in the local network are constantly linked to the grid for continuous powe
upply irrespective of the power output of the wind turbines. Rather than feeding the excess win
nergy from the turbines to the grid, a storage device is installed on the network to take up the exces
wind energy for later consumption on-site. The data of the aggregate power produced from the turbines and data of the maximum powe
demanded for the one-year period are used as the typical energy profiles of the site. During th
eriod, the base load swung around 500 kW and the peak demand was 1376 kW. The generatio
rofiles of the wind turbines, the local load profile, and the total exported electricity data are used t
stimate a suitable storage portfolio that could help achieve the objectives of maximizing sel
The data of the aggregate power produced from the turbines and data of the maximum power
demanded for the one-year period are used as the typical energy profiles of the site. During this period,
the base load swung around 500 kW and the peak demand was 1376 kW. onsumption of wind en
it
ithi
th
i
2.2. Storage Technologies ite within the same period (the power demand, the power generation, and the electricity impor
xport profiles) are used in ascertaining a suitable storage device—a storage technology that coul
meet the charge-discharge characteristics required. A cost analysis is carried out on some of th
pplicable storage technologies. 2.2. Storage Technologies
It is usually possible to find more than one suitable storage device for any storage project. Th
inal device selection could be made based on any specific storage, utility, or user requirements. Th
ccount of the characteristics of different storage technologies, including the storage that may b
uitable in a BTM application, are given in [19,20]. The technical characteristics of the different energ
torage technologies and applications are presented in [21,22]. Some storage technologies posses
nteresting characteristics Take batteries for example: they are modular—they could be combined i
It is usually possible to find more than one suitable storage device for any storage project. The final
device selection could be made based on any specific storage, utility, or user requirements. The account
of the characteristics of different storage technologies, including the storage that may be suitable in
a BTM application, are given in [19,20]. The technical characteristics of the different energy storage
technologies and applications are presented in [21,22]. Some storage technologies possess interesting
characteristics. Take batteries for example: they are modular—they could be combined in modules to
form small, medium, and large power banks. Such modularity of batteries and some other storage
devices makes them rather suitable in BTM and customer-premise storage applications. Moreover,
the battery could be sized to meet the exact user requirements, optimizing the use of resources. The
other factors that are considered in selecting the storage device for the BTM application include power
requirement, charge–discharge requirement, duration of service required, operating temperature, space
and location requirements, maintenance needs, maturity of the storage technology, and cost. nteresting characteristics. Take batteries for example: they are modular—they could be combined i
modules to form small, medium, and large power banks. onsumption of wind en
it
ithi
th
i
2.2. Storage Technologies Such modularity of batteries and some othe
torage devices makes them rather suitable in BTM and customer-premise storage application
Moreover, the battery could be sized to meet the exact user requirements, optimizing the use o
Some of the established storage options are considered for the project and a few of the most
suitable technologies meeting the desired needs are selected for economic analysis; for example,
flywheel storage and a lithium ion (Li-ion) battery are considered. 2.1. Description of Distribution Network The generation profiles of
the wind turbines, the local load profile, and the total exported electricity data are used to estimate a
suitable storage portfolio that could help achieve the objectives of maximizing self-consumption of
wind energy and providing market services. In other words, the power profiles of site within the same
period (the power demand, the power generation, and the electricity import-export profiles) are used
in ascertaining a suitable storage device—a storage technology that could meet the charge-discharge
characteristics required. A cost analysis is carried out on some of the applicable storage technologies. y
esources. The other factors that are considered in sel
li
i
i
l d
i
h
di
h
2.3. Power Flow Analysis for Determining the Effect of Storage pplication include power requirement, charge–discharge requirement, duration of service required
operating temperature, space and location requirements, maintenance needs, maturity of the storag
echnology, and cost. Some of the established storage options are considered for the project and a few of the mos
uitable technologies meeting the desired needs are selected for economic analysis; for example
To observe how the installation of the storage device will affect the distribution network, a power
flow analysis is undertaken. The network is considered operationally stable before the installation of
the device. After installing the storage device, the network is observed to verify that installing the
device has not compromised the stability and reliability of the distribution network. Energies 2020, 13, 2688
installing the devic
Given that th Energies 2020, 13, 2688
installing the devic
Given that th 5 of 17
ork. and Q Given that the real and the reactive power flowing into a bus i of a network is P and Q respectively,
the static load flow equations used for network analysis could be expressed as:
ܲ= ܸ
ܸܻcos(ߠ+ ߜ− ߜ)
ୀଵ
(2a) Pi = Vi
Xn
k=1 VkYik cos(θik + δk −δi)
(2a)
= −ܸ
ܸܻsin(ߠ+ ߜ−ߜ)
(2b) Pi = Vi
Xn
k=1 VkYik cos(θik + δk −δi)
(2a)
Qi = −Vi
Xn
k=1 VkYik sin(θik + δk −δi)
(2b)
= −ܸ
ܸܻsin(ߠ+ ߜ− ߜ)
ୀଵ
(2b)
k, Yik is the mutual admittance between the ith node and a kth node, n (2a)
( b) Qi = −Vi
Xn
k=1 VkYik sin(θik + δk −δi)
(2b)
ୀଵ
s k, Yik is the mutual admittance between the ith node and a kth node, n (2b)
de n where Vk is the voltage at bus k, Yik is the mutual admittance between the ith node and a kth node, n is
the number of buses within the network, θ represents the phase angle between current and voltage, δ
represents the load angle, and Vi represents the bus voltage. g
,
,
is the number of buses within the network, θ represents the phase angle between current and voltage,
δ represents the load angle, and Vi represents the bus voltage. A
di
A
t i
d
i
ti
f th
l
d fl
ti
Th
li
t ti l
d fl Appendix A contains a derivation of the load flow equations. The non-linear static load flow
equations are solved numerically. y
esources. The other factors that are considered in sel
li
i
i
l d
i
h
di
h
2.3. Power Flow Analysis for Determining the Effect of Storage The NEPLAN 360 modeller has a library of numerical solutions for
technical power flow analysis. The modeller takes the network elements and their electrical parameters
as inputs, uses a numerical method to analyse the power network, and outputs the electrical signals
(current, voltage, power) at the network nodes and within the elements. It also indicates whether the
numerical model converges or not and indicates where any excess power flows occur. With a model of
the distribution network created, running a power flow reveals the changes to the network as a result
of installing the storage device. Appendix A contains a derivation of the load flow equations. The non-linear static load flow
equations are solved numerically. The NEPLAN 360 modeller has a library of numerical solutions for
technical power flow analysis. The modeller takes the network elements and their electrical
parameters as inputs, uses a numerical method to analyse the power network, and outputs the
electrical signals (current, voltage, power) at the network nodes and within the elements. It also
indicates whether the numerical model converges or not and indicates where any excess power flows
occur. With a model of the distribution network created, running a power flow reveals the changes
to the network as a result of installing the storage device. 2.4. Power Management of Storage
2.4. Power Management of Storag The diagram of Figure 4 describes the final configuration of the distribution network. The diagram of Figure 4 describes the final configuration of the distribution network. Figure 4. Adding storage to distribution network. Figure 4. Adding storage to distribution network. Figure 4. Adding storage to distribution network. Figure 4. Adding storage to distribution network. Figure 4. Adding storage to distribution network. Figure 4. Adding storage to distribution network. The switch Sw1 links the distribution network to the grid. Equation (3a,b) describe how the switch
S
i t b
t d
The switch Sw1 links the distribution network to the grid. Equation (3a,b) describe how the switch
Sw1 is to be operated. e distribution network to the grid. Equation (3a,b) describe how the switch
he distribution network to the grid. Equation (3a,b) describe how the switch ܵ௪ଵ= 1, ݓℎ݁݊ ܮ+ ܼ> ܶଵ+ ܶଶ+ ܧ(୫୧୬)
(3a)
ܵ
= 0 ݓℎ݁݊ܮ+ ܼ< ܶ+ ܶ+ ܧ
(3b)
Sw1 = 1, when L + Z > T1 + T2 + E(min)
(3a)
Sw1 = 0, when L + Z < T1 + T2 + E(min)
(3b) (3a) (3a)
(3b) ܵ௪ଵ= 0, ݓℎ݁݊ ܮ+ ܼ< ܶଵ+ ܶଶ+ ܧ()
(3b)
where L denotes the energy demand by system load, Z represents the total energy expended in the
system impedance, T1 represents the energy feed from the turbine number one, T2 represents the
energy feed from the turbine number two E(min) represents the implied device discharge limit and
where L denotes the energy demand by system load, Z represents the total energy expended in the
system impedance, T1 represents the energy feed from the turbine number one, T2 represents the
energy feed from the turbine number two, E(min) represents the implied device discharge limit, and Sw1
represents switch one. energy feed from the turbine number two, E(min) represents the implied device discharge limit, and
Sw1 represents switch one. The switch Sw2 determines the time that the storage device E is to be charged or discharged; it is
operated according to a control rule set at the C
d
Equation (4) and Equation (5) describe the
The switch Sw2 determines the time that the storage device E is to be charged or discharged; it is
operated according to a control rule set at the Cnode. Equations (4) and (5) describe the operation of the
switch Sw2 and the control at the Cnode. for any discharge-limit instances t = 1, 2, 3, . . . , n. for any discharge-limit instances t = 1, 2, 3, . . . , n. for any discharge-limit instances t = 1, 2, 3, . . . , n. Switches Sw1 and Sw2 operate to ensure that the storage device is charged with a power supply from
the wind turbines only. The switches ensure that the device is discharged to maximize self-consumption
of the on-site-generated wind energy while also securing certain capacity of the device for the provision
of any commitment to ancillary services. 2.5. Assessing the Benefit of Storage A feature assessment of some storage technologies discussed in [19–22] is undertaken to identify
some of the storage options that could meet the defined objectives of maximizing self-consumption of
wind energy and providing ancillary services. A cost analysis is carried out on the identified devices. The profitability of adding the storage device is determined by taking the likely storage cost ranges,
storage efficiencies, storage capacity, the electricity market, and the potential additional storage services
as key parameters. 2.4. Power Management of Storage
2.4. Power Management of Storag cording to a control rule set at the Cnode. Equation (4) and Equation (5) describe the
the switch Sw2 and the control at the Cnode. ܧ(୫୧୬) ∝ ൣ(ܧௌை) ܣܰܦ (ܧௌ௩௦ ) ܣܰܦ (்ܶ݅݉݁) ܣܰܦ (ܶଵ) ܣܰܦ (ܶଶ)൧
(4)
E(min) ∝
h
(ESOC) AND (EServices) AND
TimeTari f f
AND (T1) AND (T2)
i
(4)
Sw2 ∝E(min) = 1 OR 0
(5) o
i g o a
o
o
u e
e a
e
Equa io
( ) a
Equa io
( )
e
i e
e
the switch Sw2 and the control at the Cnode. ܧ
ൣ(ܧ
) ܣܰܦ(ܧ
) ܣܰܦ(ܶ݅
) ܣܰܦ(ܶ) ܣܰܦ(ܶ)൧
(4)
E(min) ∝
h
(ESOC) AND (EServices) AND
TimeTari f f
AND (T1) AND (T2)
i
(4) (4) (ܧௌ௩௦ ) ܣܰܦ (்ܶ݅݉݁) ܣܰܦ (ܶଵ) ܣܰܦ (ܶଶ)൧
(4)
(
Services)
Tari f f
(
1)
(
2)
i
( )
Sw2 ∝E(min) = 1 OR 0
(5) (ܧௌ௩௦ ) ܣܰܦ (்ܶ݅݉݁) ܣܰܦ (ܶଵ) ܣܰܦ (ܶଶ)൧
(4)
Sw2 ∝E(min) = 1 OR 0
(5) (ܧௌ௩௦ ) ܣܰܦ (்ܶ݅݉݁
Sw2 ∝E(min) = 1 OR 0 (4)
(5) ܵ௪ଶ ∝ ܧ() = 1 ܱܴ 0
(5)
where T2 represents the energy feed from the turbine number two, T1 represents the energy feed
from the turbine number one, TimeTariffis the instantaneous price of electricity, Eservices is the aggregate
ancillary service demand on the storage device, ESOC is any specified charging state of the device, ܵ௪ଶ ∝ ܧ() = 1 ܱܴ 0
(5)
where T2 represents the energy feed from the turbine number two, T1 represents the energy feed
from the turbine number one, TimeTariffis the instantaneous price of electricity, Eservices is the aggregate
ancillary service demand on the storage device, ESOC is any specified charging state of the device, 6 of 17 Energies 2020, 13, 2688 Energies 2020, 13, 2688 E(min) represents the implied device discharge limit, “AND” is a summing logic, Sw2 represent switch
two and “OR” is also a logical expression. E(min) represents the implied device discharge limit, “AND” is a summing logic, Sw2 represent switch
two and “OR” is also a logical expression. E(min)(1)+ = E(min)(0)+ ± E(min)(1)−
E(min)(2)+ = E(min)(1)+ ± E(min)(2)− that is, E(min)(t)+ = E(min)(t −1)+ ± E(min)(t)− 2.5.1. Benefits of Self-Consumption of Wind Energy A benefit analysis is carried out to ascertain the gains in installing the storage device for increasing
the self-consumption of wind energy. The costs of energy storage systems are not fixed. Because
of the dynamic nature of storage economics, in estimating the cost of storage, hypothesised prices
are used to reduce the effect of random errors that could arise from the use of a static price quote. Using a price quote given at a time for an analysis invalidates any result from the analysis in a new
economic setting. Taking into cognizance the high likelihood of changes in the prices of some of the
storage technologies and with a broad study of the inconsistencies in price quotes from literature
and industry—for example, consider the different prices specified for the same storage technology
plus notes on cost inconsistencies in [14,19,22–32]—the most likely cost range for each of the storage
technologies is heuristically selected for analysis. While the analysis is not claiming that any storage option is currently economical under the
existing market arrangement, the analysis aims to identify the cost point at which the storage becomes
economically feasible with respect to the distribution network and to reveal where changes in market
conditions or storage costs could impact the profitability of the storage project. The cost range also
makes it possible to apply the results of the analysis within any reasonable future changes to the
economics of storage. Using an existing market system, the benefits of installing the storage device for increasing
self-consumption of wind energy is analysed. In the market, the price of import electricity and the
price of export electricity are in the ratio of 7 to 3 typically, the price of import electricity being often
higher: when the import electricity price is at £7/kWh, the exported electricity price will be around
£3/kWh. The prices could vary in different economic settings but have consistent relations—based on
the historical analysis of the site export-import payment data and in [33]. The benefit through self-consumption of wind energy is based on the difference between the
import and export electricity prices; the prices are fixed within days but could change when the
utility decides to review rates to reflect new economics. The total recoverable energy is obtained by
multiplying the captured (used to be exported) energy by the storage efficiency. 2.5.2. Benefits through Market Services In another case, in addition to helping to increase self-consumption of wind energy, certain
capacity of the storage device is committed to providing some services to the electricity grid through
DS3/ISEM [34–36]—“Delivering a Secure, Sustainable Electricity System” (DS3) is a programme
developed to increase the penetration of renewables like wind on the power network, whereas
the Integrated Single Electricity Market (ISEM) is a cross-border electricity market that allows the
interconnection of grids for wholesale electricity trading. The values from the actual provision of the ancillary services are not included because the actual
provision of the services is usually within very short times [18] and the exact amount of the services
provided may not be pre-determinable since the services are demanded by the electricity grid only
during special operating conditions, maintaining the stability of the grid. The values accounted for
here are only for the service “commitment,” and not for the actual performance: the value derivable
from connecting the storage device to the grid and making certain capacities available for charging or
discharging in supporting the grid during operational emergencies. The services that the storage devices could provide are selected and aggregated from the DS3
service suite given in [36]. The service suite helps in maintaining the stability and reliability of the
grid as non-synchronous power sources increase with the integration of the variable renewables. The
service products are required to guarantee a qualitative performance of the grid. The products are
described by the transmission network operators—EirGrid and the System Operator for Northern
Ireland (SONI) in [37,38]—with rates defined for specified times in [39]. The suite of services that a
typical storage device could provide is summarised in Table 1 [40–42]. Table 1. Storage eligible DS3 service suite with base rates in £/MWh (2019–2020). Products
Abbreviation
Storage Eligible
Payment Rate (£/MWh)
Fast Frequency Response
FFR
Yes
1.98
Primary Operating Reserve
POR
Yes
2.97
Ramping Margin 1
RM1
Yes
0.11
Ramping Margin 3
RM3
Yes
0.16
Ramping Margin 8
RM8
Yes
0.15
Replacement Reserve
(De-Synchronised)
RRD
Yes
0.51
Replacement Reserve
(Synchronised)
RRS
Yes
0.23
Secondary Operating Reserve
SOR
Yes
1.80
Tertiary Operating Reserve 1
TOR1
Yes
1.42 Table 1. Storage eligible DS3 service suite with base rates in £/MWh (2019–2020). 2.5.1. Benefits of Self-Consumption of Wind Energy The market value of 7 of 17 Energies 2020, 13, 2688 the recovered energy is obtained by multiplying the total recoverable energy by the market price. The
gross annual gain is the difference between the market value of the recovered energy at the import
electricity price and the market value of the exported energy at the export electricity price. 2.5.3. Potential Benefit across Electricity Supply Chain This section examines the value of the storage device installed on the described distribution
network in general, not only the device deployed to capture the wind energy produced by BTM
turbines. To account for the full range of values that could be derived from any typical installation,
a potential benefit analysis is carried out for the entire stack of services that the storage device could
potentially offer across the electricity supply chain. In accounting for the potential storage benefits, with assumptions where required, the following
approximate daily storage service values are estimated: •
DS3 services: the total suite of the DS3 service that the storage device commits to is £10/MWh, the
size of the device deployed is 2 MW/4 MWh, 40% of the device capacity has been committed to
providing the services, the storage system has 85% roundtrip efficiency—the storage has minimal
energy losses while charging and discharging. •
Increased wind self-consumption: the size of the storage device is 2 MW/4 MWh, the device is
85% efficient (roundtrip), the site data—containing the import and the export electricity prices, the
energy exports from the wind turbines, the energy generated by the turbines, and the total load
energy required—are used in calculating the gross annual gain from self-consumption of wind
energy. The daily potential gain is estimated by dividing the gross annual gain by the number of
days in a year. •
Time-of-use-bill-management: the size of the storage device is 2 MW/4 MWh; the device is 85%
efficient (roundtrip), the site data are used in calculating the mean daily import; using the Power
NI—an electricity supplier—Economy 7 (2-Rate) meter plan [44], a third of the total electricity
required is set to be imported at a low rate period (at nights) while the remaining electricity is
imported at a high rate period (during the day). •
Demand response of load shifting: the size of the storage device is 2 MW/4 MWh; the device is
85% efficient (roundtrip), the site data are used in calculating the mean daily import; using the
SSE Airtricity (an electricity supplier) KeyPad Powershift meter plan, a third of the total electricity
required for the day is imported within the “low” rate period—between 1:00 and 9:00 [45,46]
while the remaining electricity is imported at the “normal” rate period during the day. 2.5.3. Potential Benefit across Electricity Supply Chain Some of the storage services highlighted are mutually exclusive; for example, while the storage
device has been deployed for increasing self-consumption of wind energy and providing certain levels
of ancillary services, the device may no longer be fully utilisable for time-of-use-bill-management at
the same time. While inadequate policies may not allow some storage benefits to be realizable now,
the potential benefit analysis is to indicate storage-utilisation possibilities and reveal the changes in
policies that could monetise additional storage values at the distribution network. Other potential storage values could be estimated for specific sites within the distribution network. Meanwhile, any given application could require using a storage device with specific characteristics. 2.5.2. Benefits through Market Services While ancillary services were traditionally provided by equipment connected to the transmission
network; in certain instances the services could be provided through devices connected to the
distribution network—this will usually depend on locational service needs, existing interconnection
policies, and requires planning and coordination of network operations. The storage device could be
restricted within certain limits in providing the services [42,43]. For this case of presenting the device for both maximizing self-consumption of wind energy and
committing to providing certain ancillary services in stack, a new economic analysis is performed. The
new analysis is to reveal how the commitment of the device to providing stacked market services
impacts the profitability of the storage project. The total DS3 service provided is the summation of the
storage eligible DS3 service suite of Table 1—at the aggregated standard rate of £10.47/MWh. Energies 2020, 13, 2688 8 of 17 At the first instance 20% of the storage capacity is committed within less than 2% of total lifespan
of the storage device, for the estimation of Gain 1 and the payback Period 1. The same storage capacity
is committed for 25% of the device lifespan at the second instance, for the estimation of the Gain 2
and the payback Period 2. The ancillary service gain is a product of the committed capacity and the
aggregated value, £10.47/MWh. The new annual gains are estimated as the sum of the gain from
self-consumption of wind energy and the gain from the provision of ancillary services. It is assumed
that committing the storage device to providing the ancillary services comes at zero or insignificant
extra cost. 3. Results and Discussion While the on-site loads are supplied with the power generation from the wind turbines and the
grid, the installed storage device takes up any excess wind energy generation from the turbines as the 9 of 17 Energies 2020, 13, 2688 load flow converges while the network elements operate within safe limits, illustrated for a typical
windy day in Figure 5. Energies 2020, 13, x FOR PEER REVIEW
9 of 17 Figure 5. Energy profiles for an illustrative day. Figure 5. Energy profiles for an illustrative day. Figure 5. Energy profiles for an illustrative day. Figure 5. Energy profiles for an illustrative day. Figure 5. Energy profiles for an illustrative day. Figure 5. Energy profiles for an illustrative day. Figure 5. Energy profiles for an illustrative day. Figure 5. Energy profiles for an illustrative day. The energy profile reveals the charge-discharge characteristics, suggesting an applicable storage
device, Figure 5. Between midnight (00:00) and evening (18:00), the aggregate power from the two
wind turbines was close to 600 kW—a typically windy day. With the load demand rising from the
base point at 500 kW, the loads are served from the turbines (with the excess wind generation and
low demand at this time) and the storage device is discharged to meet the additional demand until
at around 4:30 when the energy generation from the turbines increases, the load demands being fully
served and the excess wind energy charging the device through to around 5:40. As the load demand
increases through the day, more energy is imported from the grid to supplement the energy
generation from the turbines while the storage device is kept at a state of charge. At about 20:00, the
wind energy generation drops; the battery is discharged as much as possible while the deficit in
energy supply is met by the grid
the import from the grid moving close to 400 kW
The energy profile reveals the charge-discharge characteristics, suggesting an applicable storage
device, Figure 5. Between midnight (00:00) and evening (18:00), the aggregate power from the two
wind turbines was close to 600 kW—a typically windy day. 3. Results and Discussion With the load demand rising from the
base point at 500 kW, the loads are served from the turbines (with the excess wind generation and
low demand at this time) and the storage device is discharged to meet the additional demand until at
around 4:30 when the energy generation from the turbines increases, the load demands being fully
served and the excess wind energy charging the device through to around 5:40. As the load demand
increases through the day, more energy is imported from the grid to supplement the energy generation
from the turbines while the storage device is kept at a state of charge. At about 20:00, the wind energy
generation drops; the battery is discharged as much as possible while the deficit in energy supply is
met by the grid—the import from the grid moving close to 400 kW. energy supply is met by the grid—the import from the grid moving close to 400 kW. The profile indicates that the deployed storage device could be subject to daily multiple rounds
of discharge cycles to achieve a maximum self-consumption of wind energy. This suggests that the
deployed storage device should have the capability for several rounds of deep discharge
The profile indicates that the deployed storage device could be subject to daily multiple rounds
of discharge cycles to achieve a maximum self-consumption of wind energy. This suggests that the
deployed storage device should have the capability for several rounds of deep discharge. deployed storage device should have the capability for several rounds of deep discharge. Within the one-year period under consideration, while a 3,720,642 kWh of energy at a market
value of £4,464,777.04 (3,720,642 kWh × £0.12/kWh) was imported from the grid, a total energy of
601,780 kWh at a market value of £31,593.45 (601,780 kWh × £0.0525/kWh) was exported to the grid. The total recoverable energy is obtained by multiplying the captured (used to be exported) energy
(601,780 kWh) by the storage efficiency. The market value of the recovered energy has been obtained
by multiplying the total recoverable energy by the market price of £0.12/kWh—the import and the
export electricity prices are approximated from the historical analysis of the export and the import
payments data. In [33], a similar price relation between the export electricity price and the import
electricity price for grid-connected wind turbines on the foregoing distribution network may be seen. 3. Results and Discussion The gross annual gain shows the difference in market value at the import electricity price of
£0 12/kWh and at the export electricity price of £0 0525/kWh Table 2
Within the one-year period under consideration, while a 3,720,642 kWh of energy at a market
value of £4,464,777.04 (3,720,642 kWh × £0.12/kWh) was imported from the grid, a total energy of
601,780 kWh at a market value of £31,593.45 (601,780 kWh × £0.0525/kWh) was exported to the grid. The total recoverable energy is obtained by multiplying the captured (used to be exported) energy
(601,780 kWh) by the storage efficiency. The market value of the recovered energy has been obtained
by multiplying the total recoverable energy by the market price of £0.12/kWh—the import and the
export electricity prices are approximated from the historical analysis of the export and the import
payments data. In [33], a similar price relation between the export electricity price and the import
electricity price for grid-connected wind turbines on the foregoing distribution network may be seen. The gross annual gain shows the difference in market value at the import electricity price of £0.12/kWh
and at the export electricity price of £0.0525/kWh, Table 2. 10 of 17 Energies 2020, 13, 2688 Table 2. Effect of storage efficiency on total recoverable energy. Efficiency of
Storage
System (%)
Total Recoverable
Energy (kWh)
Market Value of
Recovered Energy
at £0.12/kWh (£)
Gross Annual Gain at
£(0.12–0.0525)/kWh (£)
Self-consumption
of Wind Energy
(%)
95
571,691.00
68,602.92
37,009.47
48.89
90
541,602.00
64,992.24
33,398.79
48.40
85
511,513.00
61,381.56
29,788.11
47.91
80
481,424.00
57,770.88
26,177.43
47.42
75
451,335.00
54,160.20
22,566.75
46.93
70
421,246.00
50,549.52
18,956.07
46.45 The quantity of the recoverable energy is more when using a storage a device of higher
efficiency—as less of the excess wind energy is wasted through the charge-discharge cycles with the
higher efficiency storage system; for example, while a total energy of 571,691.00 kWh is recoverable
when using a 95% efficient storage system, only a 421,246.00 kWh of energy is recoverable when
using a 70% efficient storage system. In the existing market in which the import electricity price is
£0.12/kWh and the export electricity price is £0.0525/kWh—taken as typical prices—the gross annual
gain and the percentage of energy serving the loads from the storage device are more while using the
high-efficiency storage system, Table 2. 3. Results and Discussion 11 of 17
sil fuel Energies 2020, 13, 2688
average energy mix
[15] Figure 6. Energy mix of site. Figure 6. Energy mix of site. Figure 6. Energy mix of site. Figure 6. Energy mix of site. In analysing the value derived from deploying the storage device for self-consumption of wind
energy: the total storage capacity cost is a total system cost—covering any cost associated with the
acquisition, installation, and usage of the storage (including fixed cost, variable cost, capital cost,
Each of the storage technologies has been assigned a nominal storage efficiency; the values are
the overall roundtrip efficiencies of the whole system of storage. The typical lifespan of a flywheel
storage is taken to be above 20 years, the lithium ion and the sodium ion batteries are taken to have
lifespans between 10 to 15 years, and the zinc-bromine flow battery is considered to have a lifespan of
between 5 to 10 years [19,22]. The lifespans of the storage technologies are included to show where the
technologies could make economic sense around the hypothesised prices. The payback period is the
ratio of the cost of the total storage system to the gross annual gain of storage, Table 3. Table 3. Deployment of storage device to store excess wind energy only. Table 3. Deployment of storage device to store excess wind energy only. Table 3. Deployment of storage device to store excess wind energy only. Selected Energy Storage
Technologies and Costs
(£/kW; £/kWh)
Total Storage
Capacity Cost
(£ Million)
Nominated
Storage
Efficiency (%)
Life
Span
(Years)
Gross
Annual
Gain (£)
Payback
Period (Years)
Flywheel at £120/kW; at £80/kWh
0.56
90
20+
33,398.79
16.8
Flywheel at £1880/kW; at
£1715/kWh
10.62
90
20+
33,398.79
318.0
Li-ion Battery at £110/kW, at
£70/kWh
0.50
85
10–15
29,788.11
16.8
Li-ion Battery at £1580/kW, at
£1510/kWh
9.20
85
10–15
29,788.11
308.8
Na-ion Battery at £90/kW, at
£60/kWh
0.42
80
10–15
26,177.43
16.0
Na-ion Battery at £1200/kW, at
£1100/kWh
6.80
80
10–15
26,177.43
259.8
* Zn-Br Flow Battery at £105/kW,
at £65/kWh
0.47
75
5–10
22,566.75
20.8
* Zn-Br Flow Battery at £1150/kW,
at £800/kWh
5.50
75
5–10
22,566.75
243.7
* As most power equipment usually last for over 40 years, it is customary to evaluate new equipment within a
minimum of 10-year frame. Zn-Br Flow battery may not last for up to 10 years. 3. Results and Discussion The result of Table 2 suggests that, to derive more gain from
deploying a storage device for increasing self-consumption of the locally generated wind energy, a
storage technology having a higher efficiency should be used. Another important storage characteristic that should be considered is the operating temperature of
the storage device in respect of its environment. For example, some battery performances may degrade
while operating outside recommended temperature ranges. The mean annual temperatures at this site
over centuries have ranged from 8.5 ◦C to 10.0 ◦C, with a record extreme maximum temperature at
32.3 ◦C and minimum temperature at −9.0 ◦C [47,48]. The storage technologies selected can operate
well within the site temperature range. In other words, the storage technologies selected have typical roundtrip efficiencies above 65%,
could meet the charge-discharge characteristics required, are mature or demonstrated technologies,
have reasonable cost trends, have operating temperature features that make them appropriate at the site,
are applicable at the point of the distribution network, and could serve both as load and as generator. Of the considered storage technologies, flywheel storage, a lithium ion battery, sodium ion (Na-ion)
battery, and a zinc-bromine (Zn-Br) flow battery are found to meet the storage requirements [19–22]. Considering the changes to the energy mix of the site: with the storage, no on-site generated wind
energy is supplied to the grid—the storage captures the excess wind energy for self-consumption
on-site. As depicted in Figure 6, the percentage of the wind energy in the energy mix at the location
moved from 39.47% in Figure 6a to 48.32% in Figure 6b—an almost 10% increase in self-consumption
of wind energy. The other part of the energy mix came from a grid supply with an average energy mix
containing about 55% of the total energy generation coming from fossil fuel sources [15]. In analysing the value derived from deploying the storage device for self-consumption of wind
energy: the total storage capacity cost is a total system cost—covering any cost associated with the
acquisition, installation, and usage of the storage (including fixed cost, variable cost, capital cost, initial
cost, maintenance cost, and any complementary costs). The cost ranges are heuristic test-case selections. The cost options help to see where the profitability of the storage project lies for different storage cost
parameters that could typify varying market conditions, using a payback period estimation within the
life span of the storage device. 3. Results and Discussion * As most power equipment usually last for over 40 years, it is customary to evaluate new equipment within a
minimum of 10-year frame. Zn-Br Flow battery may not last for up to 10 years. The results of Table 3 suggest that with the current market conditions, the deployment of the
2 MW/4 MWh energy storage device for self-consumption of wind energy could become economically
feasible at the storage cost around £500,000. Given that the storage technologies have similar costs,
flywheel storage promises higher return on investment because of its longer lifespan, inherent
almost-unlimited cycles, and ruggedness in responding effectively to providing specialised electricity
grid services. However, its considerable self-discharge rate could make it a less desirable choice for
deferred self-consumption of stored energy [22]. A lithium-ion battery could be a better option for
being a more mature technology, being less susceptible to self-discharge, being able to withstand
several rounds of deep discharging, and like most batteries, being able to respond in time to providing
grid services [19]. 12 of 17 Energies 2020, 13, 2688 While the results of Table 3 are for the case where the storage device has been deployed only
for increasing self-consumption of wind energy, Table 4 depicts the result of deploying the device for
providing certain DS3 market services in addition to increasing self-consumption of wind energy. While the results of Table 3 are for the case where the storage device has been deployed only
for increasing self-consumption of wind energy, Table 4 depicts the result of deploying the device for
providing certain DS3 market services in addition to increasing self-consumption of wind energy. Table 4. Deployment of storage for self-consumption of wind energy and ancillary services. 3. Results and Discussion Selected Energy Storage
Technologies and Costs
(£/kW; £/kWh)
Ancillary
Services
Duration/
Lifespan (%)
New Annual
Gain 1 (£)
New
Payback
Period 1
(Years)
Ancillary
Services
Duration/
Lifespan (%)
New Payback
Period 2
(Years)
Flywheel at £120/kW; at
£80/kWh
0.42
36,150.31
15.5
25
2.8
Flywheel at £1880/kW; at
£1715/kWh
0.42
36,150.31
293.8
25
53.9
Li-ion Battery at £110/kW, at
£70/kWh
0.56–0.83
32,126.90
15.6
25
3.9
Li-ion Battery at £1580/kW, at
£1510/kWh
0.56–0.83
32,126.90
286.4
25
72.3
Na-ion Battery at £90/kW, at
£60/kWh
0.56–0.83
28,048.42
15.0
25
3.5
Na-ion Battery at £1200/kW, at
£1100/kWh
0.56–0.83
28,048.42
242.4
25
57.7
Zn-Br Flow Battery at
£105/kW, at £65/kWh
0.83–1.7
23,970.04
19.6
25
6.3
Zn-Br Flow Battery at
£1150/kW, at £800/kWh
0.83–1.7
23,970.04
229.5
25
74.2 Table 4. Deployment of storage for self-consumption of wind energy and ancillary services. With the storage deployed for the multipurpose of increasing self-consumption of wind energy
and providing the ancillary services, the results indicate a shorter payback period on investment,
suggesting increased profitability. The total DS3 service provided has been taken from the storage
eligible DS3 service suite of Table 1. The storage capacity is committed within less than 2% of total
lifespan of the storage device at the first instance: this estimates the new annual Gain 1 and the
new payback Period 1. The same capacity is committed for 25% of the device total lifespan at the
second instance: estimates a new Gain 2 and the new payback Period 2, Table 4. The new annual
gain is the sum of the gain from self-consumption of wind energy and the gain from the provision of
ancillary services. The payback periods are shorter when the storage device is committed for longer duration. This
suggests that, when deploying a storage device at the distribution network, it could be more profitable
to commit the device to providing ancillary services to an extent permissible and that does not pose a
risk to the security of other investments serving the grid. Another picture is depicted in Figure 7, where the daily potential value that the deployed energy
storage system could offer to stakeholders across the electricity supply chain has been estimated using
the approximate data described in Section 2.5.3. installed for man
ends of the elect
4. Conclusions y
gy
g ;
p ,
gy
g
g
periods of high price volatility through the Irish ISEM intra-day market [35]. Lastly, a country-wide analysis could be performed to see how storage systems could be
deployed to support renewables and bring optimal benefits to the customer, to the grid, and to the
utility; maximizing renewable energy generation in achieving key sustainability targets. 4. Conclusions
Energy generation from wind turbines connected to the distribution network could contribute
to the effort of decarbonizing electricity systems. With storage devices, more of the on-site generated
wind energy could be captured for later energy consumption. For grid-connected systems, where the
market and integration policies permit it, the storage device could—in addition to providing
customer services—be committed to providing DS3 services of active and reactive power, ramping
margins, and reserves. When a 2 MW/4 MWh storage device was deployed at a distribution network
having two 800 KW BTM wind turbines, a typical peak load under 1500 kW and a base load around
500 kW, the percentage of self-consumption of wind energy rose from 39.47% to 48.32%. Deploying
the device for providing other market services in addition to helping to achieve increased self-
consumption of wind energy makes the storage project more profitable
suggesting a mechanism
Energy generation from wind turbines connected to the distribution network could contribute to
the effort of decarbonizing electricity systems. With storage devices, more of the on-site generated
wind energy could be captured for later energy consumption. For grid-connected systems, where the
market and integration policies permit it, the storage device could—in addition to providing customer
services—be committed to providing DS3 services of active and reactive power, ramping margins, and
reserves. When a 2 MW/4 MWh storage device was deployed at a distribution network having two
800 KW BTM wind turbines, a typical peak load under 1500 kW and a base load around 500 kW, the
percentage of self-consumption of wind energy rose from 39.47% to 48.32%. Deploying the device
for providing other market services in addition to helping to achieve increased self-consumption of
wind energy makes the storage project more profitable—suggesting a mechanism through which the
storage system could be deployed to contribute to the on-going effort of maximizing the utilization
of clean energy for sustainable development. 3. Results and Discussion While some of the potential values such as demand
charge reduction and increased wind self-consumption are concrete, others—such as transmission
and distribution deferrals—could be conceptual and often require favourable integration policies and
proper grid planning or coordination to become realizable. Certain incentives could be available for generating and using more clean energy on-site; for
example, the revenue stream from the Renewable Obligation Certificate (ROC) that was in place
to promote renewable energy in Northern Ireland [33]. Similarly, some mechanisms that reduce
investment risks; for example, the power purchase agreements (PPA) could serve to guarantee the
market for the storage services. The ROC and the PPA arrangements are typical market and integration
policies that could impact the value of any energy storage project. 13 of 17 Energies 2020, 13, 2688
integration policies Figure 7. Potential daily revenue of storage across electricity supply chain. £219.40
£81.60
£128.70
£192.00
0
50
100
150
200
250
Services (£)
Value (£)
DS3 Services
Demand Response of Load Shifting
Increased Wind Energy Self-consumption
Time-of-Use-Bill-Management
Figure 7. Potential daily revenue of storage across electricity supply chain. Figure 7. Potential daily revenue of storage across electricity supply chain. £219.40
£81.60
£128.70
£192.00
0
50
100
150
200
250
Services (£)
Value (£)
DS3 Services
Demand Response of Load Shifting
Increased Wind Energy Self-consumption
Time-of-Use-Bill-Management
Figure 7. Potential daily revenue of storage across electricity supply chain. Figure 7. Potential daily revenue of storage across electricity supply chain. Figure 7. Potential daily revenue of storage across electricity supply chain. Certain incentives could be available for generating and using more clean energy on-site; for
example, the revenue stream from the Renewable Obligation Certificate (ROC) that was in place to
promote renewable energy in Northern Ireland [33]. Similarly, some mechanisms that reduce
investment risks; for example, the power purchase agreements (PPA) could serve to guarantee the
market for the storage services. The ROC and the PPA arrangements are typical market and
integration policies that could impact the value of any energy storage project. Meanwhile beyond the distribution network some other storage benefits which are also
Meanwhile, beyond the distribution network, some other storage benefits which are also typically
very site-specific could be derived while using the storage device for capturing or saving energy for
later use. 3. Results and Discussion To mention a few: to manage the output of mass wind turbines where a network congestion
would have disallowed any further grid-integration of turbines, a storage device could be installed
for managed connection. The storage device could also be installed at the higher voltage ends of the
electricity network for energy arbitrage; for example, for bulk energy trading during periods of high
price volatility through the Irish ISEM intra-day market [35]. Meanwhile, beyond the distribution network, some other storage benefits which are also
typically very site-specific could be derived while using the storage device for capturing or saving
energy for later use. To mention a few: to manage the output of mass wind turbines where a network
congestion would have disallowed any further grid-integration of turbines, a storage device could be
p
y
g
y
Lastly, a country-wide analysis could be performed to see how storage systems could be deployed
to support renewables and bring optimal benefits to the customer, to the grid, and to the utility;
maximizing renewable energy generation in achieving key sustainability targets. Acknowledgments: James Waide of the Physical Resources Department at Ulster University provided support
while collecting data. Paul Bell of the Utility Regulator Northern Ireland supported in information gathering.
The System Operator for Northern Ireland (SONI) and the Northern Ireland Electricity (NIE) Networks provided
support in data collection. Funding: This research was funded by the Science Foundation Ireland (SFI) and the Department for the Economy
(DfE) in Northern Ireland, grant number 92160R. Appendix A Static Load Flow Equations: Given that the net complex power flowing into a bus i of a network is: (A1) Si = Pi + jQi = (PGi −PDi) + j(QGi −QDi)
(A1) where PD and QD are the real power demand and the reactive power demand respectively while PG
and QG are the real power generation and the reactive power generation within the bus respectively, where PD and QD are the real power demand and the reactive power demand respectively while PG
and QG are the real power generation and the reactive power generation within the bus respectively, Pi = PGi −PDi Pi = PGi −PDi Qi = QGi −QDi, for i = 1, 2, 3, . . . , n If n represents the number of buses within the network, the flow of current through the bus i is: Ii =
Xn
k=1 YikVk, for i = 1, 2, 3, . . . , n;
(A2) (A2) where Vk is the voltage at bus k, Yik is the mutual admittance—the admittance between the ith and the
kth nodes; is the negative of the total admittances existing between the ith and kth nodes, whereas, where Vk is the voltage at bus k, Yik is the mutual admittance—the admittance between the ith and the
kth nodes; is the negative of the total admittances existing between the ith and kth nodes, whereas, Yik = Yki Similarly, the complex power flowing into a bus i is given as: Similarly, the complex power flowing into a bus i is given as: milarly, the complex power flowing into a bus i is given as: (A3) Si = Pi + jQi = ViI∗
i ; for i = 1, 2, 3, . . . , n
(A3) with I∗
i representing a complex conjugate of the current flow within the ith bus, and Vi representing the
bus voltage, S∗
i = Pi −jQi = V∗
i Ii; for i = 1, 2, 3, . . . , n;
S∗
i = Pi −jQi = V∗
i
Pn
k=1 YikVk
; for i = 1, 2, 3, . . . , n
(A4) (A4) Now, if the real and the imaginary sections of Equation (A4) are correlated, Pi = Re
V∗
i
Xn
k=1 YikVk
; Qi = −Im
V∗
i
Xn
k=1 YikVk
; for i = 1, 2, 3, . installed for man
ends of the elect
4. Conclusions The profitability of the storage system deployed at
the distribution network is dependent on the aggregate storage cost, the integration policies at the
location, and the ability to deploy the device for stacked services. Through favourable integration
and environmentally cautious policies, energy storage could provide customer and ancillary services
within the electricity supply chain. through which the storage system could be deployed to contribute to the on-going effort of
maximizing the utilization of clean energy for sustainable development. The profitability of the
storage system deployed at the distribution network is dependent on the aggregate storage cost, the
Author Contributions: Conceptualization, O.O.A., P.K. and N.J.H.; Methodology, O.O.A.; software, O.O.A.;
writing—original draft preparation and editing, O.O.A.; writing—review, P.K., M.B.M. and N.J.H.; supervision,
N.J.H. and P.K.; funding acquisition, N.J.H. All authors have read and agreed to the published version of
the manuscript. Funding: This research was funded by the Science Foundation Ireland (SFI) and the Department for the Economy
(DfE) in Northern Ireland, grant number 92160R. Acknowledgments: James Waide of the Physical Resources Department at Ulster University provided support
while collecting data. Paul Bell of the Utility Regulator Northern Ireland supported in information gathering. The System Operator for Northern Ireland (SONI) and the Northern Ireland Electricity (NIE) Networks provided
support in data collection. Energies 2020, 13, 2688 14 of 17 Energies 2020, 13, 2688 Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results. Appendix A . . , n
(A5) (A5) In polar form, Vi = Viδi; V∗
i = Vi −δi and Yik = Yikθik; while θ represents the phase angle between
current and voltage, δ represents the load angle. In polar form, Vi = Viδi; V∗
i = Vi −δi and Yik = Yikθik; while θ represents the phase angle between
current and voltage, δ represents the load angle. Substituting the polar expressions for V∗
i , Yik, and Vk in Equation (A5); the real power and the
reactive power components of the static load flow equation are respectively, Pi = Vi
Xn
k=1 VkYik cos(θik + δk −δi)
Qi = −Vi
Xn
k=1 VkYik sin(θik + δk −δi) 1.
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Devon, UK, 2019. © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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(CC BY) license (http://creativecommons.org/licenses/by/4.0/). © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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Strain-specific quantification of root colonization by plant growth promoting rhizobacteria Bacillus firmus I-1582 and Bacillus amyloliquefaciens QST713 in non-sterile soil and field conditions
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RESEARCH ARTICLE OPEN ACCESS Citation: Mendis HC, Thomas VP, Schwientek P,
Salamzade R, Chien J-T, Waidyarathne P, et al. (2018) Strain-specific quantification of root
colonization by plant growth promoting
rhizobacteria Bacillus firmus I-1582 and Bacillus
amyloliquefaciens QST713 in non-sterile soil and
field conditions. PLoS ONE 13(2): e0193119. https://doi.org/10.1371/journal.pone.0193119 Strain-specific quantification of root
colonization by plant growth promoting
rhizobacteria Bacillus firmus I-1582 and
Bacillus amyloliquefaciens QST713 in non-
sterile soil and field conditions Hajeewaka C. Mendis1, Varghese P. Thomas2, Patrick Schwientek2, Rauf Salamzade2¤a,
Jung-Ting Chien2, Pramuditha Waidyarathne3¤b, Joseph Kloepper1, Leonardo De La
Fuente1* a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 1 Department of Entomology and Plant Pathology, Auburn University, Auburn, AL, United States of America,
2 Crop Science Division, Biologics, Bayer, West Sacramento, CA, United States of America, 3 Centre for
Advanced Computational Solutions, Lincoln University, Christchurch, New Zealand ¤a Current address: Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States of
America ¤a Current address: Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States of
America
¤b Current address: Coconut Research Institute of Sri Lanka, Lunuwila, Sri Lanka
* lzd0005@auburn.edu ¤b Current address: Coconut Research Institute of Sri Lanka, Lunuwila, Sri Lanka
* lzd0005@auburn edu Abstract Bacillus amyloliquefaciens QST713 and B. firmus I-1582 are bacterial strains which are
used as active ingredients of commercially-available soil application and seed treatment
products Serenade® and VOTiVO®, respectively. These bacteria colonize plant roots pro-
moting plant growth and offering protection against pathogens/pests. The objective of this
study was to develop a qPCR protocol to quantitate the dynamics of root colonization by
these two strains under field conditions. Primers and TaqMan® probes were designed
based on genome comparisons of the two strains with publicly-available and unpublished
bacterial genomes of the same species. An optimized qPCR protocol was developed to
quantify bacterial colonization of corn roots after seed treatment. Treated corn seeds were
planted in non-sterile soil in the greenhouse and grown for 28 days. Specific detection of
bacteria was quantified weekly, and showed stable colonization between ~104–105 CFU/g
during the experimental period for both bacteria, and the protocol detected as low as 103
CFU/g bacteria on roots. In a separate experiment, streptomycin-resistant QST713 and
rifampicin-resistant I-1582 strains were used to compare dilution-plating on TSA with the
newly developed qPCR method. Results also indicated that the presence of natural micro-
flora and another inoculated strain does not affect root colonization of either one of these
strains. The same qPCR protocol was used to quantitate root colonization by QST713 and I-
1582 in two corn and two soybean varieties grown in the field. Both bacteria were quanti-
tated up to two weeks after seeds were planted in the field and there were no significant dif-
ferences in root colonization in either bacteria strain among varieties. Results presented
here confirm that the developed qPCR protocol can be successfully used to understand Editor: Sudisha Jogaiah, Karnatak University,
INDIA Introduction Plant growth-promoting rhizobacteria (PGPR) are a group of bacteria that colonize plant
roots and provide beneficial effects on plant growth and development [1]. Many diverse bacte-
rial genera such as Alcaligenes, Arthrobacter, Azoarcus, Azospirillum, Azotobacter, Bacillus, Bur-
kholderia, Clostridium, Enterobacter, Gluconacetobacter, Klebsiella, Pseudomonas, and Serratia
include specific strains which are reported as PGPR [2–5]. PGPR have direct or indirect effects
on plant growth promotion and improved crop yield. Direct effects of PGPR include providing
plants with fixed nitrogen and phytohormones, increasing the availability of nitrogen, soluble
phosphate and minerals in the soil and control or inhibition of the activity of plant pathogens
[6–9]. Some PGPR are also responsible for promoting plant growth indirectly by eliciting
Induced Systemic Resistance (ISR) [10–13]. Due to an increased public concern on the use of chemicals in agriculture [5], there has
been a growing demand for PGPR as an alternative to synthetic fertilizer and chemical pesti-
cides [14–16]. Use of PGPR in agriculture provides many benefits. PGPR have been used as a
component of an integrated management system to reduce or to replace the use of chemical
fertilizer and pesticides [3]. Members of the genus Bacillus produce heat and desiccation-resis-
tant spores and are more amenable to commercial formulation. Therefore, most of the com-
mercially available PGPR products contain Bacillus strains [10]. Many of these Bacillus spp. are
developed as biocontrol agents of plant pests [5]. Bacillus species such as B. amyloliquefaciens,
B. licheniformis, B. pumilus and B. subtilis are available in the market as biofungicide formula-
tions [17]. Bacillus amyloliquefaciens QST713 (synonymous with Bacillus subtilis QST713) and B. fir-
mus I-1582 are two key bacterial strains which are active ingredients in commercially-available
biological products Serenade1 and VOTiVO1, respectively These bacteria colonize plant
roots, promoting plant growth and offering protection against pathogens. VOTiVO1 is com-
mercially available as a seed treatment to provide protection against nematode infection [18]. In addition, B. firmus I-1582 is also the active ingredient in Nortica1 in turf grasses. B. firmus
strains reduce populations of Meloidogyne incognita in tomato roots and Radopholus similis,
Ditylenchus dipsaci and Heterodera glycines in vitro [19,20]. Serenade1 is recommended as a
soil treatment for managing soilborne and seedling diseases. B. amyloliquefaciens strains have
shown antifungal activity against Fusarium oxysporum [21], Puccinia striiformis [22], Rhizocto-
nia solani [23], and Pythium aphanidermatum [24]. Strain specific qPCR to quantify Bacillus firmus I-1582 and Bacillus amyloliquefaciens QST713 dynamics of root colonization by these bacteria in plants growing in growth chamber, green-
house and the field. dynamics of root colonization by these bacteria in plants growing in growth chamber, green-
house and the field. for supplies for authors VPT, PS, RS, JTC, HM, JK
and LD, but did not have any additional role in the
study design, data collection and analysis, decision
to publish, or preparation of the manuscript. The
specific roles of these authors are articulated in the
‘author contributions’ section. Competing interests: Authors JK and LD received
funds in the form of a research grant from Bayer
Cropsciences. The company did not interfere in the
research design nor the data interpretation. The
only restriction imposed by the company was on
the sharing of whole genome data for some
bacterial strains used for their product
development. Those genomes were used for
comparison to related genomes publicly available,
to design the specific primers characterized here. The sequences corresponding to the target region
of the primers for both strains as well as the
surrounding area, were deposited in a public
database. This does not alter our adherence to
PLOS ONE policies on sharing data and materials. Editor: Sudisha Jogaiah, Karnatak University,
INDIA Editor: Sudisha Jogaiah, Karnatak University,
INDIA Received: December 18, 2017
Accepted: February 5, 2018
Published: February 15, 2018 Copyright: © 2018 Mendis 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. Data Availability Statement: The GenBank
accession numbers for the target DNA sequences
generated here and used for strain-specific qPCR
are MG581691 for strain B. firmus I-1582 and
MG581692 for strain B. amyloliquefaciens
QST713. Funding: JK and LDLF received funding from Bayer
CropScience LP for this project. Bayer CropScience
provided support in the form of salaries and funds 1 / 19 PLOS ONE | https://doi.org/10.1371/journal.pone.0193119
February 15, 2018 PLOS ONE | https://doi.org/10.1371/journal.pone.0193119
February 15, 2018 Bacterial strains and growth conditions Bacillus amyloliquefaciens QST713 was previously described as Bacillus subtilis QST713. At the
time of filing U.S. Patent Application No. 09/074,870 in 1998, the strain was designated as B. subtilis based on classical, physiological, biochemical and morphological methods. Taxonomy
of the Bacillus species has evolved since then, especially in light of advances in genetics and
sequencing technologies, such that species designation is based largely on DNA sequence
rather than the methods used in 1998. Bayer is currently in the process of revising the B. subti-
lis designation of QST713, changing it to B. amyloliquefaciens, as would be expected currently
based solely on genome sequence comparison and inferred taxonomy. For concordance with
current taxonomic classification, the bacterial isolate formerly described as B. subtilis QST713
will be referred here as B. amyloliquefaciens QST713. B. firmus I-1582 and B. amyloliquefaciens
QST713 were grown at 28˚C with shaking at 150 rpm in Tryptic soy broth (TSB, pH 8.0) (BD
Diagnostic Systems, Sparks, MD) from frozen glycerol stocks. Streptomycin (Sm)-resistant B. amyloliquefaciens QST713 (B. amyloliquefaciens QST713 –Sm) and rifampicin-resistant (Rif)
B. firmus I-1582 (B. firmus I-1582-Rif) strains were grown in TSB or Tryptic soy agar (TSA)
plates with streptomycin at 100 μg/ml (TSASm) or rifampicin at 100 μg/ml (TSARif). Spores of
all Bacillus strains used in this study were produced by streaking 100 μL of two-day-old liquid
cultures in sporulation media plates containing 3.3 g of Proteose peptone No. 3 (Becton, Dick-
inson and Company, Sparks, MD), 1.0 g of Beef extract powder (Neogen Corporation, Lan-
sing, MI), 5.0 g of NaCl, 2.0 g of K2PO4, 1.0 g of KCl, 0.25 g of MgSO4, 0.01 g of MnSO4, 5.0 g
of Lactose, 18.0 g of Agar (Becton, Dickinson and Company, Sparks, MD) in 1 L of MiliQ
water. The sporulation plates were incubated for 2 weeks at 28˚C. Strain specific qPCR to quantify Bacillus firmus I-1582 and Bacillus amyloliquefaciens QST713 more convenient quantification method should be able to differentiate the inoculated strain
from indigenous rhizosphere bacterial community. Since standard microscopic methods and
dilution-plate counting-based methods heavily rely on morphological characteristics of the
bacteria, these methods are of limited value in quantification of PGPR root colonization where
morphology is indistinguishable from other common microflora [1,27,28]. However, PGPR
strains carrying antibiotic resistance determinants can also be used in dilution-plate method in
the presence of native microflora. The use of PGPR strains genetically engineered to express
fluorescent proteins is helpful in studying root colonization by PGPR under sterile conditions
[29,30]. However, this method is of limited use under non-sterile conditions in greenhouse or
in field, and for the use on wild-type strains. The objective of this study was to develop a strain-specific molecular technique based on
qPCR to detect and quantify root colonization of two PGPR strains B. amyloliquefaciens
QST713 and B. firmus I-1582. We were able to quantifiy root colonization of B. amyloliquefa-
ciens QST713 and B. firmus I-1582 strains in sterile soil, non-sterile soil in the greenhouse and
non-sterile soil in the field. Introduction The term “root colonization” of PGPR describes the active growth of introduced bacteria
on or around roots in the presence of indigenous microflora [1]. Research on PGPR as biocon-
trol agents shows that root colonization is a prerequisite for PGPR activity [25,26]. Therefore,
quantification of root colonization by PGPR is an essential part of discovering and evaluating
promising PGPR strains. Dilution-plate counting is a common method used in quantification
of root colonizing bacteria. This method can be used successfully in gnotobiotic systems under
sterile conditions [27]. However, dilution-plate counting could underestimate the number of
bacteria associated with roots due to some of the cells failing to grow into colonies under the
plating conditions [28]. The use of dilution-plating without a selective media is impracticable
in the presence of native microflora. Furthermore, dilution-plating could be laborious and
sample processing has to be carried out immediately after sampling. Therefore, a faster and PLOS ONE | https://doi.org/10.1371/journal.pone.0193119
February 15, 2018 2 / 19 Strain specific qPCR to quantify Bacillus firmus I-1582 and Bacillus amyloliquefaciens QST713 the bottom. The bag is then shaken to distribute spore suspension onto corn seeds. This step
was repeated until there was no visible liquid droplets in the bag. Bags were kept open in the
laminar flow hood to air dry (Breeanne Schillianskey, personal communication). Growth chamber. Corn seeds (2H723 Mycogen, Dow AgroSciences LLC, Indianapolis,
IN) were treated with 107, 105 and 103 spores of B. firmus I-1582; or 106, 104 and 102 spores of
B. amyloliquefaciens QST713 strains. Seeds were placed on sterile sand in 50 ml centrifuge
tubes covered with aluminum foil and 1 ml of sterile water with corresponding amount of
spores were drenched onto the seeds. Seeds were then covered with sterile sand and placed in
growth chamber for 3 weeks. Two corn seedlings from each treatment were removed at 14 and
21 days after planting. Seedlings were watered once in every three days and temperature and
relative humidity was maintained at 25o C and 75% in the growth chamber. Greenhouse. Corn seeds (2H723 Mycogen, Dow AgroSciences LLC, Indianapolis, IN)
were treated with 107 spores of B. firmus I-1582 and B. amyloliquefaciens QST713 strains sepa-
rately, and 107 spores of each strain together. Corn seeds were also treated with B. firmus I-
1582-Rif and B. amyloliquefaciens QST713-Sm as described above. Seeds were then planted in
sandy loam soil (pH = 7.4) in conetainers and grown in the greenhouse for 4 weeks. Three
corn seedlings from each treatment were removed from the conetainer at 3, 7, 14, 21 and 28
days after planting. Greenhouse. Corn seeds (2H723 Mycogen, Dow AgroSciences LLC, Indianapolis, IN)
were treated with 107 spores of B. firmus I-1582 and B. amyloliquefaciens QST713 strains sepa-
rately, and 107 spores of each strain together. Corn seeds were also treated with B. firmus I-
1582-Rif and B. amyloliquefaciens QST713-Sm as described above. Seeds were then planted in
sandy loam soil (pH = 7.4) in conetainers and grown in the greenhouse for 4 weeks. Three
corn seedlings from each treatment were removed from the conetainer at 3, 7, 14, 21 and 28
days after planting. Field. Seeds from two commercial maize and soybean hybrids with herbicide tolerance
traits were treated with 106 spores of either B. firmus I-1582 or B. amyloliquefaciens QST713 and
grown in research fields of Crop Science Division, Bayer in Tifton, GA and Champaign, IL. Soy-
bean and corn seedlings were carefully uprooted at 2 weeks after planting and shipped under
cold storage to the De La Fuente lab at Auburn University. Root samples were processed and
stored at -80o C in 50 ml centrifuge tubes until beadbeating and DNA extraction (see below). DNA extraction from plant roots and in vitro cultures Seedlings were removed from conetainers or conical tubes and shaken gently to remove
loosely-attached soil from the roots. Approximately 5 g of roots were cut into 2–3 cm-long
pieces and collected in a 7 ml plastic vial (BioSpec Products Inc, Bartlesville, OK) with 1.5–2 g
of 2 mm Zirconia beads (BioSpec Products Inc, Bartlesville, OK). An aliquot of 2 ml of sterile
water was added to each vial containing roots and Zirconia beads, secured in a Mini-BeadBea-
ter-96 (BioSpec Products Inc, Bartlesville, OK), and shaken at 2,400 oscillations/min for 5 min-
utes. Beadbeated vials were then briefly centrifuged at 1,500 rpm for 1 second in an Eppendorf
5810R centrifuge (Eppendorf-Netheler-Hinz GmbH, Barkhausenweg, Germany). An aliquot
of 200 μl of the supernatant was transferred to a ZR BashingBead Lysis Tube from ZR Soil
Microbe DNA miniprep kit (Zymo Research Corporation, Irvine, CA) and bacterial DNA was
extracted following manufacturer’s instructions. Quick-DNA™Fungal/Bacterial Miniprep Kit
(Zymo Research Corporation, Irvine, CA) was used to extract DNA from batch cultures. Plant inoculation with bacterial treatments Seed treatment. A spore suspension of B. amyloliquefaciens QST713 or B. firmus I-1582
was obtained by adding sterile MiliQ water into several sporulation plates made as described
above. The number of spores in the spore suspension was determined after heating the spore
suspension at 80˚C for 20 minutes in a water bath followed by dilution-plating. The spore sus-
pension was then centrifuged at 13,000 rpm for 10 minutes to prepare a spore suspension with
2.5x108 spores in 125 μl. This spore suspension and 25 corn seeds were added to a ziplock bag. A bubble was formed at the bottom of the ziplock bag by twisting the top of the bag towards PLOS ONE | https://doi.org/10.1371/journal.pone.0193119
February 15, 2018 3 / 19 Bioinformatics approach for finding unique region for Bacillus firmus I-
1582 To determine a fragment of the strain’s genome, which is unique relative to other strains in the
B. firmus species, an in-house computational protocol was developed. This protocol essentially
used NCBI’s BLAST tool (version 2.2.28+) to align whole genomes from alternate strains in B. firmus to the genome of the strain B. firmus I-1582. Genomic regions in the reference B. firmus
I-1582 were then identified by fitting a profile of having at most 60% of the bases mapped to
by any given alternate strain used in the analysis and meeting a minimum length requirement
of 5,000 bp. 4 / 19 PLOS ONE | https://doi.org/10.1371/journal.pone.0193119
February 15, 2018 Strain specific qPCR to quantify Bacillus firmus I-1582 and Bacillus amyloliquefaciens QST713 qPCR assays qPCR primers and TaqMan1 probes were designed using PrimerQuest software (Integrated
DNA Technologies, Inc) available online (Table 1). qPCR was performed in a final volume of
20 μl in the CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories Inc, Hercules,
CA). Three μl of extracted DNA, as described above, was amplified with forward and reverse
primers, Taqman1 probe (Eurofins MWG Operon LLC, Chicago, IL) and PerfeCTa Multiplex
qPCR ToughMix Low ROX (Quantabio, Beverly, MA). Final concentrations of primers and
probe were 250 nM and 150 nM respectively for both B. firmus I-1582 and B. amyloliquefaciens
QST713. PCR amplification conditions: 95o C for 15 min; 39 cycles of 95o C for 15 s and 58 o C
for 1 min. Bioinformatics approach for finding unique region for Bacillus
amyloliquefaciensQST713 To determine a unique region in the genome of the strain B. amyloliquefaciens QST713 which
is not observed in most other B. amyloliquefaciens genomes available, a slightly different algo-
rithm was devised. The reason for doing so was that the method used for finding a unique
region in B. firmus I-1582 did not work at identifying such a region in B. amyloliquefaciens
QST713. The new method devised here targeted to find the “most” unique region in B. amylo-
liquefaciens QST713 relative to other B. amyloliquefaciens. Similar to the other approach a
binary matrix was first constructed where the rows consisted of every position in the reference
genome and the columns were alternate B. amyloliquefaciens strains in our strain collection. In
such a matrix, a value of 1 was used to indicate that a certain position in B. amyloliquefaciens
QST713’s genome (row) was mapped upon by a given alternate strain (column) by a local
alignment which had an expected value of 1e-12. A value of 0 was used to show that the posi-
tion was not mapped to that alternate strain. Afterwards, the reference genome was scanned
with a sliding window of size 250 bp, to increase sensitivity, and the windows with the least
number of alternate B. amyloliquefaciens mapping to them were logged. These windows over-
lapped and a single 351 bp region was identified as being the “most” unique for the strain B. amyloliquefaciens QST713. Further searching NCBI’s nt database for the region, revealed that
only 1 of 50 publicly available genomes in the B. amyloliquefaciens species, regarding strain
G341, seemed to possess this region. Additionally, the region was also found in strain D2-2,
which is one out of 68 B. velezensis publically available genomes,. Quantification of bacteria Numbers of B. firmus I-1582 or B. amyloliquefaciens QST713 bacteria associated with corn
roots was quantified using standard curves constructed from spore suspensions of B. firmus I-
1582 or B. amyloliquefaciens QST713 spiked with corn roots and root-associated soil, as
described above. Serial dilutions of spores (101 to 109 cfu/ml spores) of B. firmus I-1582 or B. Table 1. Primers and probes used in this study specific to B. firmusI-1582 and B. amyloliquefaciensQST713. Primer/Probe name
Sequence (5’->3’)
Votivo_2F (forward)
CTCCAATTCCTAATATCCTGCAAAG
Votivo_2R (reverse)
GGAAAGTCACGGGACAGTTAT
Votivo_2P (probe)
TCAGGATGAGTATGCCATTCACCCA
Serenade_1F (forward)
GACGTATGGATACACCTCTTTAAT
Serenade_1R (reverse)
CCAAATTCCTCAGAAGAGAGAG
Serenade _1P (probe)
TTCCCATTAATATACTCAATTAGAGAACCT
https://doi.org/10.1371/journal.pone.0193119.t001
PLOS ONE | https://doi.org/10.1371/journal.pone.0193119
February 15, 2018
5 / 19 Table 1. Primers and probes used in this study specific to B. firmusI-1582 and B. amyloliquefaciensQST713. Primer/Probe name
Sequence (5’->3’)
Votivo_2F (forward)
CTCCAATTCCTAATATCCTGCAAAG
Votivo_2R (reverse)
GGAAAGTCACGGGACAGTTAT
Votivo_2P (probe)
TCAGGATGAGTATGCCATTCACCCA
Serenade_1F (forward)
GACGTATGGATACACCTCTTTAAT
Serenade_1R (reverse)
CCAAATTCCTCAGAAGAGAGAG
Serenade _1P (probe)
TTCCCATTAATATACTCAATTAGAGAACCT
https://doi org/10 1371/journal pone 0193119 t001 mers and probes used in this study specific to B. firmusI-1582 and B. amyloliquefaciensQST713. PLOS ONE | https://doi.org/10.1371/journal.pone.0193119
February 15, 2018 5 / 19 Strain specific qPCR to quantify Bacillus firmus I-1582 and Bacillus amyloliquefaciens QST713 amyloliquefaciens QST713 were added to 5 g of corn roots with 2 mm Zirconia beads in a 7 ml
plastic vial. Bacterial DNA was extracted as described above. qPCR was performed as described
above and Cq values from qPCR data for each serial dilution was plotted against the log value
of the number of spores in the serial dilution to get the standard curve. % efficiency was calcu-
lated by the Bio-Rad CFX Manager 3.1™software (Bio-Rad Laboratories Inc, Hercules, CA)
from the amplification efficiency (E) using the formula, % Efficiency = (E—1) 100. Amplifi-
cation efficiency of the qPCR reaction was calculated using the formula E = 10 (-1/slope) -1 [31]. % efficiency of 100% means perfect doubling of products with every cycle. Statistical analysis Populations of bacteria treated with three initial inoculums in growth chambers over three
weeks were compared using logistic regression analysis as bacteria counts were not normally
distributed. Data analysis for greenhouse experiments and field experiments were performed
by repeated measures analysis of variance (ANOVA) and two-way ANOVA respectively using
SPSS software (SPSS Inc., 2016). Wild-type and antibiotic resistant strains were analyzed
together when comparing both qPCR and dilution-plating. Data was normalized using natural
log transformation prior to repeated measures analysis. Two-way ANOVA were conducted
with normalized bacteria strain (natural log transformed), variety of corn and soybean, and
their interactions as factors. Tukey’s HSD post hoc test was used to compare the means of sta-
tistically significant factors. Construction of standard curves for bacterial population quantification Two standard curves were constructed to quantify B. firmus I-1582 and B. amyloliquefaciens
QST713 cells associated with corn and soybean roots. A B. firmus I-1582 spore suspension of
6.6 x 108 cfu/ml was used to construct B. firmus I-1582 standard curve. Seven serial dilutions
ranging from 6.6 x 108 to 6.6 x 102 cfu/ml, covering 6 orders of magnitude was used in the
standard curve. The efficiency was 91.2% and the coefficient of correlation (R2) was 1.000 for
B. firmus I-1582 standard curve (Fig 1). Similarly, a B. amyloliquefaciens QST713 spore suspen-
sion of 4.9 x 109 cfu/ml was used to construct B. amyloliquefaciens QST713 standard curve. Eight serial dilutions ranging from 4.9 x 109 to 4.9 x 102 cfu/ml, covering 7 orders of magni-
tude was used in the standard curve. The efficiency was 93.0% and the coefficient of correla-
tion (R2) was 0.995 for B. amyloliquefaciens QST713 standard curve (Fig 1). Nucleotide sequence accession numbers The GenBank accession numbers for the target DNA sequences generated here and used for
strain-specific qPCR are MG581691 for strain B. firmus I-1582 and MG581692 for strain B. amyloliquefaciens QST713. Quantification of specific bacterial strains in corn roots in growth chamber
assays As shown in Fig 2, bacterial populations were detected in most cases in corn roots for both B. firmus I-1582 and B. amyloliquefaciens QST713. Populations at time = 0 were calculated based
on initial inoculum applied to seeds. Both strains showed continuous root colonization up to 3
weeks after planting for all treatments. No significant differences (P = 0.318) were observed for
populations of bacteria over time considering each initial inoculum and strain separately at day
14 and day 21. The exception was the treatment with 102 spores of B. amyloliquefaciens QST713 PLOS ONE | https://doi.org/10.1371/journal.pone.0193119
February 15, 2018 6 / 19 Strain specific qPCR to quantify Bacillus firmus I-1582 and Bacillus amyloliquefaciens QST713 Fig 1. qPCR standard curves of B. firmusI-1582 and B. amyloliquefaciensQST713. Standard curves were plotted
from Cq values obtained by qPCR of spiked spore suspensions of B. firmus I-1582 and B. amyloliquefaciens QST713. Spiked spore suspensions were prepared from adding 5 g of corn roots and associated soil into 2 ml of spore
suspensions with known CFU. https://doi org/10 1371/journal pone 0193119 g001 Fig 1. qPCR standard curves of B. firmusI-1582 and B. amyloliquefaciensQST713. Standard curves were plotted
from Cq values obtained by qPCR of spiked spore suspensions of B. firmus I-1582 and B. amyloliquefaciens QST713. Spiked spore suspensions were prepared from adding 5 g of corn roots and associated soil into 2 ml of spore
suspensions with known CFU. https://doi.org/10.1371/journal.pone.0193119.g001 (BA-102), that was not detected in corn seedlings at days 14 and 21 (Fig 2). This may be due to
the presence of lower number of bacteria on the roots below the detection limit of this assay. PLOS ONE | https://doi.org/10.1371/journal.pone.0193119
February 15, 2018 Quantification of wild-type and antibiotic-resistant strains in corn roots in
greenhouse assays As shown in Fig 3, B. amyloliquefaciens QST713 and B. firmus I-1582 wild-type and antibiotic
resistant strains associated with corn roots were detectable up to 4 weeks using the qPCR pro-
tocol. Presence of indigenous microflora in soil or inoculation with another PGPR strain did
not interfere with quantification. When populations of B. firmus I-1582 were quantified with
specific primers, there was no significant difference (P = 0.548) in the number of wild-type B. firmus I-1582 bacteria inoculated alone or 1:1 mixture with B. amyloliquefaciens QST713 at all
data points. We observed that B. firmus I-1582 bacteria inoculated alone had significantly
higher B. firmus I-1582 bacteria population compared to 1:1 mixture with B. amyloliquefaciens
QST713 at day 21 based on the 95% confidence intervals at that particular time point (Fig 3A). There was no significant difference (P = 0.548) in number of B. firmus I-1582-Rif bacteria
detected in corn seedlings inoculated with this strain alone or as a mixture (1:1) with B. amylo-
liquefaciens QST713-Sm at all data points (Fig 3B). There was no significant difference (P = 0.208) in number of wild-type B. amyloliquefaciens
QST713 bacteria detected in corn seedlings inoculated alone or as a 1:1 mixture with wild-type 7 / 19 PLOS ONE | https://doi.org/10.1371/journal.pone.0193119
February 15, 2018 Strain specific qPCR to quantify Bacillus firmus I-1582 and Bacillus amyloliquefaciens QST713 Fig 2. Quantification of root associated bacteria in corn seedlings grown under sterile conditions in growth
chambers. Corn seeds were treated with 107 (BF-107), 105 (BF-105), or 103 (BF-103) spores of B. firmus I-1582; and 106
(BA-106), 104 (BA-104), or 102 (BA-102) spores of B. amyloliquefaciens QST713 per seed. Root sampling was carried
out at 14 and 21 days after planting (n = 2). Number of cells at t = 0 days was estimated based on the initial seed
treatment. ND = non-detectable. https://doi org/10 1371/journal pone 0193119 g002 Fig 2. Quantification of root associated bacteria in corn seedlings grown under sterile conditions in growth
chambers. Corn seeds were treated with 107 (BF-107), 105 (BF-105), or 103 (BF-103) spores of B. firmus I-1582; and 106
(BA-106), 104 (BA-104), or 102 (BA-102) spores of B. amyloliquefaciens QST713 per seed. Root sampling was carried
out at 14 and 21 days after planting (n = 2). Number of cells at t = 0 days was estimated based on the initial seed
treatment. ND = non-detectable. https://doi.org/10.1371/journal.pone.0193119.g002 B. firmus I-1582 (Fig 3C). PLOS ONE | https://doi.org/10.1371/journal.pone.0193119
February 15, 2018 Quantification of wild-type and antibiotic-resistant strains in corn roots in
greenhouse assays Similar results were found when antibiotic-resistant strains were
used. There was no significant difference (P = 0.208) in number of B. amyloliquefaciens
QST713-Sm bacteria detected in corn seedlings inoculated alone or as a 1:1 mixture with B. fir-
mus I-1582-Rif (Fig 3D). Comparing our qPCR protocol with dilution-plating of wild-type strains was investigated,
but it was difficult to distinguish B. firmus I-1582 or B. amyloliquefaciens QST713 colonies
from other soil bacteria colonies in TSA plates (Fig 4). Therefore, we decided to use antibiotic-
resistant strains for comparison, since population dynamics of wild-type and antibiotic-resis-
tant derivatives were similar (Fig 3A vs. 3B, 3C vs. 3D). Numbers of bacteria associated with corn roots were estimated for B. firmus I-1582-Rif and
B. amyloliquefaciens QST713-Sm strains by qPCR and dilution-plating to compare the two
techniques (Fig 5A and 5B, respectively). There was no significant difference (P = 0.138) in the
number of B. firmus I-1582-Rif bacteria estimated from qPCR quantification and dilution-
plating at all the data points. We observed that qPCR quantification estimated a significantly
higher number of B. firmus I-1582 compared to dilution-plating at day 14, and day 21 in corn
seedlings treated with B. firmus I-1582-Rif alone and as a mixture (1:1) with B. amyloliquefa-
ciens QST713-Sm based on the 95% confidence intervals at that particular time points (T1 and
T2, Fig 5A). There were no significant difference (P = 1.852) in the numbers of B. amyloliquefaciens
QST713-Sm bacteria estimated from qPCR quantification and dilution-plating at all data PLOS ONE | https://doi.org/10.1371/journal.pone.0193119
February 15, 2018 8 / 19 Strain specific qPCR to quantify Bacillus firmus I-1582 and Bacillus amyloliquefaciens QST713 Fig 3. Quantification of root associated bacteria in corn seedlings grown in natural soil in the greenhouse. Corn
seeds were treated on each case with: (A) 107 spores of B. firmus I-1582 alone (T1), mixed with 107 spores of B. amyloliquefaciens QST713 (T2) or non-treated (T3); (B) 107 spores of B. firmus I-1582-Rif alone (T1), mixed with 107
spores of B. amyloliquefaciens QST713-Sm (T2), or non-treated (T3); (C) 107 spores of wild-type B. amyloliquefaciens
QST713 alone (T1), mixed with 107 spores of B. firmus I-1582 (T2), or non-treated (T3). (D) 107 spores of B. amyloliquefaciens QST713-Sm alone (T1), mixed with 107 spores of B. firmus I-1582-Rif (T2), or non-treated (T3). In
(A) and (B) B. Quantification of wild-type and antibiotic-resistant strains in corn roots in
greenhouse assays firmus I-1582-Rif alone (T1), mixed with 107
spores of B. amyloliquefaciens QST713-Sm (T2), or non-treated (T3); (C) 107 spores of wild-type B. amyloliquefaciens
QST713 alone (T1), mixed with 107 spores of B. firmus I-1582 (T2), or non-treated (T3). (D) 107 spores of B. amyloliquefaciens QST713-Sm alone (T1), mixed with 107 spores of B. firmus I-1582-Rif (T2), or non-treated (T3). In
(A) and (B) B. firmus I-1582 specific primers and probe were used for qPCR bacterial population determination; while
in (C) and (D) B. amyloliquefaciens QST713 specific primers and probe were used for qPCR (n = 3 seedlings/time
point and error bars show SE). Repeated measures analysis of variance was performed at each data point comparing T1
and T2, Tukey’s HSD post hoc test was used to compare the means of statistically significant factors. Symbol
indicates time points that do not overlap 95% confidence intervals between T1 and T2 treatments. https://doi.org/10.1371/journal.pone.0193119.g003 https://doi.org/10.1371/journal.pone.0193119.g003 points (day 3, day 7, day 14, day 21 and day 38) when corn seedlings were treated with B. amy-
loliquefaciens QST713-Sm alone (T1). Similarly, there were no significant differences (P =
0.852) in the numbers of B. amyloliquefaciens QST713-Sm bacteria estimated from qPCR
quantification and dilution-plating at all data points when bacteria was inoculated as part of a
mixture (1:1) of B. amyloliquefaciens QST713-Sm and B. firmus I-1582-Rif (T2). We observed
that dilution-plating estimated a significantly higher number of B. amyloliquefaciens QST713-
Sm compared to qPCR quantification at day 7 and day 21 in corn seedlings treated as a mix-
ture (1:1) of B. amyloliquefaciens QST713-Sm and B. firmus I-1582-Rif (T2) based on the 95%
confidence intervals at that particular time points (Fig 5B). Quantification of wild-type and antibiotic-resistant strains in corn roots in
greenhouse assays firmus I-1582 specific primers and probe were used for qPCR bacterial population determination; while
in (C) and (D) B. amyloliquefaciens QST713 specific primers and probe were used for qPCR (n = 3 seedlings/time
point and error bars show SE). Repeated measures analysis of variance was performed at each data point comparing T1
and T2, Tukey’s HSD post hoc test was used to compare the means of statistically significant factors. Symbol
indicates time points that do not overlap 95% confidence intervals between T1 and T2 treatments. https://doi.org/10.1371/journal.pone.0193119.g003 Fig 3. Quantification of root associated bacteria in corn seedlings grown in natural soil in the greenhouse. Corn
seeds were treated on each case with: (A) 107 spores of B. firmus I-1582 alone (T1), mixed with 107 spores of B. amyloliquefaciens QST713 (T2) or non-treated (T3); (B) 107 spores of B. firmus I-1582-Rif alone (T1), mixed with 107
spores of B. amyloliquefaciens QST713-Sm (T2), or non-treated (T3); (C) 107 spores of wild-type B. amyloliquefaciens
QST713 alone (T1), mixed with 107 spores of B. firmus I-1582 (T2), or non-treated (T3). (D) 107 spores of B. amyloliquefaciens QST713-Sm alone (T1), mixed with 107 spores of B. firmus I-1582-Rif (T2), or non-treated (T3). In
(A) and (B) B. firmus I-1582 specific primers and probe were used for qPCR bacterial population determination; while
in (C) and (D) B. amyloliquefaciens QST713 specific primers and probe were used for qPCR (n = 3 seedlings/time
point and error bars show SE). Repeated measures analysis of variance was performed at each data point comparing T1
and T2, Tukey’s HSD post hoc test was used to compare the means of statistically significant factors. Symbol
indicates time points that do not overlap 95% confidence intervals between T1 and T2 treatments. https://doi.org/10.1371/journal.pone.0193119.g003 Quantification of root associated bacteria in corn seedlings grown in natural soil in the greenhouse. Corn
were treated on each case with: (A) 107 spores of B. firmus I-1582 alone (T1), mixed with 107 spores of B. Fig 3. Quantification of root associated bacteria in corn seedlings grown in natural soil in the greenhouse. Corn
7
7 Fig 3. Quantification of root associated bacteria in corn seedlings grown in natural soil in the greenhouse. Corn
seeds were treated on each case with: (A) 107 spores of B. firmus I-1582 alone (T1), mixed with 107 spores of B. amyloliquefaciens QST713 (T2) or non-treated (T3); (B) 107 spores of B. Quantification of specific bacterial strains in corn and soybean in US field
trials of Crop Science Division, Bayer According to these results, both B. firmus I-
1582 and B. amyloliquefaciens QST713 do not show a significant variety dependent or plot
level differences in corn and soybean root colonization. Fig 6 and Fig 7 confirm that our
qPCR protocol can be successfully applied to quantitate root colonization by PGPR in multiple
crops grown in the field. In addition, B. firmus I-1582-treated corn and soybean seedlings
were analyzed with B. amyloliquefaciens QST713 specific primers; and B. amyloliquefaciens
QST713-treated corn and soybean seedlings with B. firmus I-1582 specific primers, to see if
there was any cross-reaction or contamination. According to qPCR results, there was no
cross-reaction or contamination in B. firmus I-1582 treated corn and soybean seedlings and B. amyloliquefaciens QST713 treated corn and soybean seedlings. Quantification of specific bacterial strains in corn and soybean in US field
trials of Crop Science Division, Bayer Root colonization of soybean and corn by wild-type B. amyloliquefaciens QST713 and B. firmus
I-1582 was quantified in the field. There was no significant difference (P = 0.387) in the 9 / 19 PLOS ONE | https://doi.org/10.1371/journal.pone.0193119
February 15, 2018 Strain specific qPCR to quantify Bacillus firmus I-1582 and Bacillus amyloliquefaciens QST713 Fig 4. Comparison of bacteria colony morphology on TSA plates. (A) B. firmus I-1582 colonies from pure culture
(B) B. amyloliquefaciens QST713 colonies from pure culture (C) Rhizosphere bacteria from untreated corn seedlings
(D) Rhizosphere bacteria from B. firmus I-1582 treated corn seedlings (E) Rhizosphere bacteria from B. amyloliquefaciens QST713 treated corn seedlings. https://doi org/10 1371/journal pone 0193119 g004 Fig 4. Comparison of bacteria colony morphology on TSA plates. (A) B. firmus I-1582 colonies from pure culture
(B) B. amyloliquefaciens QST713 colonies from pure culture (C) Rhizosphere bacteria from untreated corn seedlings
(D) Rhizosphere bacteria from B. firmus I-1582 treated corn seedlings (E) Rhizosphere bacteria from B. amyloliquefaciens QST713 treated corn seedlings. https://doi.org/10.1371/journal.pone.0193119.g004 https://doi.org/10.1371/journal.pone.0193119.g004 number of B. firmus I-1582 or B. amyloliquefaciens QST713 bacteria associated with two corn
varieties tested in field trials (Fig 6A and 6B). Similarly, there was no significant difference
(P = 0.665) in the number of B. firmus I-1582 or B. amyloliquefaciens QST713 bacteria associ-
ated with two soybean varieties (Fig 7A and 7B). According to these results, both B. firmus I-
1582 and B. amyloliquefaciens QST713 do not show a significant variety dependent or plot
level differences in corn and soybean root colonization. Fig 6 and Fig 7 confirm that our
qPCR protocol can be successfully applied to quantitate root colonization by PGPR in multiple
crops grown in the field. In addition, B. firmus I-1582-treated corn and soybean seedlings
were analyzed with B. amyloliquefaciens QST713 specific primers; and B. amyloliquefaciens
QST713-treated corn and soybean seedlings with B. firmus I-1582 specific primers, to see if
there was any cross-reaction or contamination. According to qPCR results, there was no
cross-reaction or contamination in B. firmus I-1582 treated corn and soybean seedlings and B. amyloliquefaciens QST713 treated corn and soybean seedlings. number of B. firmus I-1582 or B. amyloliquefaciens QST713 bacteria associated with two corn
varieties tested in field trials (Fig 6A and 6B). Similarly, there was no significant difference
(P = 0.665) in the number of B. firmus I-1582 or B. amyloliquefaciens QST713 bacteria associ-
ated with two soybean varieties (Fig 7A and 7B). PLOS ONE | https://doi.org/10.1371/journal.pone.0193119
February 15, 2018 PLOS ONE | https://doi.org/10.1371/journal.pone.0193119
February 15, 2018 Discussion Corn seeds were treated with 107 spores of each strain inoculated
alone (T1), 107 spores of each strain mixed together (T2) or non-treated (T3) as described in the methods. Values
represent means and error bars show standard error (n = 3 seedlings/time point). Repeated measures analysis of
variance was performed separately for T1 and T2 for each data point comparing qPCR and plating measurements. Symbol indicates time points that do not overlap 95% confidence intervals between qPCR and plating for both T1
and T2 treatments. Symbol + indicates time points that do not overlap 95% confidence intervals between qPCR
quantification and dilution-plating only for T2. https://doi.org/10.1371/journal.pone.0193119.g005 https://doi.org/10.1371/journal.pone.0193119.g005 field under non-sterile soil. One of the common drawbacks of PCR based methods is that
these methods are not applicable to non-sterile conditions in greenhouse or in field [27]. How-
ever, dilution-plate counting is laborious, and sample processing and plating has to be carried
out immediately after sampling. Therefore, a faster and more convenient quantification
method to quantitate root and rhizosphere colonization of PGPR is required. The method we
describe here to quantitate root colonization of PGPR can successfully replace dilution-plate
counting method and has the advantage of being strain-specific. This is the first comprehensive report describing a strain-specific qPCR based protocol to
quantitate PGPR strains associated with crops grown in non-sterile soil and under field condi-
tions. Previously described PCR based quantification protocols were not strain-specific or
required laborious processing such as root washing step to minimize interference from natural
microflora associated with roots or bacteria enrichment steps [32–35]. Root washing could
potentially lead to a loss of root-associated bacteria of interest together with native microflora. With strain-specific primers and probes, we were able to bypass the requirement of root wash-
ing step. The method of recovering inoculated bacteria from roots is an important part of any
quantification technique. Presumably, soil particles loosely attached to roots may contain sev-
eral log units higher of the inoculated bacteria than in the rhizosphere soil without roots [1]. Therefore, we used a gentle root shaking in our protocol to remove loosely attached soil parti-
cles to provide a more accurate estimate of root colonization. However, increasing soil mass
attached to roots does not significantly increase estimation of inoculated bacteria population
densities [36]. Discussion The aim of this study was to develop a qPCR-based protocol to quantitate total root coloniza-
tion by two PGPR strains, B. firmus I-1582 and B. amyloliquefaciens QST713 in corn seedlings
grown in natural soil. Quantification of root colonization by PGPR is an essential part of track-
ing inoculants in the field, allowing studies of persistence and understanding of environmental
fate of introduced microbes applied in the soil, as well as discovering and evaluating potential
PGPR strains. With more interest in preserving soil health and exploiting soil microbiome,
there are more microbe-based seed and soil products being made available to the growers,
therefore differentiating strains and products is fundamental for the evaluation of the environ-
mental impact of these inoculants. Despite several microscopy and PCR based quantification
methods described recently and in the past, dilution-plate counting is still the most common
method used in quantification of root colonization by PGPR bacteria in greenhouse and in PLOS ONE | https://doi.org/10.1371/journal.pone.0193119
February 15, 2018 10 / 19 Strain specific qPCR to quantify Bacillus firmus I-1582 and Bacillus amyloliquefaciens QST713 PLOS ONE | https://doi.org/10.1371/journal.pone.0193119
February 15, 2018 11 / 19 Strain specific qPCR to quantify Bacillus firmus I-1582 and Bacillus amyloliquefaciens QST713 Fig 5. Comparison of bacteria root colonization of greenhouse grown corn seedlings by qPCR and dilution-
plating. (A), B. firmus I-1582-Rif and (B), B. amyloliquefaciens QST713-Sm were quantified by qPCR and dilution-
plating on TSARif and TSASm plates, respectively. Corn seeds were treated with 107 spores of each strain inoculated
alone (T1), 107 spores of each strain mixed together (T2) or non-treated (T3) as described in the methods. Values
represent means and error bars show standard error (n = 3 seedlings/time point). Repeated measures analysis of
variance was performed separately for T1 and T2 for each data point comparing qPCR and plating measurements. Symbol indicates time points that do not overlap 95% confidence intervals between qPCR and plating for both T1
and T2 treatments. Symbol + indicates time points that do not overlap 95% confidence intervals between qPCR
quantification and dilution-plating only for T2. h
//d i
/10 1371/j
l
0193119 005 Fig 5. Comparison of bacteria root colonization of greenhouse grown corn seedlings by qPCR and dilution-
plating. (A), B. firmus I-1582-Rif and (B), B. amyloliquefaciens QST713-Sm were quantified by qPCR and dilution-
plating on TSARif and TSASm plates, respectively. PLOS ONE | https://doi.org/10.1371/journal.pone.0193119
February 15, 2018 Discussion Therefore, gentle shaking of roots would not lead to a significant loss of bacteria
of interest and result in lower qPCR detection limit. Couillerot and Bouffaud et al. described an Azospirillum lipoferum CRT1 strain-specific
qPCR based protocol [37]. Their protocol required laborious sample processing such as lyoph-
ilization and the detection limit was 4 x 104 cells per g, which is a higher detection limit com-
pared to protocol described here. Therefore, they were only able to quantitate A. lipoferum
CRT1 in maize rhizosphere up to 10 days in non-sterile soil. Pereira et al. have recently
described a Herbaspirillum seropedicae SmR1 strain-specific qPCR protocol to quantitate root
colonization of maize in sterile conditions. However they were only able to detect H. seropedi-
cae SmR1 in maize roots in non-sterile soil and did not perform any quantification of bacteria
[38]. Von Felten et al described a qPCR protocol to detect 3 Pseudomonas fluorescens strains in
maize rhizosphere. Their protocol requires a 24-hour lyophilization step before DNA extrac-
tion from maize roots and associated soil [39]. Mosimann et al also described a qPCR protocol
to detect 2 Pseudomonas strains [40]. They have used 3 different potting mixes instead of natu-
ral soil to plant maize seedlings. Potting soil could be much more homogenious and it could
contain far less native microflora compared to natural soil [39] However, both Von Felten et al
and Mosimann have not shown that their qPCR protocols could successfully analyze and
quantitate respective PGPR strains in natural soils in field conditions. Previous studies have compared strain-specific qPCR protocols with dilution-plating [37]. However, these studies were carried out in sterile soil. Competition and interference from PLOS ONE | https://doi.org/10.1371/journal.pone.0193119
February 15, 2018 12 / 19 Strain specific qPCR to quantify Bacillus firmus I-1582 and Bacillus amyloliquefaciens QST713 Fig 6. Root colonization of field grown corn seedlings by B. firmusI-1582 and B. amyloliquefaciensQST713. C
seeds were treated with (A) 106 spores of B. firmus I-1582 (T1 and T2) and (B) 106 spores of B. amyloliquefaciens
QST713 (T1 and T2). T3 = uninoculated. qPCR quantification was performed with B. firmus I-1582 or B. Fig 6. Root colonization of field grown corn seedlings by B. firmusI-1582 and B. amyloliquefaciensQST713. Corn
seeds were treated with (A) 106 spores of B. firmus I-1582 (T1 and T2) and (B) 106 spores of B. amyloliquefaciens
QST713 (T1 and T2). T3 = uninoculated. Discussion Furthermore, we did not observe
a significant difference (P = 0.852) in estimation of B. amyloliquefaciens QST713-Sm in qPCR
quantification compared to dilution-plating at all time root samplings when corn seedlings
were treated with B. amyloliquefaciens QST713-Sm alone. However, there were few data points
where qPCR quantification resulted in significantly higher estimation of B. firmus I-1582-Rif
compared to that of dilution-plating when considered 95% confidence intervals (Fig 5A). High
qPCR estimation of B. firmus I-1582-Rif could result from amplification of DNA from dead
cells, lack of germination of spores in media used, or the possibility of a state similar to viable
but non culturable. Although less frequent than high qPCR estimation compared to dilution-
plating, there were two incidences out of ten pairwise comparisons where dilution-plating
resulted in higher estimation of B. amyloliquefaciens QST713-Sm compared to that of qPCR
quantification, only in cases where this strain was co-inoculated with B. firmus I-1582-Rif (T2,
Fig 5B). A possible explanation could be that the reduction in population of B. amyloliquefaciens
QST713-Sm is only detectable by qPCR in soils, but when growing alone in plates due to antibi-
otic selection, without competing with B. firmus I-1582-Rif, that reduction in population is lost
These aspects will be studied in future research aimed to elucidate the basis for these
differences. We were able detect as low as 103 CFU/g using our qPCR protocol (Figs 1 and 2). Even
though we investigated this protocol extensively in corn, results obtained from field-grown
soybean show that this protocol could be applied to investigate root colonization of the same
bacteria strains in other crop species without any modifications (Fig 7). Use of this method is
advantageous for large field trials, as this protocol requires minimal processing prior to long-
term storage of roots at -80o C. Furthermore, we did not observe any cross-reaction of primer
pairs or cross-contamination of the two strains used in this study. Therefore, our protocol is
suitable to investigate interactions of two or more PGPR strains in a single crop. We obtained whole root systems during sample processing whenever possible as it would
give maximum amount of information about root colonization [1]. We had to obtain repre-
sentative segments of root system during latter stages of the greenhouse experiments since
root mass was higher than 5 g. We have expressed root colonization of B. firmus I-1582 and B. Discussion qPCR quantification was performed with B. firmus I-1582 or B. Fig 6. Root colonization of field grown corn seedlings by B. firmusI-1582 and B. amyloliquefaciensQST713. Corn
seeds were treated with (A) 106 spores of B. firmus I-1582 (T1 and T2) and (B) 106 spores of B. amyloliquefaciens
QST713 (T1 and T2). T3 = uninoculated. qPCR quantification was performed with B. firmus I-1582 or B. Fig 6. Root colonization of field grown corn seedlings by B. firmusI-1582 and B. amyloliquefaciensQST713. Corn
seeds were treated with (A) 106 spores of B. firmus I-1582 (T1 and T2) and (B) 106 spores of B. amyloliquefaciens
QST713 (T1 and T2). T3 = uninoculated. qPCR quantification was performed with B. firmus I-1582 or B. PLOS ONE | https://doi.org/10.1371/journal.pone.0193119
February 15, 2018 13 / 19 Strain specific qPCR to quantify Bacillus firmus I-1582 and Bacillus amyloliquefaciens QST713 amyloliquefaciens QST713 specific primers and probe using a standard curve. Error bars show Standard Error (n = 5
seedlings). Same letters on top of bars represent non-significant differences (P = 0.387) for each variety according to
two-way ANOVA. https://doi.org/10.1371/journal.pone.0193119.g006 amyloliquefaciens QST713 specific primers and probe using a standard curve. Error bars show Standard Error (n = 5
seedlings). Same letters on top of bars represent non-significant differences (P = 0.387) for each variety according to
two-way ANOVA. https://doi.org/10.1371/journal.pone.0193119.g006 https://doi.org/10.1371/journal.pone.0193119.g006 native microflora could affect root colonization pattern of PGPR as well as detection limits of
the qPCR protocol. Therefore, we compared our qPCR results with dilution-plating with B. fir-
mus I-1582-Rif and B. amyloliquefaciens QST713-Sm strains (Fig 5). Estimation of root coloni-
zation by dilution-plating could underestimate the actual value if some of the cells fail to
develop into colonies. We did not observe a significantly different (P = 0.138) estimation of root
colonization of B. firmus I-1582-Rif with dilution-plating compared to that of qPCR estimation
for three out of five root samplings days up to 28 days when corn seedlings were treated with B. firmus I-1582-Rif alone or as a mixture (1:1) with B. amyloliquefaciens QST713-Sm. There was
no significant difference (P = 0.852) in estimation of root colonization of B. amyloliquefaciens
QST713-Sm with dilution-plating compared to that of qPCR estimation for three out of five
root samplings up to 28 days when corn seedlings were treated with a mixture (1:1) compri-
singB. amyloliquefaciens QST713-Sm and B. firmus I-1582-Rif. PLOS ONE | https://doi.org/10.1371/journal.pone.0193119
February 15, 2018 PLOS ONE | https://doi.org/10.1371/journal.pone.0193119
February 15, 2018 Discussion 15 / 19 PLOS ONE | https://doi.org/10.1371/journal.pone.0193119
February 15, 2018 Strain specific qPCR to quantify Bacillus firmus I-1582 and Bacillus amyloliquefaciens QST713 We avoided excessive soil moisture during root sampling and selected approximately 5 g of
roots whenever possible to prevent underestimation of root-associated bacteria. According to our results, we were able to quantitate root colonization of B. amyloliquefa-
ciens QST713 and B. firmus I-1582 in corn seedlings grown in greenhouse and in field in non-
sterile soil. Using dilution-plating of B. amyloliquefaciens QST713-Sm and B. firmus I-1582-Rif
strains, we were able to show that our qPCR quantification was accurate. Furthermore, this
protocol can be successfully extended to quantification of other bacteria strains in other crops. Our qPCR protocol is also suitable to quantitate both total root colonization as well as internal
and external root colonization, provided the number of bacteria falls within detection limits of
our qPCR protocol. We have studied corn root colonization of B. firmus I-1582 and B. amyloliquefaciens
QST713 strains over 4 weeks in non-sterile soil. We observed a steady root colonization in
corn roots by both B. firmus I-1582 and B. amyloliquefaciens QST713 strains over 28 days. We
have also included mixed inoculations of B. firmus I-1582 and B. amyloliquefaciens QST713
together. Presence of B. firmus I-1582 or B. amyloliquefaciens QST713 in the rhizosphere did
not affect the final population size of either B. amyloliquefaciens QST713 or B. firmus I-1582 at
day 28. We have not investigated these two strains together in a single crop in field. Further
investigation of mixed inoculations of B. firmus I-1582 and B. amyloliquefaciens QST713 in
field would provide more information on each strains root colonization pattern regarding any
distinct niches in the corn rhizosphere or if there are any antagonistic effects between two
strains. Acknowledgments We thank Mr. Reid McClenny and Mr. Elliot Ordoyne (Auburn University, USA) for their
assistance in greenhouse experiments, Mr. John McInroy (Auburn University, USA) for help-
ful suggestions throughout this work and for providing rifampicin-resistant B. firmus I-1582
and sporulation protocol, Ms. Jennifer Parker (Auburn University, USA; currently at Colorado
State University, USA) for designing B. firmus I-1582 specific primers and Taqman1 probe,
Dr. Ping Huang for his assistance with greenhouse experiments, Dr. Kathy Lawrence, Dr. Ni
Xiang and Mr. Hongyu Chen (Auburn University, USA) for their assistance with growth
chamber experiments and Ms. Qing Ge for her assistance in DNA extractions from field
grown corn and soybean roots, Mr. Kevin Bugg, Mr. Nate Bestor and Mr. Scott Russell (Crop
Science Division, Bayer) for collecting and shipping corn and soybean roots from field trials. Discussion amyloliquefaciens QST713 strains in corn and soybean roots as number of cells per gram of
roots since we were interested in quantifying total root colonization. More soil particles could
be attached to roots if the soil is moist. That could result in underestimation of bacteria popu-
lations since the root surface has higher bacterial population densities than adjacent soil [1,41]. PLOS ONE | https://doi.org/10.1371/journal.pone.0193119
February 15, 2018 14 / 19 Strain specific qPCR to quantify Bacillus firmus I-1582 and Bacillus amyloliquefaciens QST713 Fig 7. Root colonization of field grown soybean seedlings B. firmusI-1582 and B. amyloliquefaciensQST713. Soybean seeds were treated with (A) 106 spores of B. firmus I-1582 (T1 and T2) and (B) 106 spores of B. amyloliquefaciens QST713 (T1 and T2). T3 = uninoculated. qPCR quantification was performed with B. firmus I-1582
or B. amyloliquefaciens QST713 specific primers and probes using a standard curve. Error bars show Standard Error. (n = 5 seedlings). Same letters on top of bars represent non-significant differences (P = 0.665) for each variety
according to two-way ANOVA. https://doi.org/10.1371/journal.pone.0193119.g007 Fig 7. Root colonization of field grown soybean seedlings B. firmusI-1582 and B. amyloliquefaciensQST713. Soybean seeds were treated with (A) 106 spores of B. firmus I-1582 (T1 and T2) and (B) 106 spores of B. amyloliquefaciens QST713 (T1 and T2). T3 = uninoculated. qPCR quantification was performed with B. firmus I-1582
or B. amyloliquefaciens QST713 specific primers and probes using a standard curve. Error bars show Standard Error. (n = 5 seedlings). Same letters on top of bars represent non-significant differences (P = 0.665) for each variety
according to two-way ANOVA. https://doi.org/10.1371/journal.pone.0193119.g007 Fig 7. Root colonization of field grown soybean seedlings B. firmusI-1582 and B. amyloliquefaciensQST713. Soybean seeds were treated with (A) 106 spores of B. firmus I-1582 (T1 and T2) and (B) 106 spores of B. amyloliquefaciens QST713 (T1 and T2). T3 = uninoculated. qPCR quantification was performed with B. firmus I-1582
or B. amyloliquefaciens QST713 specific primers and probes using a standard curve. Error bars show Standard Error. (n = 5 seedlings). Same letters on top of bars represent non-significant differences (P = 0.665) for each variety
according to two-way ANOVA. References 1. Kloepper JW, Beauchamp CJ (1992) A review of issues related to measuring colonization of plant roots
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Leonardo De La Fuente. Author Contributions Conceptualization: Leonardo De La Fuente. Data curation: Hajeewaka C. Mendis, Patrick Schwientek, Rauf Salamzade, Jung-Ting Chien,
Pramuditha Waidyarathne. Formal analysis: Hajeewaka C. Mendis, Pramuditha Waidyarathne, Leonardo De La Fuente Formal analysis: Hajeewaka C. Mendis, Pramuditha Waidyarathne, Leonardo De La Fuente. Funding acquisition: Varghese P. Thomas, Joseph Kloepper, Leonardo De La Fuente. Investigation: Hajeewaka C. Mendis, Leonardo De La Fuente. Methodology: Hajeewaka C. Mendis, Patrick Schwientek, Rauf Salamzade, Jung-Ting Chien,
Leonardo De La Fuente. Project administration: Varghese P. Thomas, Joseph Kloepper, Leonardo De La Fuente. PLOS ONE | https://doi.org/10.1371/journal.pone.0193119
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Pramuditha Waidyarathne, Joseph Kloepper, Leonardo De La Fuente. Resources: Hajeewaka C. Mendis, Varghese P. Thomas, Patrick Schwientek, Rauf Salamzade,
Pramuditha Waidyarathne, Joseph Kloepper, Leonardo De La Fuente. Software: Patrick Schwientek, Rauf Salamzade, Jung-Ting Chien, Pramuditha Waidyarathne. Supervision: Varghese P. Thomas, Joseph Kloepper, Leonardo De La Fuente. Validation: Hajeewaka C. Mendis, Patrick Schwientek, Rauf Salamzade, Leonardo De La
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https://openalex.org/W2346381726
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https://ora.ox.ac.uk/objects/uuid:4628e505-ecd6-4647-a502-e548dac20508/download_file?safe_filename=Genetic%2Band%2Benvironmental%2Brisk%2Bfactors%2Bfor%2Batherosclerosis%2Bregulate%2Btranscription%2Bof%2Bphosphatase%2Band%2Bactin%2Bregulating%2Bgene%2BPH.pdf&file_format=application%2Fpdf&type_of_work=Journal+article
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English
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Genetic and environmental risk factors for atherosclerosis regulate transcription of phosphatase and actin regulating gene PHACTR1
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Atherosclerosis
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cc-by
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Michael E. Reschen a, Da Lin a, Anil Chalisey a, Elizabeth J. Soilleux b,
Christopher A. O’Callaghan a, * Michael E. Reschen a, Da Lin a, Anil Chalisey a, Elizabeth J. Soilleux b,
Christopher A. O’Callaghan a, * a Wellcome Trust Centre for Human Genetics, Nuffield Department of Medicine, University of Oxford, Roosevelt Drive, Oxford, OX3 7BN, United Kingdom
b Nuffield Division of Clinical Laboratory Sciences, Radcliffe Department of Medicine, University of Oxford and Department of Cellular Pathology, John
Radcliffe Hospital, Oxford, OX3 9DU, United Kingdom journal homepage: www.elsevier.com/locate/atherosclerosis journal homepage: www.elsevier.com/locate/atherosclerosis a r t i c l e i n f o Background and aims: Coronary artery disease (CAD) risk is associated with non-coding genetic variants
at the phosphatase and actin regulating protein 1(PHACTR1) gene locus. The PHACTR1 gene encodes an
actin-binding protein with phosphatase regulating activity. The mechanism whereby PHACTR1 in-
fluences CAD risk is unknown. We hypothesized that PHACTR1 would be expressed in human cell types
relevant to CAD and regulated by atherogenic or genetic factors. Article history:
Received 20 October 2015
Received in revised form
20 March 2016
Accepted 26 April 2016
Available online 2 May 2016 g
y
g
g
Methods and results: Using immunohistochemistry, we demonstrate that PHACTR1 protein is expressed
strongly in human atherosclerotic plaque macrophages, lipid-laden foam cells, adventitial lymphocytes
and endothelial cells. Using a combination of genomic analysis and molecular techniques, we demon-
strate that PHACTR1 is expressed as multiple previously uncharacterized transcripts in macrophages,
foam cells, lymphocytes and endothelial cells. Immunoblotting confirmed a total absence of PHACTR1 in
vascular smooth muscle cells. Real-time quantitative PCR showed that PHACTR1 is regulated by
atherogenic and inflammatory stimuli. In aortic endothelial cells, oxLDL and TNF-alpha both upregulated
an intermediate length transcript. A short transcript expressed only in immune cells was upregulated in
macrophages by oxidized low-density lipoprotein, and oxidized phospholipids but suppressed by lipo-
polysaccharide or TNF-alpha. In primary human macrophages, we identified a novel expression quan-
titative trait locus (eQTL) specific for this short transcript, whereby the risk allele at CAD risk SNP
rs9349379 is associated with reduced PHACTR1 expression, similar to the effect of an inflammatory
stimulus. Keywords:
Myocardial infarction
Atherosclerosis
Functional genomics
Genetic polymorphism
Genomics
Low-density lipoprotein (LDL)
Genetic disease
PHACTR1
Expression quantitative trait locus (eQTL)
Oxidized low density lipoprotein (oxLDL) Conclusions: Our data demonstrate that PHACTR1 is a key atherosclerosis candidate gene since it is
regulated by atherogenic stimuli in macrophages and endothelial cells and we identify an effect of the
genetic risk variant on PHACTR1 expression in macrophages that is similar to that of an inflammatory
stimulus. © 2016 The Authors. Published by Elsevier Ireland Ltd. This is an open access article under the CC BY
license (http://creativecommons.org/licenses/by/4.0/). Atherosclerosis 250 (2016) 95e105 Atherosclerosis 250 (2016) 95e105 Contents lists available at ScienceDirect http://dx.doi.org/10.1016/j.atherosclerosis.2016.04.025
0021-9150/© 2016 The Authors. Published by Elsevier I Abbreviations: CAD, coronary artery disease; GWAS, genome wide association
studies; SNP, single nucleotide polymorphism; PHACTR1, phosphatase and actin
regulator 1; oxLDL, oxidized low density lipoprotein; PP1, protein phosphatase 1;
PBMC, peripheral blood mononuclear cells; TSS, transcription start site; RACE, rapid
amplification of cDNA ends; HAEC, human aortic endothelial cells; LPS, lipopoly-
saccharide; NLS, nuclear localization signal; DAB, diaminobenzidine.
* Corresponding author. * Corresponding author. 1. Introduction form of chronic inflammation. Deposition of pro-inflammatory li-
poproteins in arterial walls is associated with the development of
atherosclerotic plaques that can obstruct blood flow and trigger the
formation of occlusive thrombi [1,2]. CAD is a complex disease with
a strong heritable component [3]. Genome-wide association
studies (GWAS) have identified 58 genomic regions where single
nucleotide polymorphisms (SNPs) are associated with CAD risk
[4,5]. Most of these disease-associated variants are in non-coding
DNA and likely exert their effect predominantly by altering tran-
scription factor binding to regulatory DNA elements [4,6e8]. A
number of CAD risk loci contain genes that were not previously
implicated in coronary artery disease and for which no mechanism Coronary artery disease (CAD) is caused by atherosclerosis, a Abbreviations: CAD, coronary artery disease; GWAS, genome wide association
studies; SNP, single nucleotide polymorphism; PHACTR1, phosphatase and actin
regulator 1; oxLDL, oxidized low density lipoprotein; PP1, protein phosphatase 1;
PBMC, peripheral blood mononuclear cells; TSS, transcription start site; RACE, rapid
amplification of cDNA ends; HAEC, human aortic endothelial cells; LPS, lipopoly-
saccharide; NLS, nuclear localization signal; DAB, diaminobenzidine. * Corresponding author M.E. Reschen et al. / Atherosclerosis 250 (2016) 95e105 96 inflammatory stimuli and that its expression is influenced by CAD-
associated genetic variants. After establishing the expression of
PHACTR1 protein in atherosclerotic lesions and the profile of hu-
man PHACTR1 transcripts in primary cell types involved in
atherosclerosis, we determined the responses of these transcripts
to inflammatory stimuli and to atherogenic lipid. The role of
PHACTR1 in CAD was highlighted by its in vivo abundance in
macrophages and foam cells in human atherosclerotic plaque. The
CAD risk allele at SNP rs9349379 was associated with significantly
reduced expression of a short transcript in macrophages such that
the risk genotype mirrors the effect of an inflammatory stimulus. has been identified to account for the risk associated with the re-
ported genetic variant. Understanding the effect of these genetic
risk variants on the expression and function of the genes that they
regulate will advance our understanding of atherosclerosis and
could lead to new therapeutic targets. p
g
Several studies have independently replicated an association
between CAD and SNPs at a genetic locus containing the phos-
phatase and actin regulator 1 (PHACTR1) gene [9e12]. 2.2. Cell isolation, culture and reagents Primary human coronary artery vascular smooth muscle cells
(Invitrogen, Carlsbad, CA) were cultured in Medium 231 (Invi-
trogen) and Smooth Muscle Growth Supplement (Invitrogen). Pri-
mary human aortic endothelial cells (Invitrogen) were cultured in
Medium 200 (Invitrogen) with Low Serum Growth Supplement. CD14þ monocytes, T cells (CD3þ) and B cells (CD19þ) were isolated
from the peripheral blood of healthy human volunteers. Blood was
centrifuged over Ficoll-Paque PLUS (GE Healthcare LifeSciences,
Buckinghamshire, UK) to isolate peripheral blood mononuclear
cells (PBMCs). Subsets of cells were isolated using positive selection
with magnetic bead-conjugated antibodies (Miltenyi Biotec, Ber-
gisch Gladback, Germany). Individual populations were confirmed
to be >95% pure using flow cytometry for the markers used for
positive selection. The cells remaining after CD14þ cells were
magnetically separated from PBMCs were termed CD14þ-depleted
PBMCs and constitute a mixed predominantly lymphocytic popu-
lation. CD14þ monocytes were differentiated into macrophages by
7 days of culture in RPMI 1640 medium with 10% fetal calf serum,
4 mM L-glutamine, 50 units/ml penicillin and 50 mcg/ml strepto-
mycin (Sigma, St Louis, MO), supplemented with 50 ng/ml macro-
phage colony stimulating factor (M-CSF, eBioscience, San Diego,
CA). Foam cells were generated by treating macrophages with
human oxLDL (50 mcg/ml) for 48 h. Foam cell formation was
confirmed by oil red O staining for intracellular neutral lipid by
light microscopy. Macrophage and foam cell viability was shown to
be >98% using the Invitrogen LIVE/DEAD microscopy kit. The PHACTR family consists of 4 genes encoding proteins that
interact directly with both actin and protein phosphatase 1 (PP1)
[18]. PHACTR1 was originally cloned from a rat brain cDNA library
using a yeast two-hybrid system with PP1 as bait [18]. The human
PHACTR1 gene is on chromosome 6 and a transcript with a 1743 bp
open reading frame has been cloned from human brain [19]. The
resulting 580 amino acid protein has 4 highly conserved actin-
binding RPEL domains and both mouse and rat orthologues have
been shown to bind actin [18,20]. Human PHACTR1 protein con-
tains nuclear localization signal (NLS) motifs at both ends of the
protein that, in the mouse ortholog, have been shown to facilitate
importin-dependent nuclear translocation in response to serum
stimulation [20]. Binding of the RPEL domains to G-actin maintains
PHACTR1 in a cytoplasmic location [20]. Serum induces Rho-
dependent remodeling of actin from G- to F-actin and trans-
location of PHACTR1 into the nucleus [20]. 2.2. Cell isolation, culture and reagents PP1 is part of a family of serine/threonine phosphatases that are
present in both the nucleus and cytoplasm [21]. The enzymatic
specificity of PP1 enzymes is achieved in part by their association
with accessory proteins [22]. PHACTR1 may function as an acces-
sory protein since PHACTR1 binds to PP1 and inhibits its activity
in vitro [18]. PHACTR1 may also affect actin structure as siRNA-
mediated PHACTR1 knockdown in human umbilical vein endo-
thelial cells (HUVEC) reduces F-actin filament numbers and repar-
titioning as well as increasing cell protrusion dynamics [23]. Although the precise cellular role of PHACTR1 remains to be
determined, overexpression and siRNA-mediated knockdown ex-
periments suggest a role in cell motility and vascular morphogen-
esis [20,23,24]. Ultracentrifugation of freshly isolated human plasma using a
discontinuous potassium bromide gradient was used to purify LDL
with density 1.019e1.063 g/ml [28]. LDL was extensively dialyzed
against PBS, and incubated with 25 mM CuCl2 at 37 for 18 h to
produce oxidized-LDL. To terminate oxidation 1 mM EDTA was
added and oxLDL was stored at 4. The TBARS assay was used to
confirm that LDL was oxidized (Caymen Chemical, Ann Arbor,
Michigan). Careful precautions were taken to ensure low endotoxin
levels and LDL was tested for endotoxin using the gel clot method
after heating to 75 for 15 min to remove the plasma inhibitor
(Associates of Cape Cod, East Falmouth, MA). LDL was used at levels
of <0.1 EU/ml. Atherosclerosis involves the interplay of genetic factors with
atherogenic stimuli, such as modified low-density lipoprotein, in
cell types including macrophages, lymphocytes, endothelial cells
and vascular smooth muscle cells [1,25e27]. The role of PHACTR1 in
these cells and the mechanism whereby it alters CAD risk are un-
known. Understanding the role of PHACTR1 is complicated by the
fact that the human PHACTR1 gene is predicted to encode multiple
transcripts that have not been characterized. In this study, we hy-
pothesized
that
PHACTR1
is
regulated
by
atherogenic
or 1. Introduction SNPs at the
PHACTR1 locus are associated with the specific phenotypes of early
onset myocardial infarction, coronary artery calcification [11,12]
and with an intermediate phenotype of impaired central hemo-
dynamic indices, indicating abnormal vascular stiffness [13]. The
PHACTR1 locus is pleiotropic since the protective alleles of the CAD
risk SNPs are associated with an increased risk of ischemic stroke
caused by cervical artery dissection, a form of non-atherosclerotic
vascular disease [14]. The variants reported in these studies lie in
an intronic region of PHACTR1 over 250 kb away from any other
gene. A genetic fine mapping study suggested that rs9349379 was
the most likely causative variant at the locus and it was associated
with expression of PHACTR1 mRNA in composite right coronary
artery tissue [15]. An expression quantitative trait locus (eQTL)
study assaying gene expression in diverse human tissues showed
that rs9349379 also affected PHACTR1 mRNA expression in aortic
artery and tibial artery tissue [16]. In a whole genome epigenetic
and expression study, we recently demonstrated that PHACTR1 is
one of the most highly upregulated genes in macrophages exposed
to oxLDL [17]. 2. Materials and methods 2.1. Ethical approval 2.1. Ethical approval Ethical approval for the study was obtained from the NHS
Research Ethics Committee (South Central-Hampshire B, reference
13/SC/0392)
and
all
participants
provided
written
informed
consent. 2.2. Cell isolation, culture and reagents 2.4. RNA extraction and cDNA synthesis and 50 RACE RNA was extracted using the Trizol RNA Plus extraction kit with
on-column DNAse treatment according to the manufacturer’s in-
structions (Invitrogen). cDNA was produced by reverse transcrip-
tion of 1 mcg of RNA using Bioscript reverse transcriptase (Bioline,
London, UK) with random hexamers (Qiagen, Venlow, Limburg,
Netherlands). For 50 RACE, 5 mcg of total RNA was reverse tran-
scribed
using
20
pmol
of
gene
specific
primer
(CO5441
TTTGGCTGAAGGATCTTGGG) and then poly A tailed at the 30 cDNA
end. First round PCR was performed using an inner gene specific
primer (CO5439 CCATCCATGATGTCTGACGGTTGG) and oligo(dT)
primer (with additional 50 sequence for round two PCR, CO1352) for
18 cycles using Bioscript (Bioline). Second round PCR was per-
formed
using
gene
specific
primer
(CO5400
CTTCTCTGCTTTGCCTCATAGATATTT) and a primer (CO3984 CTAG-
GAATTCTAGAGGTACCTCGAG) annealing to a 50 prime site created in
the 1st round PCR. PCR products were gel extracted and ligated into
Strataclone vector pSC-A-amp/kan using the strataclone PCR
cloning kit (Agilent, Santa Clara, CA). Colonies were cultured,
plasmid DNA extracted using a miniprep kit and sequenced using
BigDye (Invitrogen) with both inner gene-specific primer (CO5513
CTGACGTGTGTTTGAACTTTTCGC)
and
also
(CO5400
CTTCTCTGCTTTGCCTCATAGATATTT). Sequences were aligned to the
hg19 reference genome using the BLAT program in the UCSC
genome browser. 2.5. PHACTR1 cloning The long PHACTR1 transcript was amplified from an Image clone
(IRATp970E05120D) using primers CO4400 ACTGATGATCAATG-
GATTATCCCAAAATGGATTATTTTC, and CO4807 TCAGTCTCGAGT-
TAAGGTCGGTGAAACCTTGTTAAGTG and cloned into pCDNA3.1
with an N-terminal myc tag to produce plasmid pOC1259. The in-
termediate transcript was amplified from CD14þ macrophage cDNA
using primers CO4976 ACTGATGATCAATGCGTTCTGACTCCCTCGTCC
and CO4807 and cloned into pCDNA3.1 with an N-terminal myc tag
to produce plasmid pOC1256. The short PHACTR1 transcript
(ENST00000379335) was amplified from macrophage cDNA using
CO4806 AATGAGGATCCATGTATCTGCAAGGGCCGAGG and CO4807
and cloned into pCDNA3.1 with an N-terminal Myc tag to make
plasmid pOC1257. Plasmids were sequenced using BigDye. 2.8. Western blotting Cells were lyzed in RIPA buffer with protease inhibitors and
subjected to SDS-PAGE analysis using 10 mcg of protein per lane
and PageRuler Plus pre-stained marker lane. Endogenous PHACTR1
was detected using a rabbit polyclonal anti-PHACTR1 antibody
(Novus Biologicals, Littleton, CO) and secondary HRP-conjugated
goat anti-rabbit IgG antibody (Vector labs, Burlingame, CA). Chemiluminescent reactions were performed using ECL Advance
(GE Healthcare LifeSciences) and imaged using either a BioRAD gel
doc imaging station (BioRad, Hercules, CA) or Amersham hyperfilm
ECL (GE Healthcare LifeSciences). 2.6. Tissue bank and primary cell expression analysis An oligo(dT)-primed cDNA library set made from adult and fetal
human tissue was screened for expression of PHACTR1 transcripts. PHACTR1 transcripts and beta-actin controls were amplified using
BioTaq polymerase with 33 cycles and 25 cycles respectively. Products were run on a 1% agarose gel and stained using ethidium
bromide. The primer pairs used were as follows: long transcript,
CO3948
TGCTCACAGACTCTTGGATGTTG
and
CO3880
CTTCTCTGCTTTGCCTCATAGATATTT;
intermediate
transcript,
CO3879 GGAAGAAGAAAAGCGAAAAGTTCA and CO3876 GGCAG-
TAGCAGGACTGGTTTG;
short
transcript,
CO4987
ATGTATCTG-
CAAGGGCCGAG and CO3944 TGAATTCATTGAGCTCCTTTCG; control
beta-actin,
CO1851
TCCAGCCTTCCTTCCTGGGCAT
and
CO1852
GTCAAGAAAGGGTGTAACGCAACT. 2.3. Genotyping of rs9349379 Individuals providing blood samples were genotyped by the M.E. Reschen et al. / Atherosclerosis 250 (2016) 95e105 97 2.7. Quantitative PCR and eQTL analysis 2.7. Quantitative PCR and eQTL analysis Oxford Biobank and additional primary cells were genotyped using
an inventoried Taqman genotyping assay according to the manu-
facturer’s instructions (Applied Biosystems, Foster City, CA, ID
4351379). Expression levels of PHACTR1 were determined by RT-qPCR with
SYBR green reagents (Invitrogen) using either cDNA from macrophages
or CD14þ-depleted PBMCs from genotyped healthy individuals from
the Oxford Biobank. RT-qPCR primers used were: CO5399 GGAAGAA-
GAAAAGCGAAAAGTTCA and CO5400 CTTCTCTGCTTTGCCTCATAGA-
TATTT (intermediate transcripts); CO4987 ATGTATCTGCAAGGGCCGAG
and
CO5122
CTCTTGATCTCTCTCTTCTCCTCCTG
(short
transcript);
CO3744
TTGCCATCAATGACCCCTTCA
and
CO3745
CGCCCCACTT-
GATTTTGGA (control GAPDH). Melt curve analysis demonstrated a
single product for each primer pair. For each sample, triplicates were
assayed. A Kruskal-Wallis test was applied across the three possible
genotypes to identify a significant genotype-expression relationship
with significance defined as a p value < 0.05. To assess PHACTR1
expression in response to stimuli, primary human macrophages
(rs9349379 AA) were treated with 50 mcg/ml oxLDL for 48 h,10 ng/ml
LPS (Sigma) for 24 h, 10 ng/ml TNFa for 24 h (eBioscience), 30 mcg/ml
OxPAPC (Invivogen, San Diego, CA) for 48 h, or 10 mg/ml cyclodextrin-
cholesterol (Sigma) for 48 h and assayed in biological triplicates. HAEC
(rs9349379 AG) were treated similarly with 50 mg/ml oxLDL for 48 h,
10 ng/ml TNFa or 10 mg/ml cyclodextrin-cholesterol for 48 h. Coronary
artery smooth muscle cells (rs9349379 AG) were treated with 10 mg/ml
cyclodextrin-cholesterol for 48 h and assayed in triplicate. Student’s t-
test was applied with significance defined as a p value < 0.05. 2.9. Immunohistochemistry Formalin-fixed
paraffin-embedded tissue
samples
of
non-
atherosclerotic aorta (n ¼ 5), and atherosclerotic aorta (n ¼ 5),
obtained with full ethical approval from the National Research and
Ethics Service (Oxfordshire Research and Ethics Committee A:
reference 04/Q1604/21), were immunostained for PHACTR1 with a
rabbit polyclonal anti-PHACTR1 antibody (Abcam, Cambridge, UK)
detected with the NovolinkTM max polymer detection system
(Leica Microsystems, Buffalo Grove, IL), as per the manufacturer’s
instructions, or with detection reagents only, as a negative control. Slides were mounted in Aquatex mounting medium (Merck, White
House Station, NJ). Stained sections were photographed with a
Nikon DS-FI1 camera with a Nikon DS-L2 control unit (Nikon,
Tokyo, Japan) and an Olympus BX40 microscope (Olympus, Tokyo,
Japan). All immunohistochemical staining was analyzed by and
performed under the supervision of an experienced board certified
consultant pathologist (EJS). 2.6. Tissue bank and primary cell expression analysis 3.1. PHACTR1 expression is increased in human atherosclerotic
lesions To investigate the role of PHACTR1 in CAD, we used immuno-
histochemistry to evaluate human PHACTR1 protein expression M.E. Reschen et al. / Atherosclerosis 250 (2016) 95e105 98 3.3. PHACTR1 transcripts display tissue-specific differential
expression in vivo in atherosclerotic lesions in arteries from patients with
atherosclerosis (n ¼ 5) and in normal arteries from healthy controls
(n ¼ 5). In healthy arteries, PHACTR1 was expressed in endothelial
cells and in the occasional immune cells that were present, but not
in vascular smooth muscle cells (Fig. 1A). In atherosclerotic plaque
lesions, an abundance of infiltrating macrophages and foam cells
were present that all strongly expressed PHACTR1, but no expres-
sion was seen in vascular smooth muscle cells (Fig. 1B, C). Plaque
endothelial cells also expressed PHACTR1 (Fig. 1B). Lymphocytes in
peri-plaque adventitial aggregates also expressed PHACTR1 and
this was present in both the cytoplasm and nucleus (Fig. 1D). PHACTR1 was also expressed by lymphocytes in secondary
lymphoid tissue (tonsillar), but here it was predominantly in the
cytoplasm, rather than the nucleus (Fig. 1E). Overall, the immu-
nohistochemistry results highlight the marked expression of
PHACTR1 in immune cells in atherosclerosis, particularly in mac-
rophages and lipid-laden foam cells. in vivo in atherosclerotic lesions in arteries from patients with
atherosclerosis (n ¼ 5) and in normal arteries from healthy controls
(n ¼ 5). In healthy arteries, PHACTR1 was expressed in endothelial
cells and in the occasional immune cells that were present, but not
in vascular smooth muscle cells (Fig. 1A). In atherosclerotic plaque
lesions, an abundance of infiltrating macrophages and foam cells
were present that all strongly expressed PHACTR1, but no expres-
sion was seen in vascular smooth muscle cells (Fig. 1B, C). Plaque
endothelial cells also expressed PHACTR1 (Fig. 1B). Lymphocytes in
peri-plaque adventitial aggregates also expressed PHACTR1 and
this was present in both the cytoplasm and nucleus (Fig. 1D). PHACTR1 was also expressed by lymphocytes in secondary
lymphoid tissue (tonsillar), but here it was predominantly in the
cytoplasm, rather than the nucleus (Fig. 1E). Overall, the immu-
nohistochemistry results highlight the marked expression of
PHACTR1 in immune cells in atherosclerosis, particularly in mac-
rophages and lipid-laden foam cells. We designed transcript-specific primer pairs to examine the
expression pattern of the long, intermediate and short transcripts
in atherosclerosis-related cell types (Table 1). 3.1. PHACTR1 expression is increased in human atherosclerotic
lesions The long transcript
was not expressed in primary human monocytes, macrophages,
HAEC, VSMC, T cells, B cells or CD14þ-depleted PBMC (Fig. 3A). The
intermediate transcript was expressed in macrophages, HAEC and
VSMC (Fig. 3A), but was barely detectable in monocytes and B cells
and absent from T cells. The short transcript was expressed in
primary human monocytes, macrophages, B cells, CD14þ-depleted
PBMC, and to a lesser extent in T cells, but was absent from HAEC
and VSMC (Fig. 3A). We screened a panel of adult and fetal tissues and the long
transcript was only present in adult brain tissue (Fig. 3B). The in-
termediate transcript was expressed in pancreas and fetal heart
(Fig. 3B). The short transcript was expressed in adult spleen, fetal
spleen, PBMC, colon and at a low level in the thymus (Fig. 3B). Overall, the long transcript is restricted to neuronal tissue only and
the intermediate transcript is more variable, being present in HAEC,
VSMC and macrophages. The short transcript is restricted to im-
mune cells. 3.2. PHACTR1 expresses multiple transcripts from two promoters in
vascular and immune cells We next determined the expression pattern of PHACTR1 in pri-
mary human atherosclerosis-related cell types involved in athero-
sclerosis. A combined genomic analysis of human expressed
sequence tag (EST) data, chromatin state mapping based on ChIP-
seq
data
and
CAP
analysis
of
gene
expression
with
high
throughput sequencing (CAGE-seq) [29,30] suggested the presence
of a transcriptional start site (TSS) around 240 kb downstream from
that of the reported Refseq transcript (NM_030948.2) and a further
TSS close to the 30 end of this transcript (Fig. 2A). To determine the
origin of transcripts in cell types involved in atherosclerosis, we
mapped the TSS using Rapid Amplification of cDNA Ends (RACE) in
primary human macrophages, aortic endothelial cells (HAEC),
monocytes
and
peripheral
blood
mononuclear
cells
(PBMC)
depleted of CD14þ monocytes (Fig. 2A, Supplementary data 1). This
demonstrated that the known TSS of the human RefSeq transcript
(NM_030948.2) was not used, but instead the RACE clones
confirmed a TSS 240 kb downstream, that was supported by the
EST, chromatin state and CAGE-seq data (Fig. 2A). We used the
RACE results to design primers to amplify and clone a 1674 bp open
reading frame from primary human macrophages and arterial
endothelial cells. This transcript encodes a 557 amino acid
PHACTR1 isoform that matches the predicted NCBI Reference
Sequence: XP_011512694.1 (Fig. 2A/B, Supplementary data 1). On
the basis of its length, we refer to this transcript as the ‘interme-
diate transcript’. The open reading frame of the intermediate
transcript begins partway through exon 3 of NM_030948.2 and
contains all subsequent downstream exons. Additionally, the in-
termediate transcript includes an extra exon (exon 6) that is not
present in NM_030948.2 (Fig. 2B/C). In CD14þ-depleted PBMCs, the
transcriptional start site was around 600 bp upstream of that seen
in both primary arterial endothelial cells and CD14þ cells, but this
did not alter the open reading frame. 3.4. PHACTR1 protein is expressed in macrophages and HAEC but
not VSMC To assess PHACTR1 protein expression in primary human cells,
western blotting was undertaken using an antibody raised against a
region of the protein shared by all PHACTR1 isoforms. Recombinant
PHACTR1 isoforms were expressed and analyzed in parallel to
facilitate molecular size comparison (Fig. 3C). The isoform encoded
by the long transcript migrated with an apparent molecular weight
of about 75 kDa (predicted 66 kDa, pI 6.51), similar to that of the rat
brain isoform (Fig. 3C) [18]. The intermediate isoform is 23 amino
acids shorter, but migrated with an apparently higher molecular
weight, equivalent to about 80 kDa (predicted 64 kDa, pI 6.81)
(Fig. 3C). This apparently anomalous migration pattern may arise
from differences between the protein isoforms such as shape, ri-
gidity, charge or post translational modification. Notably, exon 6 is
rich in proline residues that may increase rigidity (14 of 69 residues
in exon 6 are proline). Exon 6 in the intermediate isoform does not
contain any additional predicted glycosylation motifs. PHACTR1
protein was completely absent from primary vascular smooth
muscle cells despite moderate level mRNA expression in these cells
(Fig. 3C). In primary human macrophages, foam cells, HAEC and
oxLDL-treated HAEC there was strong expression of the interme-
diate isoform (Fig. 3C). We did not detect protein expression of the
short transcript but detection of this protein was difficult even
following overexpression in 293T cells (Fig. 3C and D). Although it
was not possible to assess the endogenous short isoform reliably,
these findings are consistent with our transcript studies and indi-
cate that primary macrophages and arterial endothelial cells ex-
press the intermediate, but not the long protein isoform. The genomic analysis supported the presence of a distal TSS that
matched the beginning of ENSEMBL transcript ENST00000379335
[29,30] (Fig. 2 A/B). Additional analysis of chromatin mapping data
from our previous study of primary human macrophages demon-
strated an open chromatin site at the TSS that was flanked by
H3K27ac-marked histones consistent with promoter activity [17]. Using this information, we cloned a 435 bp transcript from mac-
rophages, which encoded a 144 amino acid protein. This ‘short’
transcript shares its last four 4 exons with the longer transcripts,
but begins with an extended version of exon 10 that starts 50 to the
exon 10 start position seen in the longer transcripts (Fig. 2B/C). 3.5. PHACTR1 expression is regulated by oxLDL and inflammatory
stimuli in macrophages and endothelial cells Given the association of the PHACTR1 locus with CAD and its
expression
in
atherosclerotic
lesions,
we
hypothesized
that
PHACTR1 would be regulated by atherogenic and inflammatory
stimuli. We used transcript-specific RTqPCR to assess expression of
PHACTR1 in response to a variety of stimuli. Lipopolysaccharide
(LPS) treatment of primary human macrophages upregulated the
intermediate
transcript
(fold
¼
4.68,
p
<
0.0001),
but g. 1. Immunohistochemistry demonstrates in vivo PHACTR1 expression in atherosclerotic plaque. (A) Healthy aorta demonstrates PHACTR1-positive endothelium (ar
ut vascular smooth muscle cells in the sub-intimal layer lack PHACTR1 staining. (B) Atherosclerotic plaque showing endothelial positivity (arrowed) and positivity in t
ntimal space, comprising predominantly foam cells. (C) Higher power view of foam cells in atherosclerotic plaque showing cytoplasmic and nuclear staining for P
Green arrows indicate examples of strongly stained foam cell nuclei. Cytoplasmic foam cell staining is present in the lighter brown areas that asymmetrically encircle the
D) Lymphocytic aggregate in aortic adventitia of atherosclerotic vessel showing strong positivity for PHACTR1. The PHACTR1 staining is present throughout the cells, in
uclear and cytoplasmic distribution. (E) Germinal centre in tonsillar lymphoid tissue; lymphocytes lack nuclear PHACTR1 as demonstrated by a thin rim of brown cyto
aining with unstained, blue nuclei (example lymphocytes arrowed). Staining for human PHACTR1, using HRP/DAB, each section representative of data from 5 individ
ondition, scale bars indicate 50 mm. M.E. Reschen et al. / Atherosclerosis 250 (2016) 95e105 99 M.E. Reschen et al. / Atherosclerosis 250 (2016) 95e105 Fig. 1. Immunohistochemistry demonstrates in vivo PHACTR1 expression in atherosclerotic plaque. (A) Healthy aorta demonstrates PHACTR1-positive endothelium (arrowed),
but vascular smooth muscle cells in the sub-intimal layer lack PHACTR1 staining. (B) Atherosclerotic plaque showing endothelial positivity (arrowed) and positivity in the sub-
intimal space, comprising predominantly foam cells. (C) Higher power view of foam cells in atherosclerotic plaque showing cytoplasmic and nuclear staining for PHACTR1
(Green arrows indicate examples of strongly stained foam cell nuclei. Cytoplasmic foam cell staining is present in the lighter brown areas that asymmetrically encircle the nuclei). (D) Lymphocytic aggregate in aortic adventitia of atherosclerotic vessel showing strong positivity for PHACTR1. The PHACTR1 staining is present throughout the cells, indicating
nuclear and cytoplasmic distribution. (E) Germinal centre in tonsillar lymphoid tissue; lymphocytes lack nuclear PHACTR1 as demonstrated by a thin rim of brown cytoplasmic
staining with unstained, blue nuclei (example lymphocytes arrowed). 3.5. PHACTR1 expression is regulated by oxLDL and inflammatory
stimuli in macrophages and endothelial cells Staining for human PHACTR1, using HRP/DAB, each section representative of data from 5 individuals per
condition, scale bars indicate 50 mm. (oxPAPC) is a mixture of oxidized phospholipids that have a
similar biological effect to minimally oxidized LDL [31]. OxPAPC
contains components of the biologically active fractions of HPLC-
separated minimally-modified LDL [31]. In macrophages OxPAPC
produced a similar directional effect to oxLDLdupregulation of the
short transcript (fold ¼ 2.28, p ¼ 0.002) and a trend towards
downregulation of the intermediate transcript (fold ¼ 0.83,
p ¼ 0.12) (Fig. 4B). To refine the lipid species responsible for the
effect on PHACTR1 expression we loaded macrophages with
cholesterol itself. Cells were treated with a cyclodextrin-cholesterol downregulated the short transcript more than 10-fold (fold ¼ 0.06. p < 0.0001) (Fig. 4A). The inflammatory cytokine Tumor Necrosis
Factor-alpha (TNFa) produced a similar, but weaker effect with
upregulation of the intermediate transcript (fold ¼ 1.41, p ¼ 0.04)
and downregulation of the short transcript (fold ¼ 0.64, p ¼ 0.02)
(Fig. 4A). OxLDL exerted a converse effect on expression of
PHACTR1 compared to LPS and TNFa; the short transcript was
upregulated 6 fold (p ¼ 0.004) and the intermediate transcript was
downregulated (fold ¼ 0.74, p ¼ 0.01) (Fig. 4B). Oxidized 1-
palmitoyl-2-arachidonyl-sn-
glycero-3-phosphorylcholine M.E. Reschen et al. / Atherosclerosis 250 (2016) 95e105 100 M.E. Reschen et al. / Atherosclerosis 250 (2016) 95e105 Fig. 2. The structure of human PHACTR1 transcripts. (A) The transcript structure for PHACTR1 is shown for RefSeq curated transcripts showing 2 transcripts in blue. Shown below
is human expressed sequence tag data, which is suggestive of a transcription start site halfway along the gene and a further possible site at the 30 end of the gene. These data were
compared to chromatin state data that uses multiple ChIP-seq markers to classify the genome into different types of regulatory element or transcription state. Chromatin state maps
are shown for 9 cell types with the chromatin state colored (red - promoter, orange - enhancer, yellow - weak enhancer, blue - insulator, green - transcribed, dark grey - not
transcribed). Red regions depicting active promoter elements are seen half way along the gene, coinciding with a TSS suggested by EST data. In one cell type, a promoter is also seen
at the 30 end. These sites coincide with precise TSSs identified by CAGE-seq in a variety of human cell types. 3.5. PHACTR1 expression is regulated by oxLDL and inflammatory
stimuli in macrophages and endothelial cells Taken together, these data suggest three main TSS in three main
promoter regions. The position of coronary artery disease risk variant, rs9349379, is indicated by the red triangle. (B) The transcript structure for the RefSeq transcript is shown
uppermost and is aligned to the intermediate transcript that was cloned from macrophages and endothelial cells. The short transcript cloned from macrophages is also shown
below. Vertical lines indicate exons. (C) Coding structure of PHACTR1 transcripts with exons drawn to scale and motifs annotated. Motif locations were predicted using the
Eukaryotic Linear Motif resource. The region of experimentally proven PP1 binding is shown in green. Table 1 The polyclonal rabbit anti-PHACTR1 antibody identifies a non-specific additional band at about 47 kDa even in untransfected cells,
which do not express PHACTR1. (D) Western blotting with over-expression in 293T cells of the short transcript cloned from macrophages. A cloned form of the intermediate
transcript skipping exons 5e9 is also shown for comparison. M.E. Reschen et al. / Atherosclerosis 250 (2016) 95e105
101 M.E. Reschen et al. / Atherosclerosis 250 (2016) 95e105 101 M.E. Reschen et al. / Atherosclerosis 250 (2016) 95e105 Fig. 3. PHACTR1 expression patterns in primary cells and tissues. (A) Expression in purified primary human cells showed that the long transcript was not expressed among the
cells tested. By contrast, the intermediate transcript showed robust expression in human monocytes, macrophages, arterial endothelial cells (HAEC) and vascular smooth muscle
cells (VSMC), but was not expressed by T cells and was barely detectable in B cells and PBMC-depleted of monocytes. (B) Analysis of a human tissue cDNA panel showed the long
transcript exclusively expressed in adult brain. The intermediate transcript showed strong expression in the pancreas, but was also detectable in fetal heart. By contrast, the short
transcript was present in PBMC and spleen, including fetal spleen. (C) Western blotting: PHACTR1 over-expression in 293T cells transfected with the long and intermediate isoforms
compared to endogenous PHACTR1 in primary human cells. The shorter intermediate isoform has an apparently greater size than the long isoform in 293T cells. Macrophages, foam
cells and HAECs expressed PHACTR1 with a similar size to the intermediate isoform. PHACTR1 protein was absent from VSMC. A band corresponding to endogenous expression of
the short isoform was not seen in any cell type. The polyclonal rabbit anti-PHACTR1 antibody identifies a non-specific additional band at about 47 kDa even in untransfected cells,
which do not express PHACTR1. (D) Western blotting with over-expression in 293T cells of the short transcript cloned from macrophages. A cloned form of the intermediate
transcript skipping exons 5e9 is also shown for comparison. PHACTR1 expression. In vascular smooth muscle cells, cholesterol
loading had no effect on expression of the intermediate PHACTR1
transcript. between the alleles of rs9349379 and expression of PHACTR1 in
macrophages and CD14þ-depleted PBMC. To do this we used
primary cells derived from healthy non-smoking Caucasian in-
dividuals controlled for age (>18, <50 years), body mass index,
cholesterol and triglyceride levels but with differing rs9349379
genotypes. Table 1 complex that promotes cholesterol loading of macrophages and
vascular smooth muscle cells [32,33]. Unlike the oxidized stimuli,
cholesterol loading had no effect on PHACTR1 expression (Fig. 4B). Overall, these data indicate that in macrophages inflammatory
pathways downregulate the short transcript and upregulate the
intermediate transcript, whereas oxidized lipid-responsive path-
ways upregulate the short transcript. In human aortic endothelial
cellsdwhich only express the intermediate transcriptdboth TNFa
and oxLDL upregulated PHACTR1 (fold ¼ 2.5, p ¼ 0.007, and
fold ¼ 1.8, p ¼ 0.009, respectively) (Fig. 4C). As with macrophages,
cholesterol loading with cyclodextrin-cholesterol had no effect on Table 1
Summary of expression pattern of 3 different PHACTR1 transcripts (mRNA) in pri-
mary human cell types relevant to atherosclerosis. Long
Intermediate
Short
Macrophages
No
Yes
Yes
Arterial endothelial cells
No
Yes
No
Vascular smooth muscle cells
No
Yes
No
PBMC depleted of CD14þ cells
No
Yes
Yes
B cells (CD19þ)
No
Barely detectable
Yes
T cells (CD3þ)
No
No
Barely detectable Summary of expression pattern of 3 different PHACTR1 transcripts (mRNA) in pri-
mary human cell types relevant to atherosclerosis. Summary of expression pattern of 3 different PHACTR1 transcripts (mRNA) in pri-
mary human cell types relevant to atherosclerosis. Fig. 3. PHACTR1 expression patterns in primary cells and tissues. (A) Expression in purified primary human cells showed that the long transcript was not expressed among the
cells tested. By contrast, the intermediate transcript showed robust expression in human monocytes, macrophages, arterial endothelial cells (HAEC) and vascular smooth muscle
cells (VSMC), but was not expressed by T cells and was barely detectable in B cells and PBMC-depleted of monocytes. (B) Analysis of a human tissue cDNA panel showed the long
transcript exclusively expressed in adult brain. The intermediate transcript showed strong expression in the pancreas, but was also detectable in fetal heart. By contrast, the short
transcript was present in PBMC and spleen, including fetal spleen. (C) Western blotting: PHACTR1 over-expression in 293T cells transfected with the long and intermediate isoforms
compared to endogenous PHACTR1 in primary human cells. The shorter intermediate isoform has an apparently greater size than the long isoform in 293T cells. Macrophages, foam
cells and HAECs expressed PHACTR1 with a similar size to the intermediate isoform. PHACTR1 protein was absent from VSMC. A band corresponding to endogenous expression of
the short isoform was not seen in any cell type. Table 1 There was a significant allelic effect on expression of
the short transcript; the A allele was associated with higher
expression (Fig. 5A). Macrophages from individuals homozygous
for the protective A allele had 2.5-fold higher expression than
macrophages from individuals homozygous for the G allele, which
is the coronary artery disease risk-associated allele (p ¼ 0.0187). There was a trend towards reduced expression of the intermediate
transcript with the A allele, but this did not reach significance
(Fig. 5B). In CD14þ-depleted PBMCs no association was detectable 3.6. Genetic regulation of PHACTR1 expression by CAD SNP
rs9349379 Significant differences are indicated by black lines with p values shown. The controls labelled on the x-axis
represent the relevant buffer controls for each treatment. Fig. 4. PHACTR1 transcripts are differentially regulated by atherogenic and inflammatory stimuli. (A) Effect of LPS (10 ng/ml) on mRNA expression in primary human macrophages. The intermediate transcript was upregulated, but the short transcript was suppressed. TNFa (10 ng/ml) produced a directionally similar effect. (B) Exposure of macrophages to
oxLDL (50 mcg/ml) caused modest downregulation of the intermediate transcript and strong upregulation of the short transcript. OxPAPC (30 mcg/ml) caused a directionally similar
effect to oxLDL on PHACTR1 expression. (C) The intermediate transcript was upregulated by oxLDL in aortic endothelial cells. TNFa (10 ng/ml) caused upregulation of PHACTR1 at 6
and 48-h time points in aortic endothelial cells. The short transcript was not expressed in aortic endothelial cells. Cyclodextrin-cholesterol (10 mcg/ml) loading of endothelial cells
and vascular smooth muscle cells had no effect on PHACTR1 expression. Significant differences are indicated by black lines with p values shown. The controls labelled on the x-axis
represent the relevant buffer controls for each treatment. 3.6. Genetic regulation of PHACTR1 expression by CAD SNP
rs9349379 The rs9349379 SNP at the PHACTR1 locus has been implicated
by GWAS in the genetic risk of CAD [11,34]. The mechanism by
which different alleles of this SNP influence CAD risk is unknown. Rs9349379 is in an intronic region of PHACTR1 and is not in high
linkage disequilibrium with any coding SNPs; it may therefore
affect transcriptional regulation of PHACTR1. Using transcript-
specific real time quantitative PCR, we tested for an association M.E. Reschen et al. / Atherosclerosis 250 (2016) 95e105 102 0
1
2
3
TNFa control
TNFa 6hrs
TNFa 48hrs
OxLDL control
OxLDL
Cholesterol control
Cholesterol
Treatment
Cell type
HAEC
VSMC
0
2
4
6
OxLDL control
OxLDL
OxPAPC control
OxPAPC
Cholesterol control
Cholesterol
Treatment
Expression relative to control
0
2
4
Treatment
Transcript
Intermediate
Short
LPS control
LPS
TNFa control
TNFa
Expression relative to control
Expression relative to control
Macrophages
Macrophages
Intermediate transcript
B
A
C
<0.0001
<0.0001
0.04
0.02
0.01
0.004
0.002
0.007
0.026
0.009
Transcript
Intermediate
Short
. 4. PHACTR1 transcripts are differentially regulated by atherogenic and inflammatory stimuli. (A) Effect of LPS (10 ng/ml) on mRNA expression in primary human macrophages. e intermediate transcript was upregulated, but the short transcript was suppressed. TNFa (10 ng/ml) produced a directionally similar effect. (B) Exposure of macrophages to
LDL (50 mcg/ml) caused modest downregulation of the intermediate transcript and strong upregulation of the short transcript. OxPAPC (30 mcg/ml) caused a directionally similar
ect to oxLDL on PHACTR1 expression. (C) The intermediate transcript was upregulated by oxLDL in aortic endothelial cells. TNFa (10 ng/ml) caused upregulation of PHACTR1 at 6
d 48-h time points in aortic endothelial cells. The short transcript was not expressed in aortic endothelial cells. Cyclodextrin-cholesterol (10 mcg/ml) loading of endothelial cells
d vascular smooth muscle cells had no effect on PHACTR1 expression. Significant differences are indicated by black lines with p values shown. The controls labelled on the x-axis
resent the relevant buffer controls for each treatment. M.E. Reschen et al. 3.6. Genetic regulation of PHACTR1 expression by CAD SNP
rs9349379 / Atherosclerosis 250 (2016) 95e105
2 0
2
4
6
OxLDL control
OxLDL
OxPAPC control
OxPAPC
Cholesterol control
Cholesterol
Treatment
Expression relative to control
0
2
4
Treatment
Transcript
Intermediate
Short
LPS control
LPS
TNFa control
TNFa
Expression relative to control
Macrophages
Macrophages
B
A
<0.0001
<0.0001
0.04
0.02
0.01
0.004
0.002
Transcript
Intermediate
Short B A Treatment Treatment 0
1
2
3
TNFa control
TNFa 6hrs
TNFa 48hrs
OxLDL control
OxLDL
Cholesterol control
Cholesterol
Treatment
Cell type
HAEC
VSMC
Expression relative to control
Intermediate transcript
C
0.007
0.026
0.009 C Expression relative to control Treatment Fig. 4. PHACTR1 transcripts are differentially regulated by atherogenic and inflammatory stimuli. (A) Effect of LPS (10 ng/ml) on mRNA expression in primary human macrophages. The intermediate transcript was upregulated, but the short transcript was suppressed. TNFa (10 ng/ml) produced a directionally similar effect. (B) Exposure of macrophages to
oxLDL (50 mcg/ml) caused modest downregulation of the intermediate transcript and strong upregulation of the short transcript. OxPAPC (30 mcg/ml) caused a directionally similar
effect to oxLDL on PHACTR1 expression. (C) The intermediate transcript was upregulated by oxLDL in aortic endothelial cells. TNFa (10 ng/ml) caused upregulation of PHACTR1 at 6
and 48-h time points in aortic endothelial cells. The short transcript was not expressed in aortic endothelial cells. Cyclodextrin-cholesterol (10 mcg/ml) loading of endothelial cells
and vascular smooth muscle cells had no effect on PHACTR1 expression. Significant differences are indicated by black lines with p values shown. The controls labelled on the x-axis
represent the relevant buffer controls for each treatment. Fig. 4. PHACTR1 transcripts are differentially regulated by atherogenic and inflammatory stimuli. (A) Effect of LPS (10 ng/ml) on mRNA expression in primary human macrophages. The intermediate transcript was upregulated, but the short transcript was suppressed. TNFa (10 ng/ml) produced a directionally similar effect. (B) Exposure of macrophages to
oxLDL (50 mcg/ml) caused modest downregulation of the intermediate transcript and strong upregulation of the short transcript. OxPAPC (30 mcg/ml) caused a directionally similar
effect to oxLDL on PHACTR1 expression. (C) The intermediate transcript was upregulated by oxLDL in aortic endothelial cells. TNFa (10 ng/ml) caused upregulation of PHACTR1 at 6
and 48-h time points in aortic endothelial cells. The short transcript was not expressed in aortic endothelial cells. Cyclodextrin-cholesterol (10 mcg/ml) loading of endothelial cells
and vascular smooth muscle cells had no effect on PHACTR1 expression. 4. Discussion between genotype and expression of either the short or inter-
mediate transcript (Fig. 5C, D). Overall, these results demonstrate
that rs9349379 is both a CAD risk-associated variant and an
expression quantitative trait locus (eQTL) for PHACTR1 in mac-
rophages, but not in CD14þ-depleted PBMC. Therefore, decreased
expression of the short transcript in macrophages is associated
with CAD. This genetic risk effect recapitulates the effect of LPS,
which also caused suppression of the short transcript (Fig. 5A). Overall, these findings demonstrate that the CAD risk effect of
genetic variants at rs9349379 may be operational in macrophages
in atherosclerotic lesions. GWAS of complex diseases have identified genomic loci con-
taining genetic variants that affect heritable disease risk [4]. For
CAD, one of the most robust reported risk loci is at the PHACTR1
gene locus where several studies have identified intronic SNPs that
are associated with the risk of myocardial infarction, coronary
stenosis and coronary calcification [9e11,34]. The same CAD risk
SNP allele is associated with protection from a non-atherosclerotic
form of vascular disease; cervical artery dissection. Unravelling the
biological pathways underpinning the pleiotropic effect of the M.E. Reschen et al. / Atherosclerosis 250 (2016) 95e105 103 Fig. 5. Genotype of CAD risk SNP rs9349379 affects expression of the short PHACTR1 transcript in macrophages. PHACTR1 expression in primary human macrophages relative
to GAPDH. (A) A significant genotype-expression level correlation was demonstrated in primary human macrophages (n ¼ 19, AA ¼ 6, AG ¼ 8, GG ¼ 5). Individuals with the AA
genotype had the highest level of expression of the short PHACTR1 transcript and subjects with the GG genotype had the lowest expression. (B) There was no significant correlation
with the intermediate transcript (n ¼ 19). (C, D) Primary human CD14þ-depleted PBMCs did not show a significant correlation between expression and genotype of rs9349379
(n ¼ 20, AA ¼ 7, AG ¼ 8, GG ¼ 5). Fig. 5. Genotype of CAD risk SNP rs9349379 affects expression of the short PHACTR1 transcript in macrophages. PHACTR1 expression in primary human macrophages relative
to GAPDH. (A) A significant genotype-expression level correlation was demonstrated in primary human macrophages (n ¼ 19, AA ¼ 6, AG ¼ 8, GG ¼ 5). Individuals with the AA
genotype had the highest level of expression of the short PHACTR1 transcript and subjects with the GG genotype had the lowest expression. 4. Discussion (B) There was no significant correlation
with the intermediate transcript (n ¼ 19). (C, D) Primary human CD14þ-depleted PBMCs did not show a significant correlation between expression and genotype of rs9349379
(n ¼ 20, AA ¼ 7, AG ¼ 8, GG ¼ 5). accumulation [35]. In endothelial cells only the intermediate transcript was
expressed and this was upregulated by oxLDL but not by non-
oxidized, non-lipoprotein cholesterol loading. In previous studies
with umbilical vein endothelial cells TNFa had no effect on
PHACTR1 expression [15,23]. However, we found that PHACTR1
was upregulated by TNFa in primary aortic arterial endothelial
cells, indicating the difference between adult arterial endothelial
cells and neonatal umbilical cord vein endothelial cells. These
findings raise the possibility that oxLDL and TNFa could influence
angiogenesis or atherosclerosis by affecting PHACTR1 expression in
arterial endothelial cells. A previous study in HUVECs reported
upregulation of PHACTR1 in response to the classic angiogenic
factor, vascular endothelial growth factor [36]. Determining the mechanisms underlying the CAD risk conferred
by GWAS-identified genetic risk variants is a major challenge for
atherosclerosis research, especially for loci containing poorly
characterized genes. We establish PHACTR1 as an important gene
expressed in atherosclerotic lesions and as the candidate gene at
the PHACTR1 CAD locus by demonstrating genetic regulation of a
novel transcript and regulation by atherogenic stimuli. Previous
molecular studies of PHACTR1 focused on a transcript which we
show is not expressed in cells involved in atherosclerotic lesions. We have identified novel intermediate and short length transcripts
as the dominant forms expressed in cells involved in atheroscle-
rosis. Our genotype-expression analysis shows that the CAD-
associated genetic variant is active in macrophages, the dominant
cell type expressing PHACTR1 in human atherosclerotic plaques. The effects of the CAD risk genotype mirror those of an inflam-
matory stimulus. g
[
]
The rs9349379 SNP is robustly associated with CAD and a fine
mapping study suggested it was the likely causal variant in the
region [15]. Previous eQTL studies have not taken account of the
different PHACTR1 transcripts and so have not been informative
about each transcript. A large-scale eQTL study in monocytes used
microarray technology with a probe that would have detected
multiple PHACTR1 transcripts [37]. An eQTL study of CAD candidate
genes, did not detect significant PHACTR1 expression in PBMC, but
the Taqman assay used would only assess the long transcript, which
is not expressed in these cells [38]. An eQTL for PHACTR1 expression
has recently been demonstrated in right coronary artery tissue, but
the cellular origin of this effect was unclear due to the mixed cell
populations in the tissue samples [15]. accumulation [35]. now established a new association in macrophages between
expression of the previously uncharacterized short PHACTR1 tran-
script and genotype at rs9349379. In PBMCs-depleted of CD14þ
cells (predominantly lymphocytes) there was a trend towards an
eQTL effect in the opposite direction to that in macrophages. A
previous study found that 4.4% of eQTL-associated SNPs produced
opposite effects in different tissues [40]. Suppression of the short PHACTR1 transcript in macrophages by
pro-inflammatory stimuli mirrors the effect of the CAD risk allele
(rs9349379-G), which is associated with reduced expression of the
short transcript compared to that seen with the protective allele
(rs9349379-A). This suggests that having the risk genotype is, in
this sense, equivalent to a persistent inflammatory stimulus, such
as that caused by LPS. As the short transcript was only expressed in
immune cells, these data imply that the short transcript could play
a role in the macrophage inflammatory response in atherosclerotic
plaques. In macrophages, oxLDL exposure caused upregulation of the
short transcript and suppression of the intermediate transcript
whereas lipopolysaccharide and TNFa caused upregulation of the
intermediate transcript and suppression of the short transcript. These findings demonstrate that PHACTR1 expression responds
differently to inflammatory stimuli and to lipid or oxidative stress
pathways activated by oxLDL. The effect of the lipid stimulus was
not dependent on the presence of lipoproteins since synthetic
oxidized phospholipids (oxPAPC) produced a similar response to
oxLDL. A non-oxidized, non-lipoprotein stimulus, comprising pure
cholesterol loading did not affect expression of PHACTR1 suggest-
ing that oxidized lipids are required. p
q
The rs9349379 SNP is located about 250 kb upstream from the
short PHACTR1 transcript. At other risk loci chromatin looping has
been established as a mechanism whereby regulatory elements
containing the SNP are brought into proximity with the gene whose
expression is altered. The DEXI gene, for example, is physically
linked by chromatin looping to a type 1 diabetes risk SNP about
150 kb away and the SNP has an eQTL for DEXI [41]. We and others
have found that GWAS risk SNPs alleles affect enhancer function by
altering transcription factor binding affinity at regulatory elements
[6,17]. We hypothesize that such a mechanism may be responsible
for the effect of rs9349379 and the G allele is predicted to reduce
binding of MEF2 transcription factors, that are expressed in mac-
rophages [17,42e44]. accumulation [35]. PHACTR1 locus is critical to harnessing the genetic knowledge
yielded by GWAS studies [14]. To better understand the role of
PHACTR1 in atherosclerosis, we investigated PHACTR1 expression
and the regulatory influence of inflammatory stimuli, atherogenic
lipid and genetic variation. The previously reported PHACTR1 transcript was isolated from
brain and encodes a 580 amino acid protein [18,19]. We only found
expression of this long PHACTR1 transcript in brain. Previous mo-
lecular and functional studies have focused on this transcript,
which may limit their relevance to understanding the role of
PHACTR1 beyond the nervous system. In cell types involved in
atherosclerotic lesions, we found that PHACTR1 was transcribed
from a more distal TSS giving rise to the intermediate transcript. This intermediate transcript was the prevalent protein isoform in
immune and endothelial cells and the encoded protein includes the
4 previously characterized actin-binding RPEL domains and NLS
regions. In addition, we found transcription of a short transcript
from an even more distal TSS. The encoded protein would only
contain two RPEL motifs and lack the N-terminal NLS. The
expression pattern of the short transcript differed markedly from Using
immunohistochemistry,
we
demonstrated
increased
expression of PHACTR1 in atherosclerotic lesions in vivo. Lipid-
laden macrophages or foam cells were clearly the most abundant
source of PHACTR1 in plaques. The distribution was both nuclear
and cytoplasmic in macrophages, foam cells and adventitial lym-
phocytes, whereas in secondary lymphoid tissue lymphocytes,
PHACTR1 was excluded from the nucleus. This differential intra-
cellular localization may reflect the accumulation in atherosclerotic
plaques of inflammatory lipids including lysophosphatidic acid
(LPA), which activates RhoA signaling causing G- to F-actin poly-
merization, a mechanism known to promote PHACTR1 nuclear M.E. Reschen et al. / Atherosclerosis 250 (2016) 95e105 104 the longer forms and expression was only detected in immune cells,
principally macrophages and B-lymphocytes. Macrophages were
notable as the only cell type with high-level expression of the in-
termediate and short transcript. The limited distribution in im-
mune cells is consistent with a role for the short transcript in
immunity and inflammation. We were unable to detect protein
expressed from the endogenous short transcript in the cell types
studied despite using an antibody raised against peptides encom-
passing most of its coding region. This may indicate post-
transcriptional effects, rapid turnover or technical limitations of
the available antibodies. A striking finding was the absence of
PHACTR1 protein in VSMC. Conflict of interest The authors declared that they do not have anything to disclose
regarding conflict of interest with respect to this manuscript. accumulation [35]. In addition, the different
transcripts were not distinguished because the qPCR primers
annealed to both the long and intermediate transcripts and the
short transcript was not studied [15]. Using RNA-seq eQTLs were
sought across a panel of human tissues and the rs9349379 SNP was
found to be an eQTL for PHACTR1 in coronary artery, tibial artery
and aortic artery tissue; the reference allele A was associated with
higher PHACTR1 expression than the alternative G allele [16]. In
this study, the composite nature of the tissue samples does not
allow identification of the cellular origin of the effect and the
transcript affected was not reported. The direction of genetic effect
is the same as that of the short transcript in the present study. An
eQTL study in endothelial cells using primers that would detect
only the longer transcripts did not find a significant association
with variants at the rs12526453 SNP, which is in linkage disequi-
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O STF e as políticas criminal, penitenciária e de segurança pública: um duplo ponto cego nas agendas de pesquisa?
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Política & Sociedade
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DOI: https://doi.org/10.5007/2175-7984.2022.e90224 DOI: https://doi.org/10.5007/2175-7984.2022.e90224 O STF e as políticas criminal,
penitenciária e de segurança pública:
um duplo ponto cego
nas agendas de pesquisa? Lígia Mori Madeira1
Guilherme Augusto Dornelles de Souza2
Leonardo Geliski3 1
Doutora em Sociologia Professora do Departamento de Ciência Política e do PPG em Políticas Públicas/UFRGS-
Coordenadora do Núcleo de Pesquisa em Desenvolvimento, Direitos, Instituições e Políticas Públicas (NEDIPP)
ligia.madeira@ufrgs.br https://orcid.org/0000-0003-3657-3153 1
Doutora em Sociologia Professora do Departamento de Ciência Política e do PPG em Políticas Públicas/UFRGS-
Coordenadora do Núcleo de Pesquisa em Desenvolvimento, Direitos, Instituições e Políticas Públicas (NEDIPP)
ligia.madeira@ufrgs.br https://orcid.org/0000-0003-3657-3153
2
Mestre em Ciências Criminais Doutorando em Políticas Públicas | UFRGS Pesquisador do NEDIPP/UFRGS
guilherme.dornelles@ufrgs.br https://orcid.org/0000-0003-2521-4294
3
Doutorando em Políticas Públicas | UFRGS Mestre em Políticas Públicas| UFRGS Pesquisador do NEDIPP/
UFRGS leonardo.geliski@ufrgs.br https://orcid.org/0000-0001-6948-761X 3
Doutorando em Políticas Públicas | UFRGS Mestre em Políticas Públicas| UFRGS Pesquisador do NEDIPP/
UFRGS leonardo.geliski@ufrgs.br https://orcid.org/0000-0001-6948-761X 2
Mestre em Ciências Criminais Doutorando em Políticas Públicas | UFRGS Pesquisador do NEDIPP/UFRGS
guilherme.dornelles@ufrgs.br https://orcid.org/0000-0003-2521-4294 Direito autoral e licença de uso: Este artigo está licenciado sob uma Licença Creative
Commons. Com essa licença você pode compartilhar, adaptar, para qualquer fim, desde que
atribua a autoria da obra, forneça um link para a licença, e indicar se foram feitas alterações. Resumo Esse artigo apresenta revisão de literatura sobre a atuação do Supremo Tribunal Federal em relação
a políticas criminais, penitenciárias e de segurança pública, mapeando temas, formas de interven-
ção da corte, bem como principais teorias e teses. A partir de um levantamento bibliográfico inicial
de 1.250 textos utilizando o software Publish or Perish, foram selecionadas 190 publicações para
análise em profundidade. Identificaram-se cinco eixos temáticos principais: “atores, discursos e
comportamento judicial”, “criminalização”, “segurança pública”, “punição e encarceramento”,
“garantias e prerrogativas processuais”. Constatou-se a prevalência de produções da área jurídi-
ca, com leituras doutrinárias e jurisprudenciais, e estudos descritivos sobre decisões de grande
repercussão, com poucos trabalhos mobilizando as literaturas de judicial politics, e os estudos
sobre crime, violência e segurança pública, cuja apropriação sugere caminhos para o avanço dessa
agenda de pesquisa. Palavras-chave: STF. Política criminal. Política penitenciária. Política de segurança pública. Revisão bibliográfica. Direito autoral e licença de uso: Este artigo está licenciado sob uma Licença Creative
Commons. Com essa licença você pode compartilhar, adaptar, para qualquer fim, desde que
atribua a autoria da obra, forneça um link para a licença, e indicar se foram feitas alterações. 255 255 – 288 O STF e as políticas criminal, penitenciária e de segurança pública: um duplo ponto cego
nas agendas de pesquisa? | Lígia Mori Madeira, Leonardo Geliski 1. Introdução A mudança nos padrões de criminalidade urbana no Brasil, a partir dos
anos 1980, com o aumento no número de mortes violentas e a expan-
são do tráfico de drogas e dos coletivos criminais a ele associados, colo-
cou a violência e a segurança pública como temas relevantes no debate
público desde então. Além de fatores explicativos desses fenômenos, pes-
quisas têm examinado as instituições voltadas ao controle do crime e seus
efeitos, como o aumento das taxas de encarceramento, das mortes provoca-
das pela ação policial, e os inefetivos padrões de funcionamento do sistema
de justiça criminal. Nesse contexto, ganham relevância os estudos que exa-
minam os papéis e os impactos do Poder Judiciário em relação às políticas
penais, bem como o tratamento dado a algumas categorias específicas de
crimes, tais como a violência contra mulheres, e, mais recentemente, os
grandes casos de corrupção. O Supremo Tribunal Federal vem sendo retratado em sua atuação em
temas criminais com foco na competência originária penal em relação a cri-
mes de agentes públicos ou mesmo em sede recursal, marcando a atividade
anticorrupção e improbidade administrativa da corte. Apesar do Mensalão
e da Operação Lava Jato despontarem como os grandes fenômenos de aná-
lise do STF em matéria criminal nas ciências sociais, autores dedicados ao
estudo do Supremo Tribunal Criminal apontam as carências e a incipiência
do debate criminal na grande agenda de estudos sobre a Suprema Corte. A
centralidade do debate sobre foro privilegiado, do acesso à corte de “casos
originários ou em sede recursal envolvendo as mesmas elites, os tipos de
comportamento dos ministros na definição de regras pelas quais a corte
opera e no processo decisório propriamente dito” revela que “a literatura
sobre o ‘STF criminal’ ainda é escassa e está em fase inicial, [sendo] pre-
ponderantemente descritiva” (TAYLOR; DA ROS, no prelo). Percebe-se lacuna ainda maior na consideração da atividade do tribu-
nal em matérias de segurança pública, políticas públicas criminais e pe-
nitenciárias. Tal constatação soa estranha, dada essa atividade pertencer à
dimensão de enforcement de direitos individuais, uma das áreas mais subs-
tantivas de investigação sobre a corte. Além disso, a segurança pública figu-
ra entre as áreas de intervenção direta dos tribunais em temas considerados 255 – 288 256 Política & Sociedade - Florianópolis - Vol. 21 - Nº 52 - Set./Dez. 1. Introdução de 2022 fundamentais da ordem política de diferentes países em processo de “judi-
cialização da mega política” (DA ROS, 2017). Apesar das lacunas na lite-
ratura, é possível apontar o papel do STF em relação a políticas criminal,
penitenciária e de segurança pública naquelas que são as três personas
identificadas de atuação do tribunal, como Corte Constitucional, Corte
Recursal e Corte Ordinária (FALCÃO et al., 2019). Soma-se a elas a ação
propriamente política, na proposição e no envolvimento em políticas pú-
blicas na área. A literatura latino-americana, apontando o legado do tipo de rela-
cionamento entre tribunais superiores e regimes autoritários presentes
no continente (SHAPIRO, 2008), auxilia na compreensão dessa falta. As
estruturas judiciais atuaram na implementação das políticas dos regimes
antidemocráticos (MOUSTAFA; GINSBURG, 2008) e no monopólio do
poder punitivo estatal a serviço das elites dominantes, por exemplo na
cassação de direitos políticos de opositores dos regimes (HELMKE, 2002,
2005; MAGALONI, 2008). Com a virada democrática, as cortes passaram a ser atores centrais da
governança dos executivos para a implementação de pautas associadas a
direitos econômicos e sociais. A trajetória do tribunal brasileiro pula da
não venue de constrangimento em matéria de política criminal em um
primeiro momento; à abertura e tomada de grande posição em relação à
criminalidade de agentes públicos, conhecendo-se pouco sobre o exercício
do STF em matérias relativas à segurança pública em sentido amplo. Adensando uma agenda de pesquisa em curso sobre judiciário e políti-
cas públicas (MADEIRA; GELISKI, [s. d.]), interessa-nos, neste trabalho,
incluir os temas da segurança pública, de política criminal e penitenciária
dentre os temas sobre os quais o STF participa como ator dos processos
de políticas públicas (policy process) e de políticas judiciais (judicial policy
process). Para isso, buscamos dar visibilidade à sua produção criminalizante
e descriminalizante, à sua atuação nas diferentes fases do ciclo dessas po-
líticas públicas, ora abrindo agenda, ora como policymaker, legislador, ora
monitorando e avaliando programas, políticas e ações em curso, para além
de sua inerente função jurisdicional. 255 – 288 257 O STF e as políticas criminal, penitenciária e de segurança pública: um duplo ponto cego
nas agendas de pesquisa? | Lígia Mori Madeira, Leonardo Geliski O STF e as políticas criminal, penitenciária e de segurança pública: um duplo ponto cego
nas agendas de pesquisa? 4
As buscas foram realizadas nos resumos combinando “Supremo” ou “STF” e alguma das seguintes palavras-
-chave: carcerária, encarceramento, pena, penais, penal, penas, penitenciária, penitenciárias, penitenciário, pri-
são, prisional, prisões, polícia, policial, polícias, crime, crimes, criminais, criminal, criminalização, criminológico,
criminosa, descriminalização, segurança pública.
5
Aplicadas as mesmas palavras-chave ao inteiro teor dos trabalhos. 5
Aplicadas as mesmas palavras-chave ao inteiro teor dos trabalhos. 1. Introdução | Lígia Mori Madeira, Leonardo Geliski Essa atuação dos tribunais tanto a partir de decisões que intervêm
em demandas deslocadas da arena política para judicial (REID, 1987),
como de políticas e estruturas especializadas (BAUM, 2011) criadas pelos
próprios tribunais em sua organização administrativa (COHEN, 2002)
é denominada pela literatura como judicial policy. A judicial policy passa,
assim, a integrar a dinâmica do próprio policy-making, deslocando o centro
decisório para os tribunais, influenciando a tomada de decisão dos gover-
nos, que devem considerar a fase de litigância e seus efeitos sobre a política,
seja no desenho ou na sua implementação (CANON; JOHNSON, 1999;
SUNKIN, 2004). A partir de um levantamento bibliográfico sobre a relação do STF
com políticas criminais, penitenciárias e de segurança pública, este artigo
objetiva mapear temas, formas de intervenção da corte, principais teorias
e teses, indicando caminhos possíveis para o avanço da agenda de pesquisa
nessa área. Interessa-nos, além de identificar as abordagens já utilizadas, re-
fletir sobre seus limites para o estudo da atuação do tribunal em relação às
políticas criminais, à segurança pública e às prisões. Além das pesquisas di-
recionadas às ações diretas, pretendemos examinar como os pesquisadores
têm integrado em suas análises os julgamentos de habeas corpus, recursos
extraordinários e outras formas de atuação da corte em matéria criminal. O software Publish or Perish (HARZING, 2007) permitiu, através da
busca por palavras-chave4 no Google Acadêmico, localizar 1.250 resultados
iniciais relacionados ao STF e às políticas objeto deste artigo. Restringimos
a análise a artigos publicados em periódicos, dissertações e teses, em portu-
guês, e que estivessem disponíveis em meio eletrônico, excluídas também
discussões típicas da dogmática jurídica. Uma busca complementar no
Scielo e no Portal de Periódicos da Capes5 resultou nos 190 trabalhos que
foram efetivamente analisados. Os trabalhos foram, então, organizados por
proximidade temática e analisados identificando-se métodos de pesquisa, 255 – 288 255 – 288 258 Política & Sociedade - Florianópolis - Vol. 21 - Nº 52 - Set./Dez. de 2022 forma de atuação do STF, sua inserção em fases do ciclo das políticas pú-
blicas, e menção a decisões emblemáticas6. 1. Introdução O artigo apresenta a seguinte estrutura: após a introdução, a próxima
seção apresenta um balanço da produção sobre STF, violência, crime, se-
gurança pública e prisões no Brasil, identificando as lacunas na intercessão
das duas áreas nessa literatura; a terceira seção apresenta o resultado da re-
visão bibliográfica realizada, distribuída em cinco eixos principais de aná-
lise (STF, segurança pública, criminalização, punição e encarceramento,
prerrogativas e garantias processuais); por fim, há a seção analítica. 6
A base de dados STF na Segurança Pública está disponível para consulta no Harvard Dataverse. 2. O visto e o não visto: os balanços das pesquisas
sobre crime, violência, segurança pública e
sobre o Supremo Tribunal Federal Em sua apresentação sobre o estado da arte das pesquisas sobre o STF,
Arantes e Arguelhes (2019) relatam que os estudos pioneiros sobre o tri-
bunal na década de 1990 estavam orientados a partir do conceito de “judi-
cialização da política”, focados em entender que tipo de questões e ações o
Supremo decidia e quem as levava ao tribunal. Koerner e Inatomi (2013)
apontam que as pesquisas iniciais se voltaram à crítica do modelo insti-
tucional, no qual tribunais teriam amplos poderes de controle, mas não
existiriam mecanismos de responsabilização de seus juízes. Posteriormente, as pesquisas passaram a se concentrar nos impactos
da atuação do STF sobre a política, no funcionamento interno da corte,
nas regras e nos atores do processo decisório (ARANTES; ARGUELHES,
2019). Entram em foco os papéis políticos dos tribunais, sua relevância
para as políticas públicas, bem como a seletividade e heterogeneidade em
seus julgamentos (KOERNER; INATOMI, 2013). Na sequência, a agen-
da de pesquisa teria se deslocado para o comportamento individual dos
ministros, buscando explicar as variações e os padrões de votação que apre-
sentavam. Desenvolveu-se, também, uma agenda crítica sobre o modo de
deliberação dos ministros e seu comportamento na produção de decisões
no Plenário e nas Turmas (ARANTES; ARGUELHES, 2019). 255 – 288 259 O STF e as políticas criminal, penitenciária e de segurança pública: um duplo ponto cego
nas agendas de pesquisa? | Lígia Mori Madeira, Leonardo Geliski O STF e as políticas criminal, penitenciária e de segurança pública: um duplo ponto cego
nas agendas de pesquisa? | Lígia Mori Madeira, Leonardo Geliski Entre os aspectos do funcionamento do tribunal já mapeados pela li-
teratura, estariam os demandantes mais frequentes, seus graus de sucesso e
fracasso em seus acionamentos, bem como as estratégias empregadas pelo
STF para minimizar as tensões democráticas decorrentes de suas decisões,
como a abertura para a participação de amicus curiae e de audiências pú-
blicas, embora com baixo impacto em seu processo decisório. Outras aná-
lises voltaram-se ao processo de indicação de ministros e se suas trajetórias
profissionais prévias e o presidente que os indicou teriam peso em suas
decisões após serem empossados (ARANTES; ARGUELHES, 2019), dei-
xando como questão em aberto na agenda de pesquisa sobre o tribunal as
motivações dos votos dos ministros, especialmente o modo como fatores
externos influenciam na decisão. 2. O visto e o não visto: os balanços das pesquisas
sobre crime, violência, segurança pública e
sobre o Supremo Tribunal Federal As análises sobre acesso ao STF e sobre o impacto da Corte exibem
conclusões relativamente claras, corroboradas por um conjunto significa-
tivo de pesquisas. As discussões sobre sua construção institucional, espe-
cialmente sobre as causas das mudanças institucionais ocorridas na Corte
desde 1988, e sobre o comportamento decisório individual dos Ministros
do STF, avançaram de forma mais lenta, constituindo agendas de pesquisa
promissoras sobre o tribunal (DA ROS, 2017). Além disso, as pesquisas
citadas nesses balanços referem de forma global a atuação do STF e de seus
ministros, sem focar em questões relativas a temas específicos que foram
submetidos ao tribunal. O debate sobre o papel do STF na efetivação de
direitos, na judicialização da política e das políticas públicas ainda não
parece ter identificado as políticas criminal, penitenciária e de segurança
pública como um objeto relevante de análise. Apesar disso, é certo que tais políticas estão na agenda do Supremo
Tribunal Federal há, pelo menos, duas décadas. Oliveira (2013) identi-
ficou que assuntos como o uso de algemas pela polícia, a atribuição do
Ministério Público para realizar investigações criminais, ou o cumpri-
mento de penas antes do trânsito em julgado da condenação, ingressam
na pauta do tribunal desde 2002. Questões relacionadas à administração
da justiça e à segurança pública se fazem presentes nas ações diretas de
inconstitucionalidade, embora possuam uma participação menor do que
assuntos relativos a agentes públicos (OLIVEIRA, 2016). Ainda assim, 255 – 288 260 Política & Sociedade - Florianópolis - Vol. 21 - Nº 52 - Set./Dez. de 2022 discussões sobre organização de carreiras e prerrogativas de agentes públi-
cos podem, ainda que indiretamente, ter impactos na segurança pública,
especialmente se relacionadas ao ciclo policial e ao ingresso nas carreiras
policiais, questões presentes nos debates sobre modernização das polí-
cias no Brasil (AZEVEDO; NASCIMENTO, 2016; BEATO FILHO;
RIBEIRO, 2016). No total de processos iniciados no STF desde 1988, aqueles com te-
mas criminais equivalem a apenas 7,3% do acervo; já, entre 2013 e 2017,
a concentração de processos penais mais que dobrou, correspondendo a
14,8% do universo de processos iniciados. A proporção de processos cri-
minais em relação ao total apresenta um crescimento consistente a partir
de 2002 e, em 2017, um a cada cinco processos iniciados no STF discutia
algum tema criminal, e 56,7% das decisões colegiadas do tribunal diziam
respeito a temas criminais (FALCÃO et al., 2019). 2. O visto e o não visto: os balanços das pesquisas
sobre crime, violência, segurança pública e
sobre o Supremo Tribunal Federal A crescente impor-
tância de temas criminais no Supremo Tribunal Federal, contudo, não se
refletiu nas pesquisas realizadas, tampouco foi suficiente para colocá-lo nas
agendas de pesquisa do campo de estudos sobre violência, crime, segurança
pública e punição. Balanços da literatura sobre violência, crime e segurança pública no
Brasil produzida a partir dos anos 2000 evidenciam a existência de eixos
consolidados nos quais pesquisas sobre o STF e seu papel nas políticas
criminal, penitenciária e de segurança pública poderiam ser encontradas. Nesse sentido, por exemplo, são estudos sobre o funcionamento dos tribu-
nais e da justiça penal, sobre as relações entre acusadores e acusados, bem
como sobre os próprios mecanismos de punição em sua reconstrução his-
tórico-institucional (BARREIRA; ADORNO, 2010). Temas como os sen-
tidos da punição, o encarceramento em massa, o recrudescimento penal no
Brasil, as continuidades e descontinuidades na penalidade contemporânea
seriam promissores em considerar o papel da Suprema Corte. Também os
estudos sobre reforma da justiça criminal, formas alternativas, tempo e flu-
xo da justiça, inquérito policial e sistemas de justiça criminal (CAMPOS;
ALVAREZ, 2016) seriam campo fértil para a inserção do papel do STF. Entre as vertentes de pesquisa sobre a política criminal, seria esperado
encontrar análises da atuação do Supremo Tribunal Federal nas discussões 255 – 288 261 O STF e as políticas criminal, penitenciária e de segurança pública: um duplo ponto cego
nas agendas de pesquisa? | Lígia Mori Madeira, Leonardo Geliski O STF e as políticas criminal, penitenciária e de segurança pública: um duplo ponto cego
nas agendas de pesquisa? | Lígia Mori Madeira, Leonardo Geliski O STF e as políticas criminal, penitenciária e de segurança pública: um duplo ponto cego
nas agendas de pesquisa? | Lígia Mori Madeira, Leonardo Geliski sobre o impacto das escolhas legislativas no funcionamento do Poder
Judiciário na área penal, as mudanças institucionais e os padrões de tomada
de decisão provocados por mudanças legais. No conjunto de pesquisas di-
recionadas a operadores judiciais e instituições, analisando a configuração
das instituições do sistema penal, as concepções e representações sociais de
seus operadores, bem como seus relacionamentos, fricções e disputas que
estabelecem (AZEVEDO; SINHORETO, 2018), o STF também poderia
ser objeto de interesse. 2. O visto e o não visto: os balanços das pesquisas
sobre crime, violência, segurança pública e
sobre o Supremo Tribunal Federal Os estudos identificados nesses balanços de pesquisas sobre crime, vio-
lência e segurança pública no Brasil, quando se voltam para a análise dos
atores da justiça criminal e suas decisões, se direcionam para aqueles que
atuam em juizados especiais criminais, varas criminais e nos tribunais de
justiça, fenômeno igualmente encontrado nos balanços sobre judiciário e
políticas sociais e seu deslocamento aos tribunais intermediários e justiça
local. Apenas dois estudos citados por Azevedo e Sinhoreto (2018) abor-
daram o STF: a tese de Diógenes (2017), que discutiu as definições sobre
crimes militares a partir da análise de mais de 2.000 acórdãos do STF e do
STJ, e o artigo de Kant de Lima e Mouzinho (2016), que analisou a dinâ-
mica das ações penais relacionadas ao Mensalão e à Operação Lava Jato. A ausência de pesquisas que abordem os impactos da atuação do Poder
Judiciário em geral, e do Supremo Tribunal Federal em particular, sobre
as políticas criminal, penitenciária e de segurança pública é, no mínimo,
curiosa, considerando, por exemplo, o papel do tribunal na manuten-
ção e posterior afastamento parcial da Lei dos Crimes Hediondos (Lei nº
8.072/1990). Tal lei é apontada em inúmeros estudos como representati-
va do processo de endurecimento penal dos anos 1990 que resultou nos
altos níveis de encarceramento nas décadas seguintes (AZEVEDO 2004;
AZEVEDO; CIFALI, 2017; CAMPOS, 2010; PINTO, 2006; TEIXEIRA,
2007), preocupação já apontada por Da Ros e Taylor (no prelo) quanto a
uma promissora direção para pesquisas sobre comportamento decisório,
explorando diferentes ideologias penais (e.g. “garantismo”, “punitivismo”)
e seu impacto sobre votações e decisões dos ministros. No balanço das pesquisas sobre as prisões no Brasil, Salla (2006) afir-
ma que um dos desafios seria o de produzir análises sobre a ação estatal na 255 – 288 262 Política & Sociedade - Florianópolis - Vol. 21 - Nº 52 - Set./Dez. de 2022 área prisional articulada às demais políticas públicas no país, definindo seus
contornos e suas especificidades quanto à formulação, à implementação e
ao impacto. Destacando que a ciência política foi uma das áreas com me-
nor concentração de estudos sobre as prisões, Lourenço e Alvarez (2018)
reiteram a constatação de Salla (2006) de que as políticas prisionais ainda
não haviam sido incorporadas como um objeto de estudo à semelhança de
outras áreas de atuação do Estado, o que ajuda a entender a ausência da
Suprema Corte nesses trabalhos. 3. Segurança pública, punição e encarceram
criminalização: novos eixos de análise da
literatura sobre o STF Seguindo a metodologia explicitada na introdução, o mapeamento da li-
teratura permitiu a classificação dos trabalhos em cinco eixos principais. Embora a busca não tenha sido direcionada a uma área específica, os resul-
tados, na sua maioria, são produções do campo do Direito, destacando-se
como características gerais a análise dogmática e a descrição sobre casos
e decisões paradigmáticas, geralmente discutindo-se sua compatibilidade
com princípios constitucionais. Poucos trabalhos extrapolam esse modelo,
quando o fazem, incluem pesquisa bibliográfica à análise de decisões, des-
crição de trajetória jurisprudencial, análise crítica do discurso. O quadro abaixo apresenta as informações gerais oriundas da coleta e
sistematização desses trabalhos. 255 – 288 263 O STF e as políticas criminal, penitenciária e de segurança pública: um duplo ponto cego
nas agendas de pesquisa? | Lígia Mori Madeira, Leonardo Geliski p
, p
g
ç p
p
p
g
nas agendas de pesquisa? | Lígia Mori Madeira, Leonardo Geliski
Quadro 1 – Eixos e casos de atuação do
Supremo Tribunal Federal em matéria criminal
Eixo/subtema
Nº de
trabalhos (*)
Casos mais citados
Atuação do STF
Atores, discursos
e comportamento
judicial
Combate à Corrupção
Temas variados
9
12
AP 470, AC 937, AC 973
HC 152.752 e outras decisões
—
Atuação clássica
Segurança Pública
Aspectos gerais
Poder de Investigação
do MP
Greve servidores
Temas variados
1
2
1
5
ADI 236, ADI 1.182, ADI
2.827
RE 205.473; RHC 81.326;
RE 535.478; HC 94.173; HC
91.613
ARE 654.432
—
Atuação clássica
Lawmaker
Atuação clássica
Criminalização
Criminalização
(antissemitismo,
homofobia e outros)
Descriminalização
(aborto e porte de
drogas)
Temas variados
20
20
20
HC 82.424
MI 4.733 e ADO 26
ADI 3.510, ADPF 442, ADPF
54, HC 124.306, ADI 5.581,
RE 635.659
ADPF 187, AP 470, HC
81.611, HC 84.412
Lawmaker
Lawmaker
Atuação clássica Quadro 1 – Eixos e casos de atuação do
Supremo Tribunal Federal em matéria criminal 255 – 288 255 – 288 264 Política & Sociedade - Florianópolis - Vol. 21 - Nº 52 - Set./Dez. Fonte: elaborado pelos autores. Fonte: elaborado pelos autores. Nota: (*) O número de trabalhos pode repetir em algumas menções dos casos mais citados. 7
Importante informar que nem todos os trabalhos referidos no Quadro 1 estão sendo referenciados nos eixos,
podendo também haver citação repetida de alguns trabalhos, por pertencerem a discussões relativas a mais de
um eixo. Priorizou-se a descrição de subtemas que concentram o maior número de trabalhos em cada eixo. 3. Segurança pública, punição e encarceram
criminalização: novos eixos de análise da
literatura sobre o STF de 2022 Eixo/subtema
Nº de
trabalhos (*)
Casos mais citados
Atuação do STF
Punição e
encarceramento
Execução provisória ou
antecipada das penas
Política penitenciária
e condições de
encarceramento
Prisão domiciliar
Prisão preventiva
Crimes contra a
humanidade
Temas variados
27
15
10
6
4
8
ADC 43, ADC 44, ADC 54,
HC 84.078, HC 126.292, HC
152.752
ADI 5.170, ADPF 347, RE
580.252 RE 641.320
HC 143.641
HC 71.361, Rcl 2.391, RHC
83.179, HC 85.237, ADI
3.112
ADPF 153
—
Atuação clássica e
lawmaker
Avaliação e
implementação
Política judicial
Atuação clássica
Atuação clássica
Atuação clássica
Prerrogativas e
Garantias Processuais
Duplo grau de jurisdição,
foro privilegiado
Foro por prerrogativa de
função
Perda de Mandato
Parlamentar
Temas variados
7
14
5
10
AP 470
AP 396, AP 937
AP 565, AP 4.039
—
Atuação clássica
Atuação clássica
Atuação clássica
Atuação clássica
Nota: (*) O número de trabalhos pode repetir em algumas menções dos casos mais citados. Fonte: elaborado pelos autores. Nota: (*) O número de trabalhos pode repetir em algumas menções dos casos mais citados. Nota: (*) O número de trabalhos pode repetir em algumas menções dos casos mais citados. 265 255 – 288 O STF e as políticas criminal, penitenciária e de segurança pública: um duplo ponto cego
nas agendas de pesquisa? | Lígia Mori Madeira, Leonardo Geliski O STF e as políticas criminal, penitenciária e de segurança pública: um duplo ponto cego
nas agendas de pesquisa? | Lígia Mori Madeira, Leonardo Geliski Apesar das diferentes acepções do termo (SILVA, 2022), nesta revisão
a categoria de judicialização mobilizada nos trabalhos mapeados restringe-
-se ao ingresso de ações e procedimentos judiciais nos tribunais. Já a judi-
cialização de políticas públicas é entendida como “[...] a crescente utiliza-
ção do sistema de justiça [...] para o questionamento de falhas ou omissões
na produção de políticas públicas (policies) por parte do Executivo, ou
inação ou falhas do Legislativo no que tange à produção de normas legais”
(OLIVEIRA, 2019, p. 18). A seguir, descrevemos cada um dos eixos7. 3.1 Atores, discursos e comportamento judicial A literatura deste eixo temático enfoca a atuação da corte de maneira gene-
ralizada, englobando diferentes objetos de pesquisa, da análise dos proces-
sos decisórios do STF em matéria criminal a estudos de caso como a Ação
Penal 470, conhecida com o mensalão, até a mobilização de categorias
próprias da ciência política, como judicialização da política, ativismo e
comportamento judicial. Cabe pontuar dois marcos importantes na produção sobre o Supremo
Tribunal Federal. O primeiro marco é a utilização dos dados consolidados do projeto
“Supremo em Números” da Fundação Getúlio Vargas que produziu di-
ferentes relatórios sobre a atuação da corte brasileira, em especial, o “V
Relatório Supremo em Números: o foro privilegiado” (FALCÃO et al.,
2017) e o “VI Relatório Supremo em Números: a realidade do Supremo
Criminal” (FALCÃO et al., 2019), utilizados tanto para a contextualização
das análises jurisprudenciais como para o emprego de descrições mais de-
talhadas sobre a atuação do tribunal. Tais trabalhos são mobilizados tanto
pelos estudos contemplados neste eixo como pelos demais. O segundo marco é a alcunha do termo “Supremo Tribunal Criminal”
ou “Supremo Criminal” (FALCÃO et al., 2017; FALCÃO et al., 2019),
que ilustra um outro olhar dado ao tribunal, outrora marcado pela atuação 255 – 288 266 Política & Sociedade - Florianópolis - Vol. 21 - Nº 52 - Set./Dez. de 2022 como ator de governança, do campo de mobilização legal de políticas sociais,
no início dos anos 2000, chegando ao STF do combate à corrupção e dos
megaprocessos criminais no fim década de 2010, do Mensalão à Operação
Lava-jato. As transformações do comportamento decisório do tribunal aos
longos dos anos, com a adoção de posturas mais restritivas do tribunal, al-
terando paradigmas consolidados da corte, em específico no julgamento de
habeas corpus e de ações penais envolvendo atores políticos apontam para
a bandeira do combate à corrupção adotada pelo tribunal e a preocupação
com a legitimidade de sua atuação perante a sociedade e a mídia. Os estudos sobre comportamento dos ministros ao julgar casos como
Mensalão ou inquéritos criminais de parlamentares e políticos (ARANTES;
MARTINS, 2022; SANTOS NETO, 2018) utilizam-se da literatura de
comportamento judicial e recrutamento de juízes, examinando se a tra-
jetória, a indicação política ou a ideologia influenciam em seus processos
de tomada de decisão. Há menção a técnicas estatísticas em trabalhos de
Vasconcellos et al. (2020) e Gomes Neto et al. 3.1 Atores, discursos e comportamento judicial (2021), que examinam o
padrão de concessão de habeas corpus pelo STF e a sua relação com as ca-
racterísticas dos demandantes desse instrumento. Os estudos que empregam análise do discurso ou de narrativa exa-
minam as decisões judiciais e os votos dos ministros, buscando associar o
conteúdo dos acórdãos com metodologias de interpretação e hermenêu-
tica jurídica; identificar o padrão discursivo dos termos utilizados pelo
tribunal; descrever os argumentos utilizados e a sua relação com o con-
texto de atuação do STF, seja no caso Mensalão (GUERRA; TARGINO;
OLIVEIRA, 2015) ou nos processos relacionados à Operação Lava-Jato
(SANTOS, 2019). No caso Mensalão, a análise de Borges (2017) examina a relação en-
tre os argumentos utilizados pelo tribunal com as posições publicadas na
imprensa, identificando a centralidade do papel da mídia na atuação do
STF, desde a adequação de ritos de publicização dos atos e da estruturação
da TV Justiça, ao tratamento dado aos réus. Na mesma linha, Fernandes
e Santana (2020) destacam os movimentos de polarização da atuação do
tribunal, dos elogios à Operação Lava-jato, ligados à ideia da “corrupção 255 – 288 267 O STF e as políticas criminal, penitenciária e de segurança pública: um duplo ponto cego
nas agendas de pesquisa? | Lígia Mori Madeira, Leonardo Geliski O STF e as políticas criminal, penitenciária e de segurança pública: um duplo ponto cego
nas agendas de pesquisa? | Lígia Mori Madeira, Leonardo Geliski enquanto tragédia brasileira” aos embates sobre legitimidade da atuação do
STF no Estado Democrático de Direito. No campo das análises de discurso e de narrativa, identifica-se que
em algumas decisões o tribunal evoca categorias externas aos objetos das
ações penais, especialmente em casos de corrupção, com recursos retóricos
para a negação ou concessão de recursos, a exemplo da aplicação de cate-
gorias sobre violência de gênero e sexual, equiparando a figura do corrupto
ao estuprador (VIEIRA; EFREM FILHO, 2020). Ou ainda, os conceitos
mobilizados pela corte, não advêm do próprio tribunal, mas de elementos
externos aos votos, como imprensa, estudos acadêmicos ou peças proces-
suais (FERREIRA, 2021). 3.1 Atores, discursos e comportamento judicial Apesar de não explicitarem o ciclo de políticas criminais ou da segu-
rança pública, os textos deste eixo sinalizam que a atuação do tribunal em
casos específicos molda o comportamento dos atores, a exemplo do que
veremos nos outros eixos com os movimentos pró-legalização do aborto,
criminalização da homofobia ou o embate entre legislativo e judiciário nos
casos de imunidade parlamentar. É interessante notar que a discussão so-
bre legitimidade retoma as preocupações presentes em outras análises, da
ideia de fortalecimento do papel do corte em matéria penal. Por fim, cabe
pontuar que as categorias relativas à política pública criminal e suas fases
não são tão latentes neste eixo, sendo mais importante aqui o processo de
tomada de decisão do que o reflexo da política judicial. 3.2 Segurança Pública Os estudos sobre a atuação do STF em torno do tema da segurança pública
compõem a menor parcela da produção encontrada neste ensaio (vide Qua-
dro 1). As análises desse eixo podem ser agrupadas em quatro temas centrais:
“segurança pública” enquanto pauta de judicialização do STF; legitimação
do poder de investigação do MP; decisão judicial e burocracias de segurança
pública; e, movimentos sociais e mobilização do direito. Nesta seção, há a
concentração na descrição do contexto da judicialização e do reflexo da de-
cisão judicial, enquanto produto de mobilização do direito ou política que
impacta no comportamento das burocracias, a exemplo das polícias. 255 – 288 268 Política & Sociedade - Florianópolis - Vol. 21 - Nº 52 - Set./Dez. de 2022 Ao longo do período democrático recente, as políticas de segurança
pública passaram por diversas transformações, como a descentralização e o
retorno do controle sobre as polícias voltando aos estados, depois de con-
centrada no Governo Federal durante a ditadura militar, e o redesenho de
atribuições dos atores do sistema de justiça criminal. Apesar das mudanças,
devido à dificuldade de promoção de uma agenda de reforma da política no
âmbito legislativo, características do período autoritário remanesceram, tais
como a militarização das polícias (GUERRA; MACHADO FILHO, 2018). O deslocamento da pauta de segurança pública para STF, com o con-
flito político transferido para a arena judicial (FEREJOHN, 2002), ini-
cia de forma tímida, com o tribunal concentrando suas discussões sobre
a interpretação do art. 144 da Constituição Federal. As discussões se li-
mitam à descrição superficial do dispositivo constitucional de modo que
abordam a segurança como problema central do poder executivo estadual,
sem adentrar precisamente no escopo da dinâmica política, sua implemen-
tação, execução e atores (MATOS, 2013), reiterando, em sede de controle
concentrado, a limitação da capacidade dos estados de redesenhar as ins-
tituições que integram as suas políticas de segurança, para além do texto
constitucional (GUERRA; MACHADO FILHO, 2018). Ainda no campo da judicialização, outro tópico presente são as ações
que buscam a responsabilização civil do Estado por omissão na adoção de
políticas de segurança pública (LEITE, 2017). De modo contrário ao da
interpretação do art. 3.2 Segurança Pública 144, está a atuação da corte legitimando mudanças
institucionais ou nos padrões de atuação dos atores do sistema de justiça,
como no caso do reconhecimento do poder de investigar do Ministério
Público (RODRIGUES; COSTA, 2015) ou mais recentemente, fruto da
articulação coletiva de movimentos sociais e diferentes instituições em res-
posta à política de segurança do Rio de Janeiro e à violência policial, a
ADPF das Favelas (OSMO; FANTI, 2021) nas restrições impostas pela
corte a operações policiais. Outros exemplos do papel do STF sobre as burocracias da área in-
cluem o debate sobre o reconhecimento do direito à greve aos servidores
públicos da segurança pública (JORGE, 2019) e sobre a capacidade de im-
plementação da Súmula Vinculante nº 11, conhecida como “súmula das 255 – 288 269 O STF e as políticas criminal, penitenciária e de segurança pública: um duplo ponto cego
nas agendas de pesquisa? | Lígia Mori Madeira, Leonardo Geliski O STF e as políticas criminal, penitenciária e de segurança pública: um duplo ponto cego
nas agendas de pesquisa? | Lígia Mori Madeira, Leonardo Geliski algemas”, nas suas práticas organizacionais (SANTOS, 2016). Seguindo a
linha de estudos sobre burocracia e judicialização, o sucesso da compliance
dessas decisões estaria ligado à permeabilidade da instituição e da existên-
cia prévia de estruturas que permitam a sua implementação, as estruturas
legais básicas (EPP, 1998). 3.3 Criminalização Conforme é esperado para um eixo de análise sobre criminalização, dois
terços dos trabalhos analisados referem-se a uma atuação do STF como
lawmaker, destacando-se discussões sobre a descriminalização do aborto e
do porte de drogas, e a criminalização da homofobia por crime de racismo. O debate orientador da maioria dos trabalhos do eixo gira em torno
do ativismo judicial e seus limites, ora defendendo-se em razão da inércia
do Poder Legislativo (como no caso da criminalização da homofobia), ora
apontando-se a necessidade de se prestar atenção aos limites na formação
de precedentes penais, dada a possível violação à separação de poderes,
legalidade, proibição de analogia in malam partem e independência judi-
cial (ARAÚJO; FIGUEIREDO, 2020). Outro fundamento teórico co-
mumente aplicado nesses trabalhos refere-se ao neoconstitucionalismo e
ao debate sobre o papel das cortes (REIS, 2020), apontando-se a tensão
entre a legitimidade democrática e o caráter contramajoritário das cortes
constitucionais (OLIVEIRA, 2020). A defesa da descriminalização do porte de drogas (FIGUEIREDO;
QUADROS, 2020, p. 29), por exemplo, vem amparada por razões prag-
máticas (fracasso da política de drogas e as consequências do superencar-
ceramento) e jurídicas (o direito à privacidade, à autonomia individual, e a
desproporcionalidade da punição), da mesma forma em que se opõe duas
ordens de discursos, de resistência e de dominação, a partir de uma leitura
do realismo marginal (FARIA, 2018). Na mesma linha, os trabalhos que dis-
cutem o uso do princípio da insignificância no tráfico, também apontando
a necessidade de descongestionamento do sistema penal e a descarcerização. A defesa da descriminalização do porte de drogas e da aplicação do
princípio da insignificância no tráfico (FIGUEIREDO; QUADROS,
2020; MUNERATI, 2021; FARIA, 2018) vem amparados em razões 255 – 288 270 Política & Sociedade - Florianópolis - Vol. 21 - Nº 52 - Set./Dez. de 2022 pragmáticas, como o fracasso da política de drogas, as consequências do
superencarceramento e a necessidade de descongestionamento do sistema
penal. Merece referência o trabalho de Garau e Pereira (2020) cuja descri-
ção de casos em que não houve a aplicação do princípio ao crime de roubo,
a despeito dos produtos do crime (sabonete íntimo, chinelo, bombons)
revela tendências de política criminal e penitenciária do STF. Já os trabalhos que sustentam posição favorável à criminalização, como
nos casos de homofobia em equiparação ao racismo, problematizam as crí-
ticas à demanda criminalizadora por parte de movimentos sociais. 3.3 Criminalização A partir
de uma perspectiva da criminologia queer, defendem uma política criminal
das classes subalternas, cuja dimensão positiva da criminalização, assim
como na Lei Maria da Penha, resultaria em efeitos secundários importantes
para as políticas públicas, dentre eles a produção de dados específicos sobre
violência dessa população (RIOS; MELLO, 2020). Trabalhos relativos tanto ao aborto quanto ao crime de estupro, mas
também em relação à descriminalização do porte de drogas, amparam-se
na epistemologia feminista para problematizar o foco da atuação da corte
em termos do comportamento moral e sexual de réus e vítimas, demons-
trando a perpetuação dos papeis de gênero e do sexismo/machismo do
sistema de justiça criminal (PAIVA; SABADELL, 2018). No último caso,
as ambiguidades jurídicas entre as categorias de traficantes e usuários re-
forçam categorias de gênero e sexualidade e de lugares familiares legítimos
nos votos dos ministros, a partir de leituras de Veena Das e Judith Butler
(MELLO; SOUZA, 2020). Voltando às áreas de comparative judicial politics citadas por Da Ros e
Ingram (2018), a produção em criminalização concentra-se em comporta-
mento decisório, sem, contudo, realmente enfrentar a preocupação quanto
à existência de relação entre a trajetória dos ministros e os posicionamentos
que adotam em questões relacionadas às políticas criminal, penitenciária
e de segurança pública, aspecto importante trazido por Arantes e Martins
(2022) para o avanço das pesquisas na área. As áreas de acesso e construção
institucional são ainda menos exploradas. Em relação ao acesso, os trabalhos permitem identificar quem provoca
o Supremo em cada um dos casos paradigmáticos. Revelando-se embates 255 – 288 271 O STF e as políticas criminal, penitenciária e de segurança pública: um duplo ponto cego
nas agendas de pesquisa? | Lígia Mori Madeira, Leonardo Geliski O STF e as políticas criminal, penitenciária e de segurança pública: um duplo ponto cego
nas agendas de pesquisa? 3.3 Criminalização | Lígia Mori Madeira, Leonardo Geliski muito conhecidos, por exemplo, nos casos de julgamento do aborto, entre
ativistas do movimento feminista e de defesa das mulheres versus enti-
dades religiosas, entidades jurídicas religiosas, partidos políticos cristãos
(MARTINS; BONISSONI, 2018; LINS, 2020); ou entre entidades da
sociedade civil e academia versus entidades ligadas a instituições policiais,
no caso da descriminalização do porte de drogas (MUNERATI, 2021),
em embate tanto em audiências públicas ou na representação como amicus
curiae, trabalhos apontam os desdobramentos político-legislativos das deci-
sões pró-aborto presentes na ADPF 442 (STEINMETZ; RECKZIEGEL,
2017), ou na criminalização da homofobia (MANEIRO; CRUZ, 2016). Através das lentes do constitucionalismo democrático (POST;
SIEGEL, 2007), aponta-se tanto a potencialidade de que movimentos
sociais participem da atualização de sentidos e valores constitucionais,
quanto o movimento de efeito contrário, que pode ser compreendido tan-
to pelas lentes do backlash, “[...] reação conservadora contra o avanço do
protagonismo judicial, resultando em retrocesso nos direitos de minorias”
(ANDRÉA, 2019). Exceção nessa amostra, destacamos trabalhos que exploram referen-
ciais teóricos do litígio estratégico e os repertórios de ação coletiva, consi-
derando o espaço judicial como arena de deliberação política (MANEIRO;
CRUZ, 2016; MELLO; SOUZA, 2020). No caso do aborto, a partir de
entrevistas em profundidade com atores-chave do processo de mobilização
judicial e através das análises das decisões sobre a matéria Ruibal (2020, p. 1169) mostra [...] como o STF se converteu numa instituição que permitiu avanços legais e uma ampla
discussão pública sobre o direito ao aborto nas últimas duas décadas no país, no contexto de
um persistente bloqueio do processo político para o tratamento deste tema, dada a crescente
ascensão de grupos religiosos conservadores nos órgãos políticos do Estado. 3.4 Punição e encarceramento As análises encontradas sobre a atuação do STF em relação ao encarce-
ramento e aos sentidos da punição na contemporaneidade podem ser
organizadas em torno de cinco temas principais: política penitenciária e
condições de encarceramento, prisão domiciliar, prisões cautelares, exe- 255 – 288 272 Política & Sociedade - Florianópolis - Vol. 21 - Nº 52 - Set./Dez. de 2022 cução provisória ou antecipada da pena, e crimes contra a humanidade. Um pequeno número de trabalhos se distribui em temas variados, como
crimes hediondos, medidas de segurança e a Lei da Reforma Psiquiátrica,
entre outros. Além de sua atuação clássica na interpretação das normas constitucio-
nais, o Supremo também aparece nesses trabalhos como lawmaker, como
elaborador de políticas para o Poder Judiciário, bem como na implementa-
ção e avaliação de políticas penais e penitenciárias. Enquanto os trabalhos
sobre prisões cautelares, execução antecipada da pena e crimes contra a
humanidade focam nos argumentos dos Ministros, analisando os entendi-
mentos do tribunal e suas mudanças, as abordagens em torno das prisões
domiciliares, das políticas penitenciárias e das condições de encarceramen-
to questionam os impactos das decisões da Corte, tanto no sistema prisio-
nal quanto no próprio Poder Judiciário. A maior parte dos trabalhos sobre execução das penas antes do trânsito
em julgado se caracteriza como uma crítica jurídica às decisões do STF,
ora analisando a trajetória da jurisprudência, ora apenas as decisões mais
recentes, como Portal e Gloeckner (2017). Embora em menor número, há
trabalhos que buscam explorar as relações entre os diferentes julgamentos
do STF sobre a execução da pena antes do trânsito em julgado, bem como
destes com fatores externos aos julgamentos. Enquanto alguns focam nos
argumentos utilizados a cada mudança de entendimento e suas relações
com os julgamentos anteriores, outros agregam às suas análises a compo-
sição da corte a cada decisão na busca de relações. Rodrigues e Arantes
(2020), além dos argumentos, examinam o modo como se operou o po-
der de agenda da Corte e os possíveis efeitos da decisão sobre os rumos
da operação Lava Jato, identificando uma atuação estratégica do tribunal
para remodelar o arranjo institucional da justiça criminal. Hartmann et al. (2018), uma exceção nesse tema, buscaram estimar o impacto que o início
da execução das penas na pendência de recursos no STJ e no STF teria para
o sistema prisional. 3.4 Punição e encarceramento Em relação à prisão preventiva, os primeiros trabalhos focaram em
decisões emblemáticas, enfatizando a atuação do STF na construção ju-
risprudencial de limites para a imposição e manutenção dessa medida 255 – 288 273 O STF e as políticas criminal, penitenciária e de segurança pública: um duplo ponto cego
nas agendas de pesquisa? | Lígia Mori Madeira, Leonardo Geliski O STF e as políticas criminal, penitenciária e de segurança pública: um duplo ponto cego
nas agendas de pesquisa? | Lígia Mori Madeira, Leonardo Geliski (ESTELLITA, 2009). Pesquisas posteriores, a partir de amostras de de-
cisões dos tribunais superiores, constataram que tais limites eram pouco
aplicados, inclusive pela própria Corte, prevalecendo o uso de fundamen-
tações vagas (RYU, 2019). As incongruências do tribunal ao não aplicar
seus próprios entendimentos também foram exploradas pelos trabalhos que
discutem o tratamento dado a crimes contra a humanidade, comparando
a interpretação dada pela Corte à Lei de Anistia e aos crimes políticos na
ADPF 153 com as decisões proferidas em casos de extradição envolvendo
crimes semelhantes (BARBOSA, 2021). (ESTELLITA, 2009). Pesquisas posteriores, a partir de amostras de de-
cisões dos tribunais superiores, constataram que tais limites eram pouco
aplicados, inclusive pela própria Corte, prevalecendo o uso de fundamen-
tações vagas (RYU, 2019). As incongruências do tribunal ao não aplicar
seus próprios entendimentos também foram exploradas pelos trabalhos que
discutem o tratamento dado a crimes contra a humanidade, comparando
a interpretação dada pela Corte à Lei de Anistia e aos crimes políticos na
ADPF 153 com as decisões proferidas em casos de extradição envolvendo
crimes semelhantes (BARBOSA, 2021). No que se refere às condições de encarceramento e às políticas peni-
tenciárias, algumas análises se diferenciam por incorporarem, em maior ou
menor medida, referenciais do campo de políticas públicas em suas discus-
sões. Enquanto alguns trabalhos buscaram examinar as repercussões da de-
cisão da medida cautelar na ADPF 347 no ciclo de políticas públicas para
as prisões e no sistema prisional como um todo (CAMPOS; DANTAS,
2020; CARMONA, 2019; LIMA, 2020), outros, como Machado e Santos
(2018), comparam essa e outras decisões da corte para o sistema prisional
com as políticas públicas formalizadas para a área. As pesquisas sobre a prisão domiciliar, por outro lado, focam princi-
palmente nas repercussões da decisão proferida pela 2ª Turma do STF no
HC coletivo 143.641 no próprio Poder Judiciário. 3.4 Punição e encarceramento Seja analisando amos-
tras de decisões posteriores do STF e do STJ, como Refosco e Wurster
(2019), seja de tribunais intermediários, como Silva (2020), os pesquisa-
dores constatam que o julgamento do HC coletivo não teve impacto nos
padrões de negativa de pedidos de substituição de prisões preventivas por
prisões domiciliares para mulheres gestantes e mães. 3.5 Garantias Processuais e Prerrogativas Diferentemente dos eixos anteriores que mobilizam os conceitos de se-
gurança pública ou dos processos de criminalização, o tema das garantias
processuais e prerrogativas se assenta em discussões próprias da dinâmica
processual brasileira e da interação entre os poderes legislativo e judiciário. Os assuntos centrais identificados são: dogmática penal e posicionamen-
to do tribunal; garantias, prerrogativas e privilégios no sistema de justiça; 255 – 288 274 Política & Sociedade - Florianópolis - Vol. 21 - Nº 52 - Set./Dez. de 2022 atuação do STF e dinâmicas da justiça criminal. Os estudos se concentram na atuação originária do Supremo em ma-
téria criminal no julgamento de ações envolvendo, em sua maioria, auto-
ridades públicas e políticos. A literatura aborda os institutos processuais a
partir de casos específicos envolvendo políticos, escândalos de corrupção
e crimes contra o sistema eleitoral, a exemplo da Ação Penal 470/MG,
conhecida como o caso Mensalão. O tema das garantias e prerrogativas processuais é retratado, em ca-
sos de combate à corrupção, em debates que questionam se a imunidade
parlamentar e o foro privilegiado agem como instrumentos produtores de
privilégios e desigualdades dentro do sistema de justiça. Para além do ma-
niqueísmo teórico sobre a produção ou não de uma seletividade punitivista
das autoridades públicas e políticas, há indícios de que os processos de
transformação da jurisprudência (FERREIRA, 2021) estariam associados
à dinâmica própria dos megaprocessos de corrupção política e eleitoral. Além dos elementos do processo decisório, o debate se estende para
a legitimação da STF frente aos outros poderes da república, refletindo
a construção da jurisprudência a partir da interação entre o congresso e
judiciário nos procedimentos de cassação de mandato político após sen-
tença condenatória e pedido de prisão de parlamentar (MALAN, 2014;
FAVETTI, 2021) ou para abertura de processo criminal contra governa-
dores estaduais (PÁDUA; PAULA, 2019). Sobre a dinâmica processual, a maioria dos trabalhos foca na evolução
da jurisprudência dentro do STF ou nas técnicas interpretativas aplicadas
na tomada de decisões em casos paradigmáticos como o Mensalão ou a pri-
são do senador Delcídio Amaral. O posicionamento majoritário da litera-
tura destaca a mudança do comportamento do STF ao julgar ações penais
originárias envolvendo escândalos de corrupção e atores políticos. 3.5 Garantias Processuais e Prerrogativas Sobre a
atuação originária do STF e a aplicação do duplo grau de jurisdição, parte
dos trabalhos questiona a atuação do STF ao delimitar os requisitos do
reconhecimento do foro por prerrogativa de função, atuando como law-
maker, ao mesmo tempo em que denuncia a mesma lógica de orientação
por casos e as frequentes alterações do posicionamento jurisprudencial. 255 – 288 275 O STF e as políticas criminal, penitenciária e de segurança pública: um duplo ponto cego
nas agendas de pesquisa? | Lígia Mori Madeira, Leonardo Geliski O STF e as políticas criminal, penitenciária e de segurança pública: um duplo ponto cego
nas agendas de pesquisa? | Lígia Mori Madeira, Leonardo Geliski Em síntese, os trabalhos dão indícios das consequências da atuação de-
cisória do tribunal focada em casos específicos para o sistema de justiça de
maneira ampla. Seja a jurisprudência produzida pós-mensalão, seja aque-
la no bojo da Operação Lava-Jato, com seus mecanismos de seletividade,
fluxo e dinâmicas prisionais, reforçam a ideia do embate entre os poderes
e da busca por legitimidade da corte na persecução criminal envolvendo
atores políticos. O foco em políticas criminais, de segurança e prisão avança por des-
crever um cenário de atuação da corte para além das ações de controle de
constitucionalidade e seu limitado rol de atores demandantes, o que preen-
che uma grande lacuna na tão questionada visão parcial promovida pelo
olhar apenas para parte do que o tribunal faz (RIBEIRO; ARGUELHES,
2019). Nesse sentido, avança-se para além das pesquisas sobre ações di-
retas, incluindo-se análises sobre habeas corpus e recursos extraordinários. Permite-se assim, avançar sobre as três personas de atuação do tribunal,
como Corte Constitucional, Corte Recursal e Corte Ordinária (FALCÃO
et al., 2019). 4 Considerações finais Apesar dos avanços em incluir os temas da segurança pública, de política
criminal e penitenciária dentre os temas sobre os quais o STF participa
como ator do policy process e do judicial policy process, entendendo-se sua
produção criminalizante e descriminalizante, são poucos os trabalhos que
realmente se debruçam sobre essa literatura reconhecida por judicial poli-
tics, embora muitos trabalhos façam uso dos conceitos de ativismo judicial,
de judicialização da política; não a partir dos autores de referência, mas de
citações em decisão e obras publicadas pelos próprios ministros do STF. Portanto, verificamos categorias teórico-analíticas transformadas em cate-
gorias nativas. Por serem oriundos da área do direito, os textos apresentam
leituras doutrinárias e jurisprudenciais sobre temas que mereceriam um
olhar aprofundado das conjunturas políticas. A abordagem de Rodrigues
e Arantes (2020) coloca em evidência como debates jurídicos se conectam
com disputas sobre políticas e poderia ser empregada em análises sobre
outros temas identificados nesse levantamento. 255 – 288 276 Política & Sociedade - Florianópolis - Vol. 21 - Nº 52 - Set./Dez. de 2022 Em relação às possibilidades e dificuldades empíricas e metodológicas
de desenvolver estudos sobre segurança pública e crime no STF, a área
avança sobre a análise de institutos pouco explorados em outros trabalhos
sobre o tribunal, deixando de lado a concentração em ações de controle
de constitucionalidade, passando a olhar para ações penais, habeas corpus,
recursos extraordinários, diversificando o olhar sobre o que a corte faz em
atuação originária e recursal. Prevalecem, todavia, os estudos descritivos
sobre decisões de grande repercussão e ainda são poucos os trabalhos ex-
plicativos ou a explorar um n maior de decisões, construindo bancos de
dados que permitam análises sobre os padrões de comportamento da cor-
te. Exceção é o esforço do Supremo em Números, da Fundação Getúlio
Vargas, em consolidar dados sobre a atuação do STF, permitindo uma aná-
lise sobre os movimentos pendulares de alteração jurisprudencial. Diante dessas constatações, é possível sugerir dimensões de atuação e
funcionamento da corte pouco exploradas pela literatura, lançando bases
para pesquisas futuras, com formulação de novas perguntas de pesquisa e
indicação de caminhos teórico-metodológicos, senão novos, de possível
transposição de análises de ciência política e de políticas públicas para a
promissora agenda de pesquisa sobre STF em políticas criminal, peniten-
ciária e de segurança pública. 4 Considerações finais A partir dos recortes das áreas de comparative judicial politcs – acesso,
comportamento decisório, impacto e construção institucional – podemos
questionar quais são os atores que, de fato, acessam o STF para discutir
questões relativas à política criminal, penitenciária e de segurança pública? Se há desigualdade no acesso ao tribunal, cabe questionar seus efeitos em
processos em que se discute um direito fundamental como a liberdade
e cujas consequências na demora das decisões são, não raramente, irre-
mediáveis. Este aspecto atrai a atenção para o duplo papel exercido pelo
Procurador-Geral da República perante a corte, ora como órgão de acu-
sação, ora como fiscal da ordem jurídica, opinando inclusive em recursos
interpostos por outros órgãos do Ministério Público brasileiro. Além disso,
a admissão de habeas corpus coletivos desde 2016 criou, pela via jurispru-
dencial, um novo acesso direto ao STF ainda não analisada na literatura. 255 – 288 277 O STF e as políticas criminal, penitenciária e de segurança pública: um duplo ponto cego
nas agendas de pesquisa? | Lígia Mori Madeira, Leonardo Geliski O STF e as políticas criminal, penitenciária e de segurança pública: um duplo ponto cego
nas agendas de pesquisa? | Lígia Mori Madeira, Leonardo Geliski O STF e as políticas criminal, penitenciária e de segurança pública: um duplo ponto cego
nas agendas de pesquisa? | Lígia Mori Madeira, Leonardo Geliski No que se refere ao comportamento decisório, considerando a forma-
ção atual da corte possuir dois ex-ministros da Justiça entre seus integran-
tes, caberia aprofundar as investigações sobre as relações entre a trajetória
profissional dos ministros e os posicionamentos que adotam em questões
relacionadas às políticas criminal, penitenciária e de segurança pública. Trabalhos descritos nos eixos do STF e da criminalização são promissores
em apontar essas relações que, sem dúvida, avançam na compreensão da
atuação da corte. Além disso, nos trabalhos sobre a atuação criminal, o
retrato sobre o STF é o de um bloco monolítico, trazendo poucas nuances
sobre a não unanimidade da corte e suas coalizões. 4 Considerações finais No entanto, a literatura
chama a atenção para a existência de diferentes Supremos, seja como ilhas ou
arquipélagos (ARGUELHES; RIBEIRO, 2018; GOMES NETO; LIMA,
2018), seja em seu papel de veto ou governança (FALCÃO; OLIVEIRA,
2013; OLIVEIRA, 2016; NUNES, 2010), seja, ainda, sobre como se dão
os processos de tomada de decisão pelos ministros da corte ou pelo órgão
colegiado (MARTINS, 2018; SILVA, 2018), abrindo-se importante pers-
pectiva de análise no âmbito do comportamento decisório criminal. Considerando a ampla exposição que alguns julgamentos recebem, mi-
nistros do STF, ou o Tribunal como um todo, também seriam pressionados
ou estimulados a serem duros contra o crime, como alguns atores do Poder
Executivo? Como compreender posturas duras em relação à corrupção, em
oposição a abrandamentos em relação a crimes tributários? Ou como se coa-
dunam posturas rígidas no julgamento do crime de furto e leves no enten-
dimento da descriminalização do porte de drogas? Que critérios o Supremo
utiliza para reconhecer ou não a existência de repercussão geral em recursos
extraordinários criminais em relação aos demais recursos extraordinários? Quais os efeitos da utilização de casos individuais, como ações penais e ha-
beas corpus, para formar precedentes com impactos coletivos? Em termos de impacto, para além da já tradicional discussão quanto
à interferência ou não da atuação da Suprema Corte na arena política,
incidindo sobre a separação de poderes e a legitimidade frente ao legisla-
tivo e executivo, merecem maiores análises as consequências das decisões
judiciais no comportamento das burocracias. Quais os efeitos dos entendi-
mentos da corte na atuação da polícia, do ministério Público, do próprio 255 – 288 278 Política & Sociedade - Florianópolis - Vol. 21 - Nº 52 - Set./Dez. de 2022 judiciário? Em termos de compliance, há diferenças nos padrões de cum-
primento pelos tribunais inferiores conforme se tratem de decisões que
afirmam ou ampliam direitos? O tema da construção e do desenvolvimento institucional, embora
seja uma das áreas mais desenvolvidas de análise sobre judicial politics,
pouco aparece, podendo-se explorar melhor como normas de organização
interna da corte influenciam em como as demandas judiciais criminais
são processadas. Ainda, avançar na compreensão das relações e dos efeitos
de conflitos entre os tribunais superiores ou entre o STF e seus órgãos de
controle, como o CNJ. 4 Considerações finais Para além desses recortes, seria muito interessante, na linha de ou-
tros trabalhos que procuraram analisar padrões de produção de política
criminal pelo Congresso Nacional, mapear essas tendências no Supremo
Tribunal Federal e suas transformações ao longo do tempo. Para isso, seria
necessário considerar em conjunto as decisões apresentadas nos diferentes
eixos. Além disso, pesquisas que analisassem se e em que medida fatores
externos ao caso concreto influenciam as chances de direitos e garantias
processuais penais serem reconhecidos pela Corte contribuiriam para tes-
tar teorias sobre as diferenças de tratamento e seletividade da justiça crimi-
nal no Brasil. Por fim, mas não menos relevante, considerando as transformações
na penalidade contemporânea que vêm sendo documentadas e analisadas
em diferentes contextos, há uma frente de pesquisas a ser explorada para
analisar, comparativamente, de que modo tais transformações estariam ou
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) N
d
CEBRAP
5 54 SILVA, J. M. Depois da “judicialização”: um mapa bibliográfico do Supremo. Revista de Sociologia
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penitenciário brasileiro. 2007. 00 f. Dissertação (Mestrado em Sociologia) – Programa de Pós-
Graduação em Sociologia, Universidade de São Paulo, São Paulo, 2007. VASCONCELLOS, V. G. et al. Habeas corpus concedidos pelo supremo tribunal federal em 2019:
pesquisa empírica e dados estatísticos. Revista Brasileira de Ciências Criminais, v. 172, p. 323-
352, 2020. VIEIRA, A. D.; EFREM FILHO, R. O rei está nu: gênero e sexualidade nas práticas e decisões no
STF. Revista Direito e Práxis, v. 11, p. 1084-1136, 2020. Recebido em 22/07/2022
Aceito em 13/10/2022
Versão final em 13/10/2022 Recebido em 22/07/2022
Aceito em 13/10/2022
Versão final em 13/10/2022 255 – 288 287 287 O STF e as políticas criminal, penitenciária e de segurança pública: um duplo ponto cego
nas agendas de pesquisa? | Lígia Mori Madeira, Leonardo Geliski Abstract This paper presents a literature review on the role of the Brazilian Federal Supreme Court concerning
criminal, penitentiary, and security policies, mapping themes, types of court intervention, and
leading theories and theses. From an initial bibliographic survey of 1250 texts using the Publish
or Perish software, 190 publications were selected for in-depth analysis. Five main thematic axes
were identified: “actors, discourses and judicial behavior,” “criminalization,” “public security,”
“punishment and incarceration,” and “procedural guarantees and prerogatives.” There was a
prevalence of productions in the legal area, with doctrinal and jurisprudential readings and
descriptive studies on decisions of great repercussion, with few works mobilizing as literature of
judicial policy. And studies on crime, violence, and public security, whose paths suggest advancing
this research agenda. Keywords: STF. Criminal. Penitentiary and public security policies. Bibliographic review. 255 – 288 288
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Tropheryma whipplei endocarditis in Spain
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Members of Spanish Collaboration on Endocarditis-Grupo de Apoyo al Manejo de la Endocarditis Infecciosa en España Group: Hospital Costa del Sol (Marbella):
Fernando Fernández Sánchez, Mariam Noureddine, Gabriel Rosas, Javier de la Torre Lima; Hospital Universitario de Cruces (Bilbao): José Aramendi, Elena Bereciartua,
María Victoria Boado, Marta Campaña Lázaro, Josune Goikoetxea, Juan José Goiti, José Luis Hernández, José Ramón Iruretagoyena, Josu Irurzun Zuazabal, Leire
López-Soria, Miguel Montejo, Pedro María Pérez, Regino Rodríguez, Roberto Voces; Hospital Universitario Virgen de la Victoria (Málaga): Mª Victoria García López,
Radka Ivanova Georgieva, Manuel Márquez Solero, Isabel Rodríguez Bailón, Josefa Ruiz Morales; Hospital Universitario Donostia-Policlínica Gipuzkoa (San Sebastián):
Ana María Cuende, Tomás Echeverría, Ana Fuerte, Eduardo Gaminde, Miguel Ángel Goenaga, Pedro Idígoras, José Antonio Iribarren, Alberto Izaguirre Yarza, Xabier
Kortajarena Urkola, Carlos Reviejo; Hospital General Universitario de Alicante (Alicante): Rafael Carrasco, Vicente Climent, Patricio Llamas, Esperanza Merino, Joaquín
Plazas, Sergio Reus; Complejo Hospitalario Universitario A Coruña (A Coruña): Nemesio Álvarez, José María Bravo-Ferrer, Laura Castelo, José Cuenca, Pedro Llinares,
Enrique Miguez Rey, María Rodríguez Mayo, Efrén Sánchez, Dolores Sousa Regueiro; Complejo Hospitalario de Especialidades Juan Ramón Jiménez (Huelva):
Francisco Javier Martínez; Hospital Universitario de Canarias (Canarias): Mª del Mar Alonso, Beatriz Castro, Dácil García Marrero, Mª del Carmen Durán, Mª Antonia
Miguel Gómez, Juan La Calzada, Ibrahim Nassar; Hospital Regional Universitario de Málaga (Málaga): Antonio Plata Ciezar, José Mª Reguera Iglesias; Hospital
Universitario Central Asturias (Oviedo): Víctor Asensi Álvarez, Carlos Costas, Jesus de la Hera, Jonnathan Fernández Suárez, Lisardo Iglesias Fraile, Víctor León
Arguero, José López Menéndez, Pilar Mencia Bajo, Carlos Morales, Alfonso Moreno Torrico, Carmen Palomo, Begoña Paya Martínez, Ángeles Rodríguez Esteban,
Raquel Rodríguez García, Mauricio Telenti Asensio; Hospital Clínic-IDIBAPS, Universidad de Barcelona (Barcelona): Manuel Almela, Juan Ambrosioni, Yolanda Armero,
Manuel Azqueta, Mercé Brunet, Ramón Cartañá, Carlos Falces, Guillermina Fita, David Fuster, Cristina García de la Maria, José M. Gatell, Jaume Llopis Pérez,
Christian Manzardo, Francesc Marco, José M. Miró, Asunción Moreno, Salvador Ninot, Eduardo Quintana, Carlos Paré, Daniel Pereda, Juan Manuel Pericás, José L.
Pomar, José Ramírez, Irene Rovira, Marta Sitges, Dolors Soy, Adrián Téllez, Bárbara Vidal, Jordi Vila; Hospital General Universitario Gregorio Marañón (Madrid): Javier
Bermejo, Emilio Bouza, Gregorio Cuerpo, Viviana de Egea, Alia Eworo, Ana Fernández Cruz, Mª Eugenia García Leoni, Marcela González del Vecchio, Víctor González
Ramallo, Martha Kestler Hernández, Mercedes Marín, Manuel Martínez-Sellés, Mª Cruz Menárguez, Patricia Muñoz, Cristina Rincón, Hugo Rodríguez-Abella, Marta
Rodríguez-Créixems, Blanca Pinilla, Ángel Pinto, Maricela Valerio, Eduardo Verde Moreno; Hospital Universitario La Paz (Madrid): Isabel Antorrena, Belén Loeches, Mar
Moreno, Ulises Ramírez, Verónica Rial Bastón, María Romero, Araceli Saldaña; Hospital Universitario Marqués de Valdecilla (Santander): Carlos Armiñanzas Castillo,
Ana Arnaiz, José Berrazueta, Sara Bellisco, Manuel Cobo Belaustegui, Raquel Durán, Mª Carmen Fariñas, Concepción Fariñas-Álvarez, Carlos Fernández Mazarrasa,
Rubén Gómez Izquierdo, Claudia González Rico, José Gutiérrez Díez, Rafael Martín Durán, Marcos Pajarón, José Antonio Parra, Ramón Teira, Jesus Zarauza; Hospital
Universitario Puerta de Hierro (Madrid): Pablo García Pavía, Jesus González, Beatriz Orden, Antonio Ramos, Elena Rodríguez González; Hospital Universitario Ramón y
Cajal (Madrid): Tomasa Centella, José Manuel Hermida, José Luis Moya, Pilar Martín-Dávila, Enrique Navas, Enrique Oliva, Alejandro del Río, Soledad Ruiz; Hospital
Universitario Virgen de las Nieves (Granada): Carmen Hidalgo Tenorio; Hospital Universitario Virgen Macarena (Sevilla): Antonio de Castro, Marina de Cueto, Pastora
Gallego, Juan Gálvez Acebal, Jesus Rodríguez Baño; Hospital Universitario Virgen del Rocío (Sevilla): Arístides de Alarcón, Emilio García, Juan Luis Haro, José Antonio
Lepe, Francisco López, Rafael Luque; Hospital San Pedro (Logroño): Luis Javier Alonso, Pedro Azcárate, José Manuel Azcona Gutiérrez, José Ramón Blanco, Lara
García-Álvarez, José Antonio Oteo, Mercedes Sanz; Hospital de la Santa Creu i Sant Pau (Barcelona): Natividad de Benito, Mercé Gurguí, Cristina Pacho, Roser
Pericas, Guillem Pons; Complejo Hospitalario Universitario de Santiago de Compostela (A Coruña): M. Álvarez, A.L. Fernández, Amparo Martínez, A. Prieto, Benito
Regueiro, E. Tijeira, Marino Vega; Hospital Santiago Apóstol (Vitoria): Andrés Canut Blasco, José Cordo Mollar, Juan Carlos Gainzarain Arana, Oscar García Uriarte,
Alejandro Martín López, Zuriñe Ortiz de Zárate, José Antonio Urturi Matos; Hospital SAS Línea de la Concepción (Cádiz): Mª Belén Nacle, Antonio Sánchez-Porto, Luis
Vallejo; Hospital Clínico Universitario Virgen de la Arrixaca (Murcia): José Mª Arribas Leal, Elisa García Vázquez, Alicia Hernández Torres, Ana Blázquez, Gonzalo de la
Morena Valenzuela; Hospital de Txagorritxu (Vitoria): Ángel Alonso, Javier Aramburu, Felicitas Elena Calvo, Anai Moreno Rodríguez, Paola Tarabini-Castellani; Hospital
Virgen de la Salud (Toledo): Eva Heredero Gálvez, Carolina Maicas Bellido, José Largo Pau, Mª Antonia Sepulveda, Pilar Toledano Sierra, Sadaf Zafar Iqbal-Mirza;
Hospital Rafael Méndez (Lorca-Murcia): Eva Cascales Alcolea, Pilar Egea Serrano, José Joaquín Hernández Roca; Hospital Universitario San Cecilio (Granada): Eduardo
Moreno Escobar, Alejandro Peña Monje, Valme Sánchez Cabrera, David Vinuesa García; Hospital Son Llátzer (Palma de Mallorca): Carmen Cifuentes Luna, Juana
Nuñez Morcillo, Mª Cruz Pérez Seco. Tropheryma whipplei endocarditis in Spain
Case reports of 17 prospective cases M Carmen Fariñas (MD, PhD) , Miguel Montejo (MD) , Josune Goikoetxea (MD) ,
Raquel Rodríguez García (MD)g, Arístides de Alarcón (MD)h, Manuel Almela (MD)i,
Nuria Fernández-Hidalgo (MD, PhD)j, María del Mar Alonso Socas (MD)k, Miguel Ángel Goenaga (MD)l, Nuria Fernández-Hidalgo (MD, PhD)j, María del Mar Alonso Socas (MD)k, Miguel Ángel Goenaga (MD)l, The authors have no funding and conflicts of interest to disclose. a Departamento de Enfermedades Infecciosas, Hospital San Pedro-Centro de Investigación Biomédica de La Rioja (CIBIR), b Departamento de Enfermedades
Infecciosas, Hospital San Pedro, Logroño, c Servicio de Microbiología Clínica y Enfermedades Infecciosas, Hospital General Universitario Gregorio Marañón, Instituto de
Investigación Sanitaria Gregorio Marañón, Madrid, d Servicio de Enfermedades Infecciosas, Hospital Universitario Marqués de Valdecilla, Universidad de Cantabria,
Santander, e Unidad de Enfermedades Infecciosas, Hospital Universitario de Cruces, Universidad del País Vasco, f Unidad de Enfermedades Infecciosas, Hospital
Universitario de Cruces, Bilbao, g Servicio de Medicina Intensiva, Hospital Universitario Central de Asturias, Universidad de Oviedo, Oviedo, h Unidad de Gestión Clínica
de Enfermedades Infecciosas, Microbiología y Medicina Preventiva, Hospital Universitario Virgen del Rocío, Sevilla, i Servicio de Microbiología y Parasitología, Hospital
Clinic de Barcelona, Barcelona, j Servicio de Enfermedades Infecciosas, Hospital Universitario Vall d’Hebron, Universidad Autónoma de Barcelona, Barcelona, k Servicio
de Enfermedades Infecciosas, Hospital Universitario de Canarias, Tenerife, l Servicio de Enfermedades Infecciosas, Hospital Universitario Donosti, San Sebastián,
m Servicio de Enfermedades Infecciosas, Hospital Universitario Ramón y Cajal, Madrid, n Servicio de Anatomía Patológica, Hospital Clínico Universitario Virgen de la
Victoria, Málaga, Spain. ∗Correspondence: José A. Oteo, Departamento de Enfermedades Infecciosas, Hospital San Pedro-CIBIR, C/Piqueras 98-7a NE, 26006 Logroño (La Rioja), Spain
(email: jaoteo@riojasalud.es) Copyright © 2016 the Author(s). Published by Wolters Kluwer Health, Inc. All rights reserved. Copyright © 2016 the Author(s). Published by Wolters Kluwer Health, Inc. All rights reserved. This is an open access article distributed under the Creative Commons Attribution License 4.0
any medium, provided the original work is properly cited. uthor(s). Published by Wolters Kluwer Health, Inc. All rights reserv Copyright © 2016 the Author(s). Published by Wolters Kluwer Health, Inc. All rights reserved. This is an open access article distributed under the Creative Commons Attribution License 4.0 (CCBY), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited. Medicine
® Medicine
® Medicine
® Observational Study OPEN 2.1. Patients’ recruitment All but 1 patients diagnosed of T. whipplei endocarditis were
recruited from a registry of the “Spanish Collaboration on
Endocarditis—Grupo de Apoyo al Manejo de la Endocarditis
infecciosa en España” (GAMES). In this registry, consecutive
patients with IE were included between January 1, 2008 and
December 31, 2014 in 25 Spanish hospitals. Multidisciplinary
teams prospectively completed a standardized case report form
containing epidemiological, clinical, biological (including main
hematological and biochemical values), and therapeutical data
for each patient. Regional and local ethics committees approved
the study (Comité Ético para la Investigación Clínica-Regional de
la Consejería de Sanidad de la Comunidad de Madrid, code: 18/
07; Date: January 11, 2008) and patients gave their informed
consent for entering the cohort.[3] Tropheryma whipplei, formerly Tropheryma whippelii, is
an intracellular gram-positive Actinobacteria ubiquitous in
the
environment
that
is
involved
in a
large
variety of
clinical forms.[7,8] First implication of T. whipplei as causative
agent of infective endocarditis was reported from Switzerland
in 1997, in a patient with BCNE using a broad-range
polymerase chain reaction (PCR) followed by sequencing.[9]
First
stable
cultivation
of
the
bacterium
of
Whipple
disease (WD) was carried out in 2000, from the mitral
valve of a patient with BCNE.[7] The knowledge of the genome
of T. whipplei has permitted the development of specific
and
sensible
tools
for
diagnosis
and
have
involved
this
microorganism
in
a
broad
spectrum
of
clinical
conditions.[10,11] A routinary protocol that includes serology to Coxiella
burnetii, Bartonella spp., Legionella spp., and Brucella spp. was completed in all patients with BCNE. When surgery was
performed, all hospitals had the opportunity to send the valves
to the referral centers located at Madrid, Catalonia and La Rioja
for molecular studies. Abstract Tropheryma whipplei endocarditis is an uncommon condition with very few series and <90 cases reported in the literature. The aim of
the study was to analyze the epidemiological, clinical, and outcome characteristics of 17 cases of T. whipplei endocarditis recruited in
our country from a multicentric cohort from 25 Spanish hospitals from the Spanish Collaboration on Endocarditis—Grupo de Apoyo
al Manejo de la Endocarditis infecciosa en España. j
p
From a total of 3165 cases included in the cohort, 14.2% were diagnosed of blood culture negative endocarditis (BCNE) and 3.5%
of these had T. whipplei endocarditis. This condition was more frequent in men. The average age was 60.3 years. Previous cardiac
condition was present in 35.3% of the cases. The main clinical manifestation was cardiac failure (76.5%) while fever was only present
in the 35.3%. Ecocardiography showed vegetations in 64.7% of patients. Surgery was performed in all but 1 cases and it allowed the
diagnosis when molecular assays were performed. A broad range rRNA 16S polymerase chain reaction was used for first instance in
all laboratories and different specific targets for T. whipplei were employed for confirmation. A concomitant Whipple disease was
diagnosed in 11.9% of patients. All patients received specific antimicrobial treatment for at least 1 year, with no relapse and complete
recovery. y
T. whipplei endocarditis is an uncommon condition with an atypical presentation that must be considered in the diagnosis of
BCNE. The prognosis is very good when an appropriate surgical management and antimicrobial-specific treatment is given. Abbreviations: BCNE = blood culture negative endocarditis, BID = “bis in die” (twice a day), CSF = cerebrospinal fluid, GAMES =
grupo de apoyo al manejo de la endocarditis infecciosa en España, IE = infectious endocarditis, IHC = immunohistochemistry, PAS =
periodic acid-Schiff, SXT = trimethoprim-sulfamethoxazole, TOE = transesophageal echocardiography, TTE = transthoracic
echocardiography, WD = Whipple disease. Keywords: blood culture negative endocarditis, infectious endocarditis, T. whipplei endocarditis, Tropheryma whipplei 2. Patients and methods Blood culture negative endocarditis (BCNE) is a relative frequent
condition among patients affected by infectious endocarditis (IE)
representing 5% to 30% in great series.[1–3] The main reasons for
this condition are the previous administration of antimicrobials
and fastidiously culture microorganisms. Anyway, in the past
decades, the application of certain tools as automated blood
cultures, molecular assays, immunohistochemistry (IHC), and
serology has improved the diagnosis of this condition and has
involved new agents.[4] These facts have been incorporated in
new guidelines.[5,6] Tropheryma whipplei endocarditis in Spain
Case reports of 17 prospective cases py g
( )
y
,
g
This is an open access article distributed under the Creative Commons Attribution License 4.0 (CCBY), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited. py g
( )
y
g
This is an open access article distributed under the Creative Commons Attribution License 4.0 (CCBY), which permits unrestricted
any medium, provided the original work is properly cited. ributed under the Creative Commons Attribution License 4.0 (CCBY), which permits unrestricted use, distribution, and reproduction
work is properly cited. Received: 22 February 2016 / Received in final form: 18 April 2016 / Accepted: 29 May 2016 Received: 22 February 2016 / Received in final form: 18 April 2016 / Accepted: 29 May 2016 http://dx.doi.org/10.1097/MD.0000000000004058 1 Medicine García-Álvarez et al. Medicine (2016) 95:26 Enrique Navas (MD, PhD)m, Luis Vicioso (MD, PhD)n, José Antonio Oteo (MD, PhD)a,∗, Enrique Navas (MD, PhD)m, Luis Vicioso (MD, PhD)n, José Antonio Oteo (MD, PhD)a,∗,
the Spanish Collaboration on Endocarditis-Grupo de Apoyo al Manejo de la Endocarditis Infecciosa
en España (GAMES) Enrique Navas (MD, PhD)m, Luis Vicioso (MD, PhD)n, José Antonio Oteo (MD, PhD)a,∗,
the Spanish Collaboration on Endocarditis-Grupo de Apoyo al Manejo de la Endocarditis Infecciosa
en España (GAMES) Enrique Navas (MD, PhD) , Luis Vicioso (MD, PhD) , José Antonio Oteo (MD, PhD)
,
the Spanish Collaboration on Endocarditis-Grupo de Apoyo al Manejo de la Endocarditis Infecciosa
en España (GAMES) 3. Results was diagnosed because of valve cardiac involvement in the
context of classical WD. was diagnosed because of valve cardiac involvement in the
context of classical WD. 3.3. Microbiological diagnosis Culture of the valves was negative in all the cases. PAS staining
was performed in valves from 6 patients (35.3%) with a positive
result in 5 of them (83.3%). PCR against T. whipplei were
positive in all studied valves with at least 2 different targets. An
rRNA 16S PCR was used for first instance in all laboratories. Then, different targets were employed for confirmation (Table 2). PCRs were also positive in the intestinal biopsy in the 2 patients
with classical WD and in the pericardial fluid of the patient with
chronic pericarditis. In 1 of the patients affected by classical WD,
a positive PCR in an adenopathy was also obtained. The patient
in which cardiac valve surgery was not performed had a positive
PCR in samples from small bowel, cerebrospinal fluid (CSF), and
synovial fluid. The mean age of T. whipplei endocarditis was 60.3 years
(48–79 years) and most of cases were men, 14 (82.4%). Twelve
patients (70.6%) were from the North of Spain, 1 from the Center
(5.9%), 3 from the South (17.6%), and 1 (5.9%) from the Canary
Islands. 3.2. Clinical features According to the medical history, 6 patients had associated
pathological cardiac conditions (35.3%): 4 had aortic insuffi-
ciency, 1 atrial fibrillation with mitral insufficiency, and 1 chronic
pericarditis. One patient suffered a previous IE and carried a
prosthetic valve. Of 17 patients, 5 had hypertension (29.4%), 4
patients (23.5%) suffered a cerebrovascular disease, and 2
patients (11.7%) chronic lung disease. Immunosuppressive
therapy had been given only to 1 of the 17 patients (5.9%). One patient was HIV-positive (no AIDS) (5.9%). One had
diabetes mellitus (5.9%) and 1 suffered of chronic renal
insufficiency. Another 1 presented hypothyroidism and had
been operated for knee osteoarthritis. Alcohol intake (>60g/d)
was referred by the 23.5% of the patients. 2.2. Definitions IE was defined according to the modified Duke criteria.[17]
Definitive T. whipplei endocarditis was considered if positive
results of periodic acid-Schiff (PAS) staining and/or specific IHC
test using specific antibodies against T. whipplei and/or 2 positive
results of PCR assays targeting 2 different sequences in a cardiac
valve specimen were met.[18] Sporadic cases of T. whipplei endocarditis have been reported
from different countries,[12] but there are few published series of
T. whipplei endocarditis.[13,14] In this article, we describe the
epidemiological, clinical, and outcome characteristics of 17 cases
of T. whipplei endocarditis diagnosed in several hospitals from
Spain. Some cases have been previously reported.[15,16] 2 García-Álvarez et al. Medicine (2016) 95:26 www.md-journal.com 3.4. Treatment and outcome Eleven patients (64.7%) started treatment with ceftriaxone (2g/
IV at least for 2 weeks as initial therapy followed of different
antimicrobials). Twelve patients (70.6%) received trimethoprim-
sulfamethoxazole (SXT) 160/800mg “bis in die” (BID) at least
during 1 year (3 only SXT without another antimicrobial; 1 used
gentamicin during the 1st week and other hydroxychloroquine). In 5 patients (29.4%), the elected antimicrobial drug was
doxycycline 100mg BID plus hydroxychloroquine 600mg/d (1 of
them first started with SXT and continued with doxycycline plus
hydroxychloroquine). Details of the combinations are shown in
Table 1. All regimens were administered during at least 1 year
(average of treatment: 15.8 months). Cardiac failure was the main presenting form of T. whipplei
endocarditis described in our patients. It was present in 13 of the
17 patients (76.5%). Another patient developed cardiac failure
during the course of the illness. Chronic arthralgia was related in
9 of 17 patients (53%). Asthenia and malaise lasting more than 6
months were reported in 7 patients (41.2%). Fever was only
recorded in 6 patients (35.3%). Only 2 patients presented
classical WD (11.8%). One of them was diagnosed during the
endocarditis process and the other, 1 month before. The course during the treatment was satisfactory in all but 1
patients who suffered a new IE caused by an Enterococcus
faecalis. None of the patients died during the IE process or during
the follow-up after the end of treatment (Table 1). The follow-up
after finishing the treatment has been from 2 to 65 months, with
an average of 28.8 months. Echocardiography was performed for all 17 patients: trans-
thoracic echocardiography for 14 patients (82.4%) and trans-
esophageal
echocardiography
for
the
same
number. Echocardiography showed vegetations in 11 patients (64.7%). The valve involved was the aortic in 16 cases (94.1%), although 6
of them (37.5%) also presented mitral valve involvement. In 1 of
the patients, the unique valve involved was the mitral. Native
valve was affected in 16 patients (94.1%) while prosthetic valve
was affected only in 1 case (5.9%). The size of the vegetations was
among 5 and 33mm but embolic phenomena were rare: only 1
patient (5.9%) developed a peripherical embolism (limb) in the
course of the illness. 3.1. Epidemiological data A total of 3165 cases of IE were recorded in the GAMES Cohort
between 2008 and 2014. From the total, 451 (14.2%) were
diagnosed of BCNE and 16 (3.5%) of these had IE by T. whipplei. One case was added from a hospital not included in the GAMES
group. Main epidemiological, clinical, and outcome character-
istics are shown in Table 1. 4. Discussion Although the suspicion and diagnosis of T. whipplei endocarditis
can be difficult, more than 80 cases have been reported in the
literature since 1997.[12] Here we report 17 patients affected by T. whipplei endocarditis from a prospective cohort in Spain. This
series joined to the French and German ones is the largest series of
patients with T. whipplei endocarditis.[13,14] Diagnosis of T. whipplei endocarditis remains a challenge due to this endocardi-
tis does not exhibit the typical sings and blood cultures used to be
negative. According to BCNE series, the rate of T. whipplei as
causative agent of this condition is around the 0.6% to 2.6% of
all the studied cases.[4,24] These data are consistent with those
presented in this article. Nevertheless, the prevalence of T. whipplei endocarditis could be underestimated due to the
difficulties that involve the identification of T. whipplei. We
do not know if the data shown in this article show the true
incidence of T. whipplei endocarditis in our country, as it could
happen in other countries, but we know that is a good
approximation since the molecular study of removed valves is
the rule in all hospitals of the GAMES cohort when IE is
suspected. As in other studies, males (82.4%) are more frequently
affected than females. The age of presentation (60.3 years) is also The main laboratory recording abnormalities at the time of the
diagnosis were anemia which was detected in 88.2% of the
patients (hemoglobin level average of 11g/dL with a range
between 9.7 and 12.4g/dL) and an increasing of C-reactive
protein with an average level of 51.8mg/L (range: 2.3–136.7mg/
L). The glomerular filtration rate average was 66mm/h (range:
26–98). Surgery was performed in 16 patients (94.1%) and gave the
definitive diagnosis of IE in 16/17 patients (94.1%) since none of
the patients met the criteria for IE according to the Duke’s
university. The reasons of surgery were heart failure in 9 patients
(56.3%) and severe regurgitation in 7 (43.8%). The other patient 3 Medicine García-Álvarez et al. Medicine (2016) 95:26 Table 1
Main epidemiological, clinical, and outcome characteristics of the 17 patients with T whipplei endocarditis. Patient No. 4. Discussion 1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
Age, y
57
62
64
62
56
62
71
48
79
59
62
49
54
52
70
68
50
Gender
F
M
M
M
M
M
M
M
M
F
M
M
M
M
M
F
M
Geographical area
Madrid
Basque
Country
Basque
Country
Basque
Country
Basque
Country
Basque
Country
La
Rioja
La
Rioja
Canary
Islands
Andalucía
Andalucía Andalucía Catalonia
Catalonia
Catalonia
Asturias Asturias
Year of diagnose
2008
2009
2010
2012
2013
2010
2014
2014
2009
2013
2014
2014
2014
2014
2014
2014
2012
Cardiac history
N
AVI PIE PV
AF MVI
CP
N
AVI
N
N
AVI
N
N
N
N
N
N
N
AVI
Alcohol intake
N
N
N
N
Y
N
Y
Y
N
N
Y
N
N
N
N
N
N
Other historical conditions
IS DM
CVA
CVA HT CLD
HT
HT CLD
HIV
N
HT
HT CRI
CVA
N
N
N
N
HP
N
CVA
Cardiac failure
Y
Y
Y
Y
Y
Y
Y
Y
Y
N
Y
Y
N
Y
N
Y
Y
Asthenia–malaise
N
N
N
N
N
N
N
N
Y
Y
Y
Y
N
Y
Y
Y
N
Fever
N
N
Y
N
N
N
N
Y
N
Y
N
N
N
N
Y
Y
Y
Arthralgia
Y
N
Y
Y
N
Y
N
Y
N
Y
N
N
N
Y
Y
Y
N
Affected valve/s
AV
AV + MV
AV + MV
AV + MV
AV
AV
AV
AV
AV
AV
AV
AV + MV
MV
AV
AV + MV
AV
AV + MV
Vegetations
Y
N
Y
Y
Y
N
Y
Y
N
Y
N
Y
Y
Y
N
Y
N
Classical WD
N
N
N
N
N
N
N
N
N
Y
N
N
N
Y
N
N
N
Antibiotic duration, mo
C/SXT
18 mo
C/SXT
12 mo
C/SXT
12 mo
SXT
12 mo
C/SXT
12 mo
C/SXT
12 mo
C/D + H
13 mo
C/D + H
15 mo
G/SXT
12 mo
C/SXT D + H
24 mo
SXT
12 mo
C/SXT
12 mo
H +
SXT OT
C + SXT/
D + H OT
C/D + H OT
D +
H OT
SXT
40 mo
Surgery
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
N
Y
Y
Cardiac valve analysis/
PCR/PAS
PCR+
PCR+/PAS+ PCR+/PAS+ PCR+/PAS PCR+
PCR+ PCR+/PAS+ PCR+/PAS+ PCR+
PCR+
PCR+
PCR+
PCR+
PCR+/PAS+
N
PCR+
PCR+
Other analysis
PCR+ liquid
pericardial
N
N
N
N
N
N
N
N
PCR+ intestinal
biopsy and in
nodal biopsy
N
N
N
PCR+
synovial fluid and CSF/
PAS+ synovial fluid
and duodenal biopsy
PCR+
adenopathy
and duodenal
biopsy
N
N
Outcome
TF/NR
TF/NR
TF/NR
TF/NR
TF/NR
TF/NR
TF/NR
TF/NR
TF/NR
OT/new IE by
Enterococcus faecalis
TF/NR
TF/NR
OT
OT
OT
OT
TF/NR
AF=atrial fibrillation, AV=aortic valve, AVI=aortic valve insufficiency, C=ceftriaxone, CLD=chronic lung disease, CP=chronic pericarditis, CRI=chronic renal insufficiency, CSF=cerebrospinal fluid, CVA=cerebrovascular accident, D=doxycycline, DM=diabetes mellitus,
F=female, G=gentamicin, H=hydroxychloroquine, HIV=human immunodeficiency virus, HP=hipotiroidism, HT=hypertension, IE=infectious endocarditis, IS=immunosuppressive therapy, M=male, MV=mitral valve, MVI=mitral valve insufficiency, N=no, OT=on treatment,
PAS=periodic acid-Schiff, PCR=polymerase chain reaction, PIE=previous infective endocarditis, PV=prosthetic valve, SXT=trimethoprim and sulfamethoxazole, TF/NR=treatment finished/not relapse, V=vancomycin, WD = Whipple disease, Y=yes. 4. Discussion alve insufficiency, C=ceftriaxone, CLD=chronic lung disease, CP=chronic pericarditis, CRI=chronic renal insufficiency, CSF=cerebrospinal fluid, CVA=cerebrovascular accident, D=doxycycline, DM=diabetes mellitus,
IV=human immunodeficiency virus, HP=hipotiroidism, HT=hypertension, IE=infectious endocarditis, IS=immunosuppressive therapy, M=male, MV=mitral valve, MVI=mitral valve insufficiency, N=no, OT=on treatment,
eaction, PIE=previous infective endocarditis, PV=prosthetic valve, SXT=trimethoprim and sulfamethoxazole, TF/NR=treatment finished/not relapse, V=vancomycin, WD = Whipple disease, Y=yes. and outcome characteristics of the 17 patients with T whipplei endocarditis. 4 García-Álvarez et al. Medicine (2016) 95:26 www.md-journal.com Primers used in this study. pplication
Target
Primer name
Primer sequence (50 →30)
Reference
onventional PCR
rRNA 16S
fD1, rP2
AGAGTTTGATCCTGGCTCAG; ACGGCTACCTTGTTACGACTT
[19]
onventional PCR
rRNA 16S specific for T. whipplei
W3FE, W2RB
GGAATTCCAGAGATACGCCCCCCGCAA; CGGGATCCCATTCGCTCCACCTTGCGA
[20]
YBER green PCR
rRNA 16S
PSL, P13P
AGGATTAGATACCCTGGTAGTCCA; AGGCCCGGGAACGTATTCAC
[21]
YBER green PCR
Repeated sequence
TW27F, TW182R
TGTTTTGTACTGCTTGTAACAGG; TCCTGCTCTATCCCTCCTATCAT
[18]
YBER green PCR
Repeated sequence
TW13F, TW163R
TGAGTGATGGTATGTCTGAGAGATATGT; TCCATAACAAAGACAACAACCAATC
[18]
YBER green PCR
rpoB
TwrpoB-F, TwsrpoB-R
CTCGGTGTTGATGTTGATCCAA; GCACCGCAACCTCGGAGAAA
[22]
aqman PCR
Repeated sequence
TW27F, TW182R, Probe 27F-182R
TGTTTTGTACTGCTTGTAACAGGATCT; TCCTGCTCTATCCCTCCTATCATC;
6-FAM-AGAGATACATTTGTGTTAGTTGTTACA-TAMRA
[23]
aqman PCR
Repeated sequence
TW13F, TW163R, Probe 13F-163R
TGAGTGATGGTAGTCTGAGAGATATGT; TCCATAACAAAGACAACAACCAATC;
6-FAM-AGAAGAAGATGTTACGGGTTG-TAMRA
[18] in the range of other forms of IE[1] although less than in the
registry of the total IE of the GAMES series (69 years).[1] In the
data shown in this work, an aggregation of cases can be observed
in the North of Spain. A high prevalence of asymptomatic
colonization that has been demonstrated in this area[25] could
explain this fact, but we do not know what can be the prevalence
in other regions and a potential bias may occurs. However, such
irregular distribution has been described in other countries where
genetic host factors have been involved.[14] As other authors have published, modified Duke criteria are
ineffective for diagnosis before heart valve analysis.[14] In our
series, all but 1 patients were diagnosed after the affected valve
was removed. Cardiac failure used to be the main presenting
clinical debut form in T. whipplei endocarditis.[12–14] Signs and
symptoms in this entity are not the typical ones, and therefore,
fever was present only in 35% of patients and embolic events in
5.9%. The presence of arthralgia as a prominent symptom varies
depending on the series, showing values from 31% to 75% of the
described cases.[12–14] In our series, it has been 47% although in
many cases we have looked for it when the diagnosis was made. This symptom is, sometimes, weak and only detected after an
exhaustive clinical questioning. Medicine [9] Goldenberger D, Kunzli A, Vogt P, et al. Molecular diagnosis of bacterial [9] Goldenberger D, Kunzli A, Vogt P, et al. Molecular diagnosis of bacterial
endocarditis by broad-range PCR amplification and direct sequencing. J
Clin Microbiol 1997;35:2733–9. treatment strategy of 12 months with SXT (160/800mg BID) or,
at least, 18 months of doxycycline (100mg BID) plus hydroxy-
chloroquine (600mg/d).[10,29,30] Treatment of 2 weeks with
ceftriaxone followed by 1 year with SXT has been the most
recommended
line,[31]
although
in
the
recently
published
European guidelines[6] for the management of infective endocar-
ditis recommends doxycycline (100mg BID) plus hydroxychlor-
oquine (200–600mg/24h) orally for ≥18 months. This fact could
be in relationship with the resistance observed in vitro of T. whipplei to trimethoprim[32] and the reported case of a patient
with clinically acquired resistance to SXT.[33] In our series, most
of patients have been treated with ceftriaxone for 2 weeks
followed by SXT or with doxycycline plus hydroxychloroquine
with good outcomes. Other combinations have been also
employed with satisfactory results and none of our patients
died during the IE process and neither had a relapse in the follow
up. endocarditis by broad-range PCR amplification and direct sequencing. J
Clin Microbiol 1997;35:2733–9. [10] Raoult D, Ogata H, Audic S, et al. Tropheryma whipplei Twist: a human
pathogenic Actinobacteria with a reduced genome. Genome Res
2003;13:1800–9. [11] Bentley SD, Maiwald M, Murphy LD, et al. Sequencing and analysis of
the genome of the Whipple’s disease bacterium Tropheryma whipplei. Lancet 2003;361:637–44. [12] García-Álvarez L, Sanz MM, Oteo JA. Tropheryma whipplei endocar-
ditis. An epidemiological, clinical and treatment review. Aperito J Infect
Dis Vaccines 2015;1:103. [13] Geissdörfer W, Moos V, Moter A, et al. High frequency of Tropheryma
whipplei
in
culture-negative
endocarditis. J
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F, Célard
M, Lagier
JC,
et al. Tropheryma
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whipplei. Rev Esp Cardiol 2010;63:250–1. [16] Montejo M, Goikoetxea J, Mularoni A, et al. Grupo GAMES. Tropheryma whipplei endocarditis: a report of 3 cases. Rev Esp
Quimioter 2013;26:168–70. After the end of treatment, some authors[14] recommend the
checking for the presence of T. whipplei in blood, saliva, and
fecal samples every 6 months for 2 years and every year for the
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Whipple’s disease. N Engl J Med 2000;342:620–5. [33] Fenollar F, Rolain JM, Alric L, et al. Medicine If colonization is detected, they
recommend treating again, but there is not still evidence for this
procedure. [17] Li JS, Sexton DJ, Mick N, et al. Proposed modifications to the Duke
criteria for the diagnosis of infective endocarditis. Clin Infect Dis
2000;30:633–8. [18] Fenollar F, Laouira S, Lepidi H, et al. Value of Tropheryma whipplei
quantitative polymerase chain reaction assay for the diagnosis of
Whipple disease: usefulness of saliva and stool specimens for first-line
screening. Clin Infect Dis 2008;47:659–67. In summary, T. whipplei IE is an infrequent condition that
could be diagnosed with specific procedures (mostly molecular
tests and thereafter PAS coloration) when culture negative IE
undergo cardiac surgery. An early and appropriate diagnosis is
required since this condition has a very good course and
prognosis when the appropriate treatment is started. In our series,
all patients who have finished the treatment have had good
outcome and tolerance to the antimicrobial regimen used. Furthermore, we believe that in patients with unexplained valve
destruction which requires cardiac surgery, an exhaustive clinical
investigation must be performed and removed valves should be
studied by molecular tools for to rule out an underlying IE. [19] Weisburg WG, Barns SM, Pelletier DA, et al. 16S ribosomal DNA
amplification for phylogenetic study. J Bacteriol 1991;173:697–703. [20] Relman DA. Persing DH, Smith TF, Tenover FC, et al. PCR-based
detection of the uncultured bacillus of Whipple’s diseases. Diagnostic
Molecular Microbiology: Principles and Applications. Washington, DC:
American Society for Microbiology; 1993. 496–500. [21] Xu J, Millar BC, Moore JE, et al. Employment of broad-range 16S rRNA
PCR to detect aetiological agents of infection from clinical specimens in
patients with acute meningitis. J Appl Microbiol 2003;94:197–206. [22] Drancourt M, Carlioz A, Raoult D. rpoB sequence analysis of cultured
Tropheryma whippelii. J Clin Microbiol 2001;39:2425–30. p
y
pp
J
[23] Raoult D, Fenollar F, Birg ML. Culture of T. whipplei from the stool of a
patient with Whipple’s disease. N Engl J Med 2006;355:1503–5. [24] Houpikian P, Raoult D. Blood culture-negative endocarditis in a
reference center: etiologic diagnosis of 348 cases. Medicine (Baltimore)
2005;84:162–73. [25] García-Álvarez L, Pérez-Matute P, Blanco JR, et al. High prevalence of
asymptomatic carriers of Tropheryma whipplei in different populations
from the North of Spain. Enferm Infecc Microbiol Clin 2015;Nov 13. pii:
S0213-005X(15)00343-2,
doi:
10.1016/j.eimc.2015.09.006. [Epub
ahead of print]. 4. Discussion So, in patients with subacute
endocarditis with negative blood cultures and low-grade fever
(or not fever), if arthralgias are present, T. whipplei as causative
agent should be suspected.[14,26] Primer sequence (50 →30) Ecocardiography features are 1 of the most valuable tools for
suspecting IE. In our series, echocardiography allowed the
diagnosis of IE in 70.6%: visualization of vegetations in 11 and
indirect signs in 1. In a previous report, we analyzed all the
published cases in which these data were recorded and
vegetations were seen in 64.3% of cases.[12] In the French
series,[14] echocardiography showed vegetations in 78.6% of the
patients, but these data are not recorded in the German one.[13] In
the reviewed literature, only 4 (4.7%) of the cases reported a
classic WD concomitant with endocarditis[12] and we have
noticed it for 2 (11.8%) of cases. One of them was diagnosed
during the IE process. In any case, to perform an echocardiogram
should be made to these patients since endocarditis in the context
of classic WD is more frequent than in other diseases and than in
general population. The histological study by PAS staining is considered a good
tool for demonstrating WD. In our series, this technique was
made in 6 patients and 5 of them demonstrate the PAS positive
inclusions (sensitivity of 83.3%). Data of literature recorded PAS
staining in 48 patients with similar results.[12] Diagnosis of T. whipplei endocarditis in our series has been
carried out with molecular tools on heart valve tissue. Different
targets have been used for molecular analyses. PCR based on the
16S rRNA amplification and subsequent sequencing has been
widely used and has been the first-line screening in our series. However, some authors alert that this broad-spectrum PCR
could have a limited sensitivity (value sensitivity 60%, specificity
100%),[27] while specific qPCR for T. whipplei have showed
higher sensitivities.[18,28] So, if 16S rRNA PCR has been negative,
specific targets should be used in highly suspected cases of T. whipplei. At least 2 of the PCRs must be positive and their
sequences have to show higher identity with the bacterium
studied. Current management of T. whipplei endocarditis is based from
the experience acquired in the treatment of classical WD and in
Q fever endocarditis.[29,30] Most treatments used in T. whipplei
endocarditis include 2 weeks of parenteral high dose of
meropenem, penicillin G or ceftriaxone followed by an oral 5 García-Álvarez et al. 4. Discussion Medicine (2016) 95:26 References Resistance to trimethoprim/
sulfamethoxazole and Tropheryma whipplei. Int J Antimicrob Agents
2009;34:255–9. [8] Dutly F, Altwegg M. Whipple’s disease and “Tropheryma whippelii”. Clin Microbiol Rev 2001;14:561–83. 6 6
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Urokinase Receptor Promotes Skin Tumor Formation by Preventing Epithelial Cell Activation of Notch1
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Cancer research
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cc-by
| 1,709
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5
7
9
11
15
Days of TPA treatment
+/+
-/-
15
+/+
-/-
0
20
40
60
80
100
120
140
160
180
0
2
5
7
9
11
15
microvessel number/ area
Days of TPA treatment
+/
-/-
C Wt
0
0,5
1
1,5
K14
K5
k10
involucrin
loricrin
Fold change
(uPAR-/- over
uPAR+/+ skin )
differentiation
proliferation
C 0
2
4
6
8
10
12
0
14
28
Time (days)
+/+
+/+ muPAR
+/+ empty vector
-/-
-/- muPAR
-/- empty vector
A
α6lowCD34-Sca1+
0
2
4
6
8
10
12
0
14
28
# of cells (x106)
Time (days)
α6highCD34-Sca1+
7
7
14
14
B
Freshly isolated skin cells
0
0,5
1
1,5
2
2,5
3
Fold change vs. Wt
α6lowCD34-Sca1+
+/+
-/-
3
+/+ A 0
2
4
6
8
10
12
0
14
28
Time (days)
+/+
+/+ muP
+/+ emp
-/-
-/- muP
-/- empt
A
α6lowCD34-Sca1+
0
2
4
6
8
10
12
0
14
28
# of cells (x106)
Time (days)
α6highCD34-Sca1+
7
7
14
14
B
Freshly isolated skin cells
0
0,5
1
1,5
2
2,5
3
Fold change vs. Wt
α6lowCD34-Sca1+
+/+
-/-
0,5
1
1,5
2
2,5
3
Fold change vs. Wt
α6highCD34-Sca1+
+/+
-/-
0
Fold change vs. Wt α6lowCD34-Sca1+
α6highCD34-Sca1+
B
Freshly isolated skin cells
0
0,5
1
1,5
2
2,5
3
Fold change vs. Wt
α6lowCD34-Sca1+
+/+
-/-
0,5
1
1,5
2
2,5
3
Fold change vs. Wt
α6highCD34-Sca1+
+/+
-/-
0
Fold change vs. Wt B 0
0,5
1
1,5
2
2,5
3
Fold change vs. Wt
α6lowCD34-Sca1+
+/+
-/-
0,5
1
1,5
2
2,5
3
Fold change vs. Wt
α6highCD34-Sca1+
+/+
-/-
0
Fold change vs. Wt
0
0,5
1
1,5
K14
K5
k10
involucrin
loricrin
Fold change
(uPAR-/- over
uPAR+/+ skin )
differentiation
proliferation
C 0,5
1
1,5
2
2,5
3
Fold change vs. Wt
α6highCD34-Sca1+
+/+
-/-
1,5
vs
Wt
Freshly isolated skin cells
D
14 days post-plating primary keratinocytes
1,5
0
Fold change vs. Wt
0
0,5
1
1,5
K14
K5
k10
involucrin
loricrin
Fold change
(uPAR-/- over
uPAR+/+ skin )
differentiation
proliferation
C 0,5
1
1,5
2
2,5
3
Fold change vs. Wt
α6highCD34-Sca1+
+/+
-/-
0
Fold change vs. 5
7
9
11
15
Days of TPA treatment
+/+
-/-
15
+/+
-/-
0
20
40
60
80
100
120
140
160
180
0
2
5
7
9
11
15
microvessel number/ area
Days of TPA treatment
+/
-/-
C 5
7
9
11
15
Days of TPA treatment
+/+
-/-
15
+/+
-/-
0
20
40
60
80
100
120
140
160
180
0
2
5
7
9
11
15
microvessel number/ area
Days of TPA treatment
+/
-/-
C 5
7
9
11
15
s of TPA treatment 5
7
9
11
15
of TPA treatment 5 0
20
0
2
5
mic
Days 7
9
11
15
TPA treatment
+/+
-/-
+/+
-/-
20
40
60
80
100
120
140
160
180
crovessel number/ area
C 2 7
TPA 00
00
00 6w
10w
+/+
-/-
12w 6w
10w
+/+
-/-
12w 6w
10w
12w ctin
P2
P3
α6-RPE
CD34-Alexa64
D
P3
Gated on P4
a700
Sca1-Alexa700
CD34-Alexa647
interfollicular epidermal cells
ncluding cells of the
ibulum region of the HF
interfollicular epidermal cells Sca1-Alexa700
CD34-Alexa647
follicular epidermal cells
ing cells of the
m region of the HF
interfollicular epidermal cells r epidermal cells Sca1-Alexa700
pidermal cells
f the
of the HF
interfollic α6-RPE
CD3
D
Gated on P4
CD34-Alexa647 a700 00
f lli
l 00
t
f lli
l P2 0
2
4
6
8
10
12
0
14
28
Time (days)
+/+
+/+ muPAR
+/+ empty vector
-/-
-/- muPAR
-/- empty vector
A
α6lowCD34-Sca1+
0
2
4
6
8
10
12
0
14
28
# of cells (x106)
Time (days)
α6highCD34-Sca1+
7
7
14
14
B
Freshly isolated skin cells
0
0,5
1
1,5
2
2,5
3
Fold change vs. Wt
α6lowCD34-Sca1+
+/+
-/-
0,5
1
1,5
2
2,5
3
Fold change vs. Wt
α6highCD34-Sca1+
+/+
-/-
Suppl. Fig. S4
0
0,5
1
1,5
Fold change vs
untreated Wt
Freshly isolated skin cells
α6lowCD34-
Sca1+
α6highCD34high
Sca1-
Total
GFP+ cells
α6highCD34-
Sca1+
D
14 days post-plating primary keratinocytes
0
0,5
1
1,5
α6lowCD34-
Sca1+
α6highCD34-
Sca1+
α6highCD34high
Sca1-
Total
GFP+ cells
α
+/+
-/-
+/+ after TPA
-/- after TPA
α6highCD34-
Sca1+
E
0
5
10
15
20
25
30
# of cells at
day 14 (x105)
α6lowCD34-
Sca1+
+/+
*
*
*
UN
Ad-Notch1
AdGFP
0
10
20
30
40
# of cells at
day 14
(x105)
α6lowCD34-
Sca1+
-/-
*
*
Not transfected
AS-Notch1
pcDNA
F
0
Fold change vs. 5
7
9
11
15
Days of TPA treatment
+/+
-/-
15
+/+
-/-
0
20
40
60
80
100
120
140
160
180
0
2
5
7
9
11
15
microvessel number/ area
Days of TPA treatment
+/
-/-
C Wt D
0
0,5
1
1,5
K14
K5
k10
involucrin
loricrin
Fold change
(uPAR-/- over
uPAR+/+ skin )
differentiation
proliferation
C C 0
0,5
1
1,5
K14
K5
k10
involucrin
loricrin
Fold change
(uPAR-/- over
uPAR+/+ skin )
differentiation
proliferation D 0
0,5
1
1,5
Fold change vs
untreated Wt
Freshly isolated skin cells
α6lowCD34-
Sca1+
α6highCD34high
Sca1-
Total
GFP+ cells
α6highCD34-
Sca1+
D
14 days post-plating primary keratinocytes
0
0,5
1
1,5
α6lowCD34-
Sca1+
α6highCD34-
Sca1+
α6highCD34high
Sca1-
Total
GFP+ cells
+/+
-/-
+/+ after TPA
-/- after TPA
α6highCD34-
Sca1+
differentiation
proliferation Suppl. Fig. S4
Sca1-
E
0
5
10
15
20
25
30
# of cells at
day 14 (x105)
α6lowCD34-
Sca1+
+/+
*
*
*
UN
Ad-Notch1
AdGFP
0
10
20
30
40
# of cells at
day 14
(x105)
α6lowCD34-
Sca1+
-/-
*
*
Not transfected
AS-Notch1
pcDNA
F Suppl. Fig. 5
7
9
11
15
Days of TPA treatment
+/+
-/-
15
+/+
-/-
0
20
40
60
80
100
120
140
160
180
0
2
5
7
9
11
15
microvessel number/ area
Days of TPA treatment
+/
-/-
C S4
E
0
5
10
15
20
25
30
# of cells at
day 14 (x105)
α6lowCD34-
Sca1+
+/+
*
*
UN
Ad-Notch1
AdGFP *
0
10
20
30
40
# of cells at
day 14
(x105)
α6lowCD34-
Sca1+
-/-
*
*
Not transfected
AS-Notch1
pcDNA
F E F Proliferative area
K5, proliferation marker
Proliferative area
Proliferative area
Involucrin,
differentiation marker Proliferative area mark +/+
NICD
+/+ TPA
-/- TPA
-/- TPA
DAPT
+/+
-/-
F
E
+ TPA
0
5
10
15
NICD/Notch
-/-
0
20
40
# of cells at
day 14 (x105)
-/-
+ TPA
+ TPA
H
I
-/-
+/+ TPA
-/- TPA
DL-1
Jag-1
F
0
1000
2000
3000
4000
TACE activity
( RFU/ mg
proteins)
-/-
* *
+ TPA
E
0
20
40
60
# of cells at
day 14 (x105)
*
*
-/-
+ TPA
ADAM10
K1
Lor
Inv
actin + TPA
0
20
40
day 14 (x105)
-/-
+ TPA
0
20
40
60
*
*
-/-
+ TPA -/- C
NICD
+/+ TPA
-/- TPA
-/- TPA
DAPT
F
+
0
5
10
15
-/-
0
20
40
# of cells at
day 14 (x105)
+ TPA
+
I
F
0
0
0
0
0
-/-
* *
+ TPA
0
20
40
60
# of cells at
day 14 (x105)
+ C
NICD
+/+ TPA
-/- TPA
-/- TPA
DAPT
F
0
5
0
5
-/-
f
ll
+ TPA
I
F
-/-
* *
+ TPA
# of cells at
day 14 (x105) 0
5
0
5
/
+ TPA
I
-/-
* *
# of ce #
f -/- 0
5
10
15
NICD/Notch
H
Jag-1
0
ADAM10
K1
Lor
Inv
actin +/+
/
+/+
-/-
E
-/-
+/+ TPA
-/- TPA
2000
3000
4000
E activity
FU/ mg
oteins)
E 0 + TPA
+ TPA
*
*
*
*
C
- TPA
+ TPA
50
# of cells at day
*
*
*
*
0
10
20
30
40
# of cells at day
14 (x105) + TPA
+ TPA
*
*
NICD
Notch1
0
1000
2000
3000
4000
(RFU/ mg
proteins)
*
*
C
- TPA
+ TPA
50
# of cells at day
4
5
6
7
8
9
Bottom
crose density
+ TPA
*
*
*
*
0
10
20
30
40
# of cells at day
14 (x105)
+ β-MCD
afts
Non-Rafts
4
5
6
7
8
9 + TPA
*
* + TPA
+ TPA
*
*
*
*
C
- TPA
50
# of cells at day
*
*
0
10
20
30
40
# of cells at day
14 (x105) TPA
+ TPA
*
*
C
- TPA
50
# of cells at day
*
*
0
10
20
30
40
# of cells at day
14 (x105) 000
000
000
000 000
000
000
000 -0.520
0.273
0.057
1.589
0.297
0.000
-0.013
0.002
0.000
responding standard errors and P-values of the fitted zero-inflated
: Zero Inflated Poisson) used in Fig.1D to analyze the daily
s in each uPAR+/+ and uPAR-/- mouse, as described in Methods. 5
7
9
11
15
Days of TPA treatment
+/+
-/-
15
+/+
-/-
0
20
40
60
80
100
120
140
160
180
0
2
5
7
9
11
15
microvessel number/ area
Days of TPA treatment
+/
-/-
C -0.013
0.002
0.000
-0.005
0.002
0.028
odel coefficients (Binomial regression model)
Estimate
Std. Error
P-value
6.883
0.624
0.000
-0.093
0.010
0.000 s, correspon
(ZIP: Zero
umors in ea
n model co
E s, correspon
(ZIP: Zero
umors in ea
n model co
E shows the number of uPAR+/+ and uPAR-/-hair
±SD ) in the telogen (T), anagen (A) and catagen
, 23, 28, and at weeks 6, 7, 10 and 12 after birth. eriments are shown, with 3 mice per genotype for
back skin samples per mouse. hows the numb
SD ) in the telog
3, 28, and at we
ments are show
ack skin samples hows the numb
SD ) in the telog
3, 28, and at we
ments are show
ack skin samples hows the numb
SD ) in the telog
3, 28, and at we
ments are show
ack skin samples
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https://bg.copernicus.org/articles/6/819/2009/bg-6-819-2009.pdf
|
English
| null |
Ammonia fluxes in relation to cutting and fertilization of an intensively managed grassland derived from an inter-comparison of gradient measurements
|
Biogeosciences
| 2,009
|
cc-by
| 15,283
|
Ammonia fluxes in relation to cutting and fertilization of an
intensively managed grassland derived from an inter-comparison of
gradient measurements C. Milford1,*, M. R. Theobald1, E. Nemitz1, K. J. Hargreaves1, L. Horvath2, J. Raso2, U. D¨ammgen3, A. Neftel4,
S. K. Jones1,4, A. Hensen5, B. Loubet6, P. Cellier6, and M. A. Sutton1 Milford1,*, M. R. Theobald1, E. Nemitz1, K. J. Hargreaves1, L. Horvath2, J. Raso2, U. D¨amm
K. Jones1,4, A. Hensen5, B. Loubet6, P. Cellier6, and M. A. Sutton1 1Centre for Ecology and Hydrology (Edinburgh Research Station), Bush Estate, Penicuik, Midlothian, EH26 0QB, UK
2Institute of Atmospheric Physics, P.O. Box 39, 1675 Budapest, Hungary Centre for Ecology and Hydrology (Edinburgh Research Station), Bush Estate, Penicuik, Midlothian
nstitute of Atmospheric Physics, P.O. Box 39, 1675 Budapest, Hungary 3Institut f¨ur Agrar¨okologie, Bundesforschungsanstalt f¨ur Landwirtschaft (FAL), Bundesallee 50,
38116 Braunschweig, Germany 3Institut f¨ur Agrar¨okologie, Bundesforschungsanstalt f¨ur Landwirtschaft (FAL), Bundesallee 50,
38116 Braunschweig, Germany 4Agroscope Reckenholz-T¨anikon Research Station ART, Reckenholzstrasse 191, 8046 Zurich, Switzerland
5Energy research Centre of the Netherlands (ECN), Postbus 1, 1755 ZG Petten, The Netherlands
6Institut National de la Recherche Agronomique (INRA), UMR Environnement et Grandes Cultures,
Thiverval-Grignon, 78850, France 4Agroscope Reckenholz-T¨anikon Research Station ART, Reckenholzstrasse 191, 8046 Zurich, Switzerland
5Energy research Centre of the Netherlands (ECN), Postbus 1, 1755 ZG Petten, The Netherlands
6Institut National de la Recherche Agronomique (INRA), UMR Environnement et Grandes Cultures,
Thiverval-Grignon, 78850, France ow at: Institute of Earth Sciences “Jaume Almera”, CSIC, C/Lluis Sol´e i Sabar´ıs s/n, Barcelona, 08 Received: 5 September 2008 – Published in Biogeosciences Discuss.: 4 December 2008
Revised: 23 April 2009 – Accepted: 10 May 2009 – Published: 15 May 2009 Abstract. Quantification
of
ammonia
(NH3)
land-
atmosphere exchange is required for atmospheric modelling
and assessment of nitrogen deposition, yet flux measurement
methods remain highly uncertain. To address this issue, a
major inter-comparison of ammonia fluxes over intensively
managed grassland was conducted during the GRAMINAE
Integrated Experiment held in Braunschweig, Germany. In
order to provide a robust dataset of ammonia exchange with
the vegetation, four independent continuous flux gradient
systems were operated. Three independently operated con-
tinuous wet denuders systems (AMANDA) were compared
with a Wet Effluent Diffusion Denuder (mini-WEDD) sys-
tem. Measurements were made at two distances from an
adjacent livestock farm, allowing effects of advection to be
quantified in a real landscape setting. Data treatment in-
cluded filtering for instrument failure, disturbed wind sectors
and unsuitable micrometeorological conditions, with correc-
tions made for storage and advection errors. poorer agreement on particular days. Ammonia fluxes in relation to cutting and fertilization of an
intensively managed grassland derived from an inter-comparison of
gradient measurements However, by using four
systems, the inter-comparison was able to provide a robust
mean estimate of continuous ammonia fluxes through the ex-
periment. The observed fluxes were: a) small bi-directional
fluxes prior to cutting (−64 to 42 ng NH3 m−2 s−1), b)
larger diurnally-varying emissions following cutting (−49 to
703 ng NH3 m−2 s−1) and c) much larger emissions follow-
ing fertilizer application (0 to 3820 ng NH3 m−2 s−1). The
results are a salutary reminder of the uncertainty in unrepli-
cated ammonia flux measurements, while the replication of
the present study provides a uniquely robust dataset for the
evaluation of ammonia exchange processes. It is clear that
consistently reliable determination of ammonia concentra-
tions remains the major measurement challenge. Biogeosciences, 6, 819–834, 2009
www.biogeosciences.net/6/819/2009/
© Author(s) 2009. This work is distributed under
the Creative Commons Attribution 3.0 License. Biogeosciences, 6, 819–834, 2009
www.biogeosciences.net/6/819/2009/
© Author(s) 2009. This work is distributed under
the Creative Commons Attribution 3.0 License. Biogeosciences, 6, 819–834, 2009
www.biogeosciences.net/6/819/2009/
© Author(s) 2009. This work is distributed under
the Creative Commons Attribution 3.0 License. Biogeosciences 1
Introduction The inter-comparison demonstrated good agreement in
measured ammonia concentrations and fluxes (relative stan-
dard error <20%) for some periods, although the perfor-
mance of the ammonia analyzers were variable, with much Measurements of ammonia exchange with vegetation us-
ing micrometeorological methods have been conducted since
the 1970s (e.g. Denmead et al., 1974, 1976; Dabney and
Bouldin, 1985; Harrison et al., 1989; Flechard et al., 1998;
Warland et al., 2001; Phillips et al., 2004). The measure-
ments have been conducted over a range of agricultural
systems as well as semi-natural systems (e.g. Erisman and
Wyers, 1993; Sutton et al., 1993b; Duyzer, 1994; Nemitz et Correspondence to: C. Milford
(cmilford@inm.es)
ments have been co
systems as well as s
Wyers, 1993; Sutton
Published by Copernicus Publications on behalf of the European Geosciences Union. Correspondence to: C. Milford
(cmilford@inm.es) Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. 820 C. Milford et al.: Ammonia fluxes over intensively managed grassland NH3 exchange with grasslands across Europe (Sutton et al.,
2001a, 2007). al., 2004), including grasslands (e.g. Horvath 1982; Harper
et al., 1983; Horvath et al., 2005). Initially, the motiva-
tion for measurement of ammonia was to quantify N losses
from agricultural systems. The first measurements used tech-
niques such as bubblers/acid traps (Denmead et al., 1974),
filter packs (Harrison and Kitto, 1990) or denuder tubes (e.g. Ferm 1979). All these methods operated at a time reso-
lution of at least 1 h and required significant operator ef-
fort for changing over samples and off–line analysis. As a
consequence, most early measurements were for short field-
campaigns only, of a few days or weeks (e.g. Sutton et al.,
1993b; Duyzer 1994). In addition to the interest in contrasting ecosystem types
(e.g. Horvath et al., 2005; Walker et al., 2006; Kugler
et al., 2008), large rates of NH3 emission have been ob-
served following cutting of intensively managed grassland,
from the sward itself (Sutton et al., 1997, 2001a; Milford
et al., 2001b), with this effect being modelled by Riedo et
al. (2002). There is, however, a shortage of studies inves-
tigating this emission source, while Bussink et al. (1996)
observed the opposite, i.e. an apparent increase in deposi-
tion flux immediately after cutting. 1
Introduction Emission of NH3 from
senescing and decomposing vegetation has also been investi-
gated (Whitehead and Lockyer, 1989; Mannheim, 1997), but
this is rather distinct from the enhanced emissions from a re-
growing canopy following cutting noted above. Emission of
volatile organic compounds (VOCs) has been detected from
cut grassland and attributed to a “wounding” of the vegeta-
tion (Gouw et al., 1999). Other work has investigated the
effect of cutting regimes on the health, yield and species di-
versity of swards (Smith et al., 1996a, b; Blum et al., 1997;
Evans et al., 1998), but not the effect on NH3 exchange. Much research has been conducted on NH3 volatilisation fol-
lowing N fertilization applied to grassland (e.g. Pain et al.,
1989; Thompson et al., 1990a, b, 1991; van der Weerden and
Jarvis, 1997; Sommer et al., 2004), but little research has
been conducted to quantify the contributions from direct fer-
tilizer emission of NH3 and indirect NH3 emission from the
plants themselves. y
As interest in ammonia (NH3) as an atmospheric pollu-
tant has grown, so too has the range of measurement tech-
niques available. Developments in continuous measurement-
techniques enabled much longer time periods of measure-
ments to be conducted. In particular, the continuous flow
denuder (AMANDA) developed by Wyers et al. (1993),
achieved the balance between reasonable cost, a wide op-
erating concentration range (0.02–100 µg m−3) and on-line
analysis with short time resolution (<5 min). The AMANDA
utilises wet chemistry techniques, stripping ammonia from
the air in an acidic capture solution. Other similar in-
struments have been developed which also operate via wet
chemistry methods, for example the Wet Effluent Diffusion
Denuder (WEDD) technique (Vecera and Dasgupta, 1991),
as well as recent more automated implementations of the
AMANDA technique (Trebs et al., 2006; Kruit et al., 2007). Although these techniques have been proven to measure NH3
successfully, there are limitations; in particular they cannot
be used for eddy correlation measurements where a sensor
with a time response of typically at least 4 Hz is needed. Shaw et al. (1998), Famulari et al. (2004) and Whitehead
et al. (2008) have reported eddy correlation measurements of
NH3 fluxes, using a tandem mass spectrometer and tunable
diode laser technology, but substantial further developments
(and cost reductions) are required before eddy correlation be-
comes more widely used for NH3 flux measurement. Biogeosciences, 6, 819–834, 2009 2.1
Micrometeorological theory The horizontal flux divergence ∂Fx/∂x may be more sig-
nificant for NH3, particularly where large NH3 sources exist
in the vicinity, causing large local horizontal concentration
gradients in the direction of the wind (∂χa/∂x) (Loubet et al.,
2001, 2006; Milford et al., 2001a). Estimates of the differ-
ence in the vertical flux due to advection (the advection error,
1Fz,adv) are usually not made, due to lack of information on
∂χa/∂x. This issue was, however, given special attention in
the Braunschweig Experiment, with both measurements and
modelling of ∂χa/∂x being conducted (Loubet et al., 2009). On this basis, where relevant, derived 1Fz,adv were applied
to correct the measured fluxes at 1 m (Fz(1 m)) and provide
estimates of the fluxes at the canopy surface (Fz (zo)). The NH3 flux measurements were made using the aerody-
namic gradient method, following the approaches described
in Fowler and Duyzer (1989), Monteith and Unsworth (1990)
and Sutton et al. (1993). It should be noted that recent re-
search (Flesch et al., 2002; McNaughton, 2006) has ques-
tioned the validity and accuracy of the aerodynamic gradient
method. The flux (Fz) is calculated from the friction velocity
(u∗) and concentration scaling parameter (χ∗): Fz = −u∗χ∗
(1) (1) Fz = −u∗χ∗ with emission fluxes denoted as being positive. χ∗is calcu-
lated as: χ∗= k
dχ
d[ ln (z −d) −9H( z−d
L )]
(2) Chemical production or consumption (Qchem) may also be
significant for ammonia where either evaporation of ammo-
nium containing aerosol, or production of aerosol, respec-
tively, occur in the surface layer. The difference in the verti-
cal flux due to these effects (1Fz,che) is assessed separately
by Nemitz et al., 2009a. (2) where k is von Karman’s constant (=0.41), z is height
above the surface, d is zero plane displacement, χ is NH3
concentration and 9H is the integrated stability correction
function for scalar properties, calculated from the Monin-
Obukhov length (L) according to the description of Sutton
et al. (1993). Although u∗can be calculated from profiles
of windspeed in an equation analogous to Eq. (2), in this
instance it was obtained from ultrasonic anemometry using
eddy covariance (Moncrieff et al., 1997). u∗and all other
meteorological parameters were measured by several insti-
tutes in the experiment. These estimates have been compared
and evaluated by Nemitz et al. (2009b), who provided a con-
sensus dataset for use in the flux calculations here. 2
Materials and methods which is approximately equal to: which is approximately equal to: 1Fz,sto = (z −d)∂χa
∂t
(5) (5) 1
Introduction p
To address some of these issues, the GRAMINAE Inte-
grated Experiment was held in Braunschweig, Germany, over
intensively managed grassland (Sutton et al., 2009b). In or-
der to provide a robust dataset of NH3 exchange with the
vegetation, four independent continuous flux gradient sys-
tems were operated. Although there have been many inter-
comparisons of ammonia concentration measurements (e.g. Gras, 1984; Appel et al., 1988; Harrison and Kitto, 1990;
Wiebe et al., 1990; Sutton et al., 2001b; Schwab et al., 2007)
there have been much fewer inter-comparisons of ammo-
nia flux measurements (Sutton et al., 2000; Whitehead et
al., 2008). This paper presents the results of a major inter-
comparison of continuous NH3 flux measurements over in-
tensively managed grassland. The best estimates of NH3
flux and concentration from this inter-comparison are sub-
sequently used to investigate the effect of cutting and fertil-
izing on NH3 exchange fluxes. These best estimates of NH3
concentrations and fluxes were also provided to other par-
ticipants in the experiment as a basis for further analyses,
for example in quantifying advection fluxes (Loubet et al.,
2009), assessing the relaxed eddy accumulation technique
(Hensen et al., 2008), modelling the dynamics of ammonia
fluxes (Burkhardt et al., 2009; Personne et al., 2009; Sutton
et al., 2009a) and quantifying production of particulate am-
monium aerosol (Nemitz et al., 2009a). An alternative micrometeorological flux measurement
technique is the relaxed eddy accumulation method (REA). This method has the advantage of not requiring a fast re-
sponse analyser. REA flux measurements of NH3 have been
conducted in recent years (e.g. Neftel et al., 1999; Nemitz
et al., 2001a; Meyers et al., 2006) and constitute an area of
ongoing research. Ammonia has been recognised as contributing to eutroph-
ication and acidification of ecosystems (Fangmeier et al.,
1994; Krupa et al., 2003). To assess these effects, reli-
able models of ammonia deposition are needed (Hertel et al.,
2006). Currently, there are only crude parameterisations of
NH3 exchange present in national and European deposition
models and measurements are needed to improve these pa-
rameterisations. A European project GRAMINAE (GRass-
land AMmonia INteractions Across Europe) was therefore
initiated to improve quantification and parameterisation of Biogeosciences, 6, 819–834, 2009 www.biogeosciences.net/6/819/2009/ C. Milford et al.: Ammonia fluxes over intensively managed grassland C. Milford et al.: Ammonia fluxes over intensively managed grassland 821 2.2
Ammonia measurement techniques and implemen-
tation However, the resulting differ-
ence in the vertical flux due to storage (the storage error,
1Fz,sto) may be found from: 1Fz,sto =
z−d
Z
0
∂χa
∂t dz
(4) (4) www.biogeosciences.net/6/819/2009/ www.biogeosciences.net/6/819/2009/ Biogeosciences, 6, 819–834, 2009 C. Milford et al.: Ammonia fluxes over intensively managed grassland 822 Table 1. Summary of the different continuous ammonia concentration profile sampling systems used to calculate ammonia fluxes in
resent study. System acronym
System description
Location
FRI
AMANDA (3 point profile) operated by the Hungarian Forest Research Institute (FRI)
Site 1
in collaboration with the Hungarian Institute of Atmospheric Physics (IAP). FAL-CH
Mini-WEDD (3 point profile) operated by the former Swiss Federal Research Station for
Site 1
Agroecology and Agriculture (FAL-CH). FAL-D
AMANDA (3 point profile) operated by the German Federal
Site 1
Agricultural Research Institute (FAL-D)
CEH
AMANDA (3 point profile) operated by the Centre for Ecology and Hydrology (CEH) UK. Site 2 System acronym
System description A second continuous ammonia measurement technique
utilised miniaturised Wet Effluent Denuders (mini-WEDD),
which are silica-coated glass tubes (length 125 mm) posi-
tioned vertically, with continuous flow of a stripping solution
in a membrane tube analysed online by a four-channel fluo-
rescent analyser (e.g. Neftel et al., 1998; Vecera and Das-
gupta, 1991). The mini-WEDDs were maintained by ART
the former Swiss Federal Research Station for Agroecol-
ogy and Agriculture (FAL-CH) and placed at four heights
(0.15 m, 0.3 m, 0.6 m and 1.2 m above ground). However, the
lowest mini-WEDD concentration was not used in the flux
calculations as it was judged to be too close to the canopy. An air-flow rate of 600 ml min−1 and a liquid flow rate of
0.12 ml min−1 were used. The detection limit was 0.1 µg
NH3 m−3. Calibration of both systems was conducted with
aqueous standards of 0, 50 and 500 µg kg−1 NH+
4 . A sum-
mary of the different NH3 measurement systems applied and
acronyms used in the intercomparison is provided in Table 1. denuder inlets linked to a common ammonium detector) were
deployed at Site 1 (FAL-D, FRI), as well as the mini-WEDD
system (FAL-CH), while one gradient AMANDA (CEH) was
deployed at Site 2. The field was cut for silage on the morn-
ing of 29 May 2000 (starting at 06:00 GMT), with grass re-
moved from the field on the morning of 31 May 2000. 2.2
Ammonia measurement techniques and implemen-
tation The
field was fertilized with 108 kg N ha−1 calcium ammonium
nitrate on the morning of 5 June 2000 (06:00–07:00 GMT). 2.4
Data processing procedures The complexities of data processing are increased by the
availability of the 4 independent estimates of NH3 concen-
tration profiles. The following procedure was applied: i)
Any periods of calibration or obvious malfunctioning
of each instrument were removed from the dataset of
measured concentrations. During the experiment, aqueous calibration standards
were prepared centrally and distributed amongst the ammo-
nia analyser operators. In addition, unknown quality control
standards were distributed on 3 occasions to test the accu-
racy of the analysers. On each occasion, 2 unknown stan-
dards were distributed and the difference in concentration
measured by each analyser was compared with the actual
concentration difference. ii)
Fluxes Fz(1 m) of NH3 and concentrations at 1 m
(χ(1 m)) were calculated according to Sect. 2.1. Flux
measurements were rejected during periods when the
fetch was obstructed by other equipment or by the edge
of the field (see below for details). iii) Once Fz(1 m) and χ(1 m) estimates were available for
the different systems, these were compared to iden-
tify any further periods where one system had malfunc-
tioned or underperformed which had not yet been iden-
tified. These data were then removed. 2.2
Ammonia measurement techniques and implemen-
tation Ammonia concentrations were determined in gradient con-
figuration at four locations using two different measurement
techniques. At three out of the four sites the continuous
flow wet denuder system “AMANDA” (Ammonia Measure-
ment by ANnular Denuder sampling with online Analysis)
was deployed (Wyers et al., 1993). This technique captures
gaseous ammonia in a continuous-flow horizontal annular
denuder using a stripping solution of 3.6 mM sodium hy-
drogen sulphate (NaHSO4) and determines the aqueous am-
monium concentration online by conductivity analysis. The
time resolution of this method can be set to 1 min, however,
in this instance, concentrations were measured sequentially
for 150 s at each of three heights (including liquid-flow delay
loops), resulting in a full profile measurement every 450 s. These concentrations were averaged to 15 min periods for
flux calculation. The air-flow rate of the AMANDAs was
approximately 25 l min−1 and the liquid flow rate through
the denuders was approximately 1 ml min−1; the detection
limit was about 0.02 µg NH3 m−3. The heights of the con-
centration measurements were varied throughout the mea-
surement campaign according to the canopy height, but the
maximum height and minimum heights above ground were
2.37 m and 0.32 m respectively. The three AMANDA sys-
tems were maintained by the Centre for Ecology and Hydrol-
ogy (CEH) (UK), the German Federal Agricultural Research
Institute (FAL-D) and the Hungarian Forest Research Insti-
tute (FRI), the last being in collaboration with the Hungarian
Institute of Atmospheric Physics (IAP). The basic calculations of NH3 fluxes derived here assume
stationarity and homogeneity of the atmospheric conditions,
such that the vertical flux results entirely from turbulent
transfer in the vertical and that errors related to local advec-
tion (horizontal heterogeneity), storage (temporal instation-
arity) and chemical production or consumption in the surface
layer are ignored. Strictly, this assumption cannot always be
maintained, with the result that the vertical flux is not con-
stant with height: ∂Fz
∂z = −∂χa
∂t −∂Fx
∂x + Qchem
(3) (3) In the case of ammonia, where exchange fluxes are rel-
atively large in proportion to concentration, the flux diver-
gence due to storage (∂χa/∂t) can generally be ignored as
trivial (Sutton et al., 1993a). C. Milford et al.: Ammonia fluxes over intensively managed grassland No corrections to the data for the potential
effect of chemical production or consumption are made
here, as this effect is assessed by Nemitz et al. (2009a). Loubet et al. (2009) for the Site 1 and 2 measurements
and applied to the measured fluxes, resulting in an es-
timate of the fluxes at the canopy surface (Fz (zo)) (see
Sect. 3.3). No corrections to the data for the potential
effect of chemical production or consumption are made
here, as this effect is assessed by Nemitz et al. (2009a). and then interpolating this ratio. This interpolated ratio was
then multiplied by the available mean estimate to fill in miss-
ing data. This method propagates the deviations from the
mean present at the start and end of the gap, and limits the
occurrence of step changes in the flux when individual anal-
ysers fail or are restored in the dataset. Only gaps of <6 h
were filled. vi) The “mean gradient estimate” (subscript mg) was cal-
culated for (Fz (zo)) and χ(1 m), as the arithmetic mean
of all the remaining individual measurements. At the
same stage, corrections for storage errors were also ap-
plied. Finally, the data were filtered according to micrometeoro-
logical criteria to identify periods where the fluxes are esti-
mated with less certainty. These micrometeorological crite-
ria were: u (1 m) <0.8 m s−1, |L|<5 m and cumulative nor-
malised footprint function (CNF) <67%. The cumulative
normalised footprint function was calculated using the Ko-
rmann and Meixner (2001) formulation as described in Ne-
mitz et al. (2009b). vii) The data were filtered according to the passing or fail-
ing of a set of defined micrometeorological criteria (see
below). Data failing these criteria were retained in the
dataset, but distinguished as being of lower reliability. There was a substantial amount of measurement equip-
ment at Site 1, concentrated on a N-S axis, as well as three
mobile laboratories in the N direction (see site description in
Sutton et al., 2009b). As a result, flux measurements from
the FAL-D and FRI gradient systems were rejected for wind
directions from both the N and S direction (0◦to 20◦and
180◦to 190◦rejected), whilst flux measurements were re-
jected for winds from the NNE direction (10◦to 45◦) for the
FAL-CH gradient system. C. Milford et al.: Ammonia fluxes over intensively managed grassland Due to the close proximity of the
edge of the field to the east of Site 2, flux measurements were
rejected at this site for wind direction 0◦to 170◦. C. Milford et al.: Ammonia fluxes over intensively managed grassland 823 Table 2. Results of processing procedures for the ammonia flux data, from 21 May 2000, 10:00 GMT–15 June 2000, 12:00 GMT. Proceedure
No. of valid (15-min) Fz data points remaining
Data coverage (%)
Instrument
CEH
FRI
FAL-D
FAL-
CHa
Mean
estimate
CEHb
FRIb
FAL-Db
FAL-
CHc
Mean
estimate
Field Site number
2
1
1
1
2
1
1
1
i) Periods of calibration or ob-
vious malfunctioning of the
instruments removed. 1912
1510
1893
1037
79
63
79
69
ii)
Measurements
from
obstructed
wind
sectors
removed
1400
1451
1841
1012
58
60
76
68
iii) gap-filling
No. of valid data points re-
maining after gapfilling
1496
1531
1968
1372
62
64
82
92
Number of gaps filled
23
13
31
71
Median gap length (mins)
90
30
15
30
Std. dev. of gap length (mins)
67
107
87
83
iv) Mean gradient estimate,
all data
−
−
−
−
2350
−
−
−
−
97.5
v) Data passing micromet cri-
teria
−
−
−
−
1652
−
−
−
−
68.5
a FAL-CH gradient data commences on 30 May 2000, 22:15 GMT, up until 30 May the system measured within-canopy profiles. b % data coverage calculated for period 21 May 2000, 10:00 GMT–15 June 2000, 12:00 GMT. c % data coverage calculated for period 30 May 2000, 22:15 GMT–15 June 2000, 12:00 GMT. rocessing procedures for the ammonia flux data, from 21 May 2000, 10:00 GMT–15 June 2000, 12:00 GMT. a FAL-CH gradient data commences on 30 May 2000, 22:15 GMT, up until 30 May the system measured within-canopy profiles. b % data coverage calculated for period 21 May 2000, 10:00 GMT–15 June 2000, 12:00 GMT. c % data coverage calculated for period 30 May 2000, 22:15 GMT–15 June 2000, 12:00 GMT. Loubet et al. (2009) for the Site 1 and 2 measurements
and applied to the measured fluxes, resulting in an es-
timate of the fluxes at the canopy surface (Fz (zo)) (see
Sect. 3.3). No corrections to the data for the potential
effect of chemical production or consumption are made
here, as this effect is assessed by Nemitz et al. (2009a). Loubet et al. (2009) for the Site 1 and 2 measurements
and applied to the measured fluxes, resulting in an es-
timate of the fluxes at the canopy surface (Fz (zo)) (see
Sect. 3.3). 2.3
Field site and instrument locations A full site description and diagram are given in Sutton et
al. (2009b). The field site was intensively managed grass-
land of approximately 12 ha and the principal micrometeo-
rological measurement location (Site 1) was 380 m from the
western edge of the field, this being the main wind direction
during the experiment. There was a second micrometeoro-
logical measurement location (Site 2), which was 210 m east
of Site 1 and 36 m from the eastern edge of the field. Mea-
surements were made at Site 2 in order that any advection
of NH3 emitted from a farm located 610 m west of Site 1
(Hensen et al., 2009) could be identified and quantified (see
Sect. 2.5). Two gradient AMANDAs (each consisting of 3 iv) Given the different estimates of the 4 systems, gaps in
the data create an artificial change in the mean estimate
when one system goes offline or comes back online. To
avoid this artefact, gaps of <6 h in each instrument were
filled for Fz(1 m) and χ(1 m) according to the technique
described below. iv) Given the different estimates of the 4 systems, gaps in
the data create an artificial change in the mean estimate
when one system goes offline or comes back online. To
avoid this artefact, gaps of <6 h in each instrument were
filled for Fz(1 m) and χ(1 m) according to the technique
described below. v) As the flux measurements were made at two sites, dif-
ferent vertical flux divergence will apply due to horizon-
tal advection where local sources are present. The ad-
vection corrections for 1 m above d were calculated by v) As the flux measurements were made at two sites, dif-
ferent vertical flux divergence will apply due to horizon-
tal advection where local sources are present. The ad-
vection corrections for 1 m above d were calculated by v) As the flux measurements were made at two sites, dif-
ferent vertical flux divergence will apply due to horizon-
tal advection where local sources are present. The ad-
vection corrections for 1 m above d were calculated by Biogeosciences, 6, 819–834, 2009 www.biogeosciences.net/6/819/2009/ C. Milford et al.: Ammonia fluxes over intensively managed grassland www.biogeosciences.net/6/819/2009/ C. Milford et al.: Ammonia fluxes over intensively managed grassland C. Milford et al.: Ammonia fluxes over intensively managed grassland Table 3. Results of the blind testing of aqueous ammonium Quality Control standards. The blind standards were prepared by a separate
laboratory (ECN, Netherlands). On each occasion two standards were distributed and the difference in concentration measured by each
analyser was compared with the actual concentration difference to give an indication of the performance across the full measurement range. The % difference shown was calculated as (100–(1001[NH+
4 ]lab/1[NH+
4 ]QCstd)). Table 3. Results of the blind testing of aqueous ammonium Quality Control standards. The blind standards were prepared by a separate
laboratory (ECN, Netherlands). On each occasion two standards were distributed and the difference in concentration measured by each
analyser was compared with the actual concentration difference to give an indication of the performance across the full measurement range. The % difference shown was calculated as (100–(1001[NH+
4 ]lab/1[NH+
4 ]QCstd)). Date of test
[NH+
4 ]aq of
[NH+
4 ]aq of
% difference in concentration compared
QC standard 1 (µg l−1)
QC standard 2 (µg l−1)
with the unknown standards FRI FAL-D FAL-CH CEH
25 May 2000
22
84
44∗
13
21
16
31 May 2000
16
98
16
41
n/a
10
6 June 2000
273
38
4
21
n/a
−3
* This test was conducted on 22 May 2000 for FRI and a contaminated stripping solution container was found to be the cause;
n/a: not available. * This test was conducted on 22 May 2000 for FRI and a contaminated stripping solution container was found to be the cause;
n/a: not available. 06 jun
07 jun
08 jun
09 jun
χ(1m) (µg m-3)
0
10
20
30
31 may
01 jun
02 jun
03 jun
χ(1m) (µg m-3)
0
5
10
15
20
25 may
26 may
27 may
28 may
χ(1m) (µg m-3)
0
2
4
6
8
10
12
14
Pre-cut
Post-cut
Post-fert
CEH
FRI
FAL-D
CEH
FRI
FAL-D
FAL-CH
CEH
FRI
FAL-D
FAL-CH
Figure 1. Examples of χ(1m) for NH3 from the four different systems for pre-cutting, p
cutting and post-fertilizing periods. Centre for Ecology and Hydrology (CEH), Hunga
Forest Research Institute (FRI), German Federal Agricultural Research Institute (FAL
Swiss Federal Research Station for Agroecology and Agriculture (FAL-CH). FAL-CH
gradient data comences on 30 May 2000. Tick labels mark 00:00 GMT. Fig. 1. 3.2
Temporal inter-comparison of gradient measure-
ments The range of NH3 concentrations at 1 m and fluxes from the
4 different systems are shown for example days from the pre-
cutting, post-cutting and post-fertilizing periods (Figs. 1 and
2). These figures show that there were periods of close agree-
ment (e.g. 6 and 7 June 2000) and periods of substantial di-
vergence (e.g. 8 June 2000). It can be seen that for certain
periods (31 May to 2 June 2000) there were consistent con-
centration differences between the different instruments with
FAL-D generally reading higher concentrations than CEH
and FAL-CH. 06 jun
07 jun
08 jun
09 jun
χ(1m) (µg m-3)
0
10
20
30
Post-fert
CEH
FRI
FAL-D
FAL-CH The blind testing of the aqueous ammonium Quality Con-
trol standards by the different analysers did indicate periods
of significant concentration differences (Table 3). In partic-
ular, the result of FAL-D over-reading by 41% on 31 May
2000 is consistent with Fig. 1b. However, the small num-
ber of aqueous quality tests meant that it was not possible to
adjust the concentrations in an objective manner and so the
quality tests were used for interpretation rather than adjust-
ment. The flux intercomparison highlights the changing pattern
of NH3 exchange during the experiment; before the cutting
of the grass the flux was predominantly deposition to the sur-
face. After cutting of the grass, the NH3 exchange changed
to predominantly emission with emission fluxes of up to
760 ng m−2 s−1, whilst after fertilization the fluxes accord-
ing to individual analyzers increased up to 6000 ng m−2 s−1. The emission fluxes peaked in the daytime and were gener-
ally close to zero during nighttime. As with the comparison
of ammonia air concentrations, there were periods of close
agreement (e.g. 6 and 7 June 2000) and periods of substan-
tial disagreement (e.g. 1 and 8 June 2000). However, gen- Figure 1. Examples of χ(1m) for NH3 from the four different systems for pre-cutting, p
cutting and post-fertilizing periods. Centre for Ecology and Hydrology (CEH), Hungar
Forest Research Institute (FRI), German Federal Agricultural Research Institute (FAL
Swiss Federal Research Station for Agroecology and Agriculture (FAL-CH). FAL-CH
gradient data comences on 30 May 2000. Tick labels mark 00:00 GMT. Fig. 1. Examples of χ(1 m) for NH3 from the four different systems
for pre-cutting, post-cutting and post-fertilizing periods. C. Milford et al.: Ammonia fluxes over intensively managed grassland Examples of χ(1 m) for NH3 from the four different systems
for pre-cutting, post-cutting and post-fertilizing periods. Centre for
Ecology and Hydrology (CEH), Hungarian Forest Research Insti-
tute (FRI), German Federal Agricultural Research Institute (FAL-
D) and Swiss Federal Research Station for Agroecology and Agri-
culture (FAL-CH). FAL-CH gradient data comences on 30 May
2000. Tick labels mark 00:00 GMT. windspeed, stable conditions) were the main limitation to ob-
taining a complete flux dataset. Data on the gapfilling pro-
cedure revealed that FAL-CH had the greatest number of
gaps filled and that the median gap length varied from 15 min
(FAL-D) to 90 min (CEH). 3.1
Data processing The data processing procedures ensured that high data cov-
erage at individual sites was achieved even after periods of
malfunctioning and obstructed wind sectors were removed
(Table 2). Having four estimates of the flux lead to a
“mean gradient estimate” of the flux with an overall data
coverage of 98%. This highlights the advantage of hav-
ing a number of independent systems to achieve a robust
estimate of the flux. The data coverage was reduced to
69% if the fluxes which were calculated with less certainty
were removed, showing that meteorological conditions (low The gapfilling technique applied for each instrument in-
volved calculating the ratio of the individual flux measure-
ment to the mean estimate at the start and the end of the gap Biogeosciences, 6, 819–834, 2009 www.biogeosciences.net/6/819/2009/ 824 C. Milford et al.: Ammonia fluxes over intensively managed grassland C. Milford et al.: Ammonia fluxes over intensively managed grassland 825 06 jun
07 jun
08 jun
09 jun
Fz (ng m-2 s-1)
0
1000
2000
3000
4000
5000
6000
31 may
01 jun
02 jun
03 jun
Fz (ng m-2 s-1)
-200
0
200
400
600
800
22 may
23 may
24 may
25 may
Fz (ng m-2 s-1)
-75
-50
-25
0
25
50
Pre-cut
Post-cut
Post-fert
CEH
FRI
FAL-D
FAL-CH
FAL-CH
FAL-D
FRI
CEH
CEH
FRI
FAL-D
Figure 2. Example of NH3 fluxes (Fz) from the 4 different systems for pre-cutting,
cutting and post-fertilizing periods. Centre for Ecology and Hydrology (CEH), Hung
Forest Research Institute (FRI), German Federal Agricultural Research Institute (FAL-D
Swiss Federal Research Station for Agroecology and Agriculture (FAL-CH). FA
gradient data comences on 30 May 2000. Tick labels mark 00:00 GMT. Fig. 2. Example of NH3 fluxes (Fz) from the 4 different systems
for pre-cutting, post-cutting and post-fertilizing periods. Centre for
Ecology and Hydrology (CEH), Hungarian Forest Research Insti-
tute (FRI), German Federal Agricultural Research Institute (FAL-
D) and Swiss Federal Research Station for Agroecology and Agri-
culture (FAL-CH). FAL-CH gradient data comences on 30 May
2000. Tick labels mark 00:00 GMT. 06 jun
07 jun
08 jun
09 jun
Fz (ng m-2 s-1)
0
1000
2000
3000
4000
5000
6000
31 may
01 jun
02 jun
03 jun
Fz (ng m-2 s-1)
-200
0
200
400
600
800
22 may
23 may
24 may
25 may
Fz (ng m-2 s-1)
-75
-50
-25
0
25
50
Pre-cut
Post-cut
Post-fert
CEH
FRI
FAL-D
FAL-CH
FAL-CH
FAL-D
FRI
CEH
CEH
FRI
FAL-D Fig. 3. Comparison of (a) χ(1 m) and (b) NH3 flux at zo (corrected
for advection) measurements. German Federal Agricultural Re-
search Institute (FAL-D), Centre for Ecology and Hydrology (CEH)
and Swiss Federal Research Station for Agroecology and Agricul-
ture (FAL-CH). Fig. 3. Comparison of (a) χ(1 m) and (b) NH3 flux at zo (corrected
for advection) measurements. German Federal Agricultural Re-
search Institute (FAL-D), Centre for Ecology and Hydrology (CEH)
and Swiss Federal Research Station for Agroecology and Agricul-
ture (FAL-CH). t-
n
d
relatively few periods of advection from the farm were ob-
served due to winds occurring directly from the W occurring
for only a small period of the time, and accounted on aver-
age for 32% of the measured flux. 53.4.1
Comparisons of χ(1 m) and Fz(zo) against FAL-
CH FAL-CH was chosen as the reference for a preliminary anal-
ysis because it was present at Site 1 and also because it
helps to illustrate the variation in the response of the FAL-
D analyzer. Comparisons of χ(1 m) and Fz (zo) for each
instrument versus FAL-CH were conducted (FAL-CH data
not available for the pre-cutting period). FAL-D, χ(1 m) and
Fz (zo) agreed well with FAL-CH across the data range for
some of the time (Fig. 3). However, there were a consider-
able number of data points which greatly overestimated the
concentration and flux compared with FAL-CH. The fact that
this was not evident for the whole period suggests that there
was some variation in the accuracy of the FAL-D analyser
throughout the measurement period. For example, this could
be due to variation in the accuracy of the calibration. In-
accuracies in the measurement could be exacerbated in the
large concentration range, where the calibration is no longer
as robust. In addition, temperatures inside the analysers
reached 40 ◦C on some days. Although the concentration
measurements were corrected for temperature, inaccuracies
in the temperature correction could lead to overestimation 2000) were generally coupled with larger concentrations at
1 m, although this was not always the case (e.g. FAL-D and
FRI in the early hours of 8 June 2000). Scatter plots compar-
ing the NH3 concentration and flux for the different systems
are presented in Sect. 3.4. C. Milford et al.: Ammonia fluxes over intensively managed grassland As well as advection due
to the farm emissions, advection errors also occurred due to
NH3 emissions from the field itself, which were largest fol-
lowing fertilization of the field. Although these were larger in
absolute terms (−209 to 13 ng m−2 s−1), they represented a
smaller percentage of the measured fluxes at 1 to 2%. For the
week following 29 May 2000 (post-cut period), the farm and
field advection errors amounted on average to +3% and −4%
of the measured fluxes, respectively (Loubet et al., 2009). Figure 2. Example of NH3 fluxes (Fz) from the 4 different systems for pre cutting,
cutting and post-fertilizing periods. Centre for Ecology and Hydrology (CEH), Hung
Forest Research Institute (FRI), German Federal Agricultural Research Institute (FAL-D
Swiss Federal Research Station for Agroecology and Agriculture (FAL-CH). FA
gradient data comences on 30 May 2000. Tick labels mark 00:00 GMT. Fig. 2. Example of NH3 fluxes (Fz) from the 4 different systems
for pre-cutting, post-cutting and post-fertilizing periods. Centre for
Ecology and Hydrology (CEH), Hungarian Forest Research Insti-
tute (FRI), German Federal Agricultural Research Institute (FAL-
D) and Swiss Federal Research Station for Agroecology and Agri-
culture (FAL-CH). FAL-CH gradient data comences on 30 May
2000. Tick labels mark 00:00 GMT. 3.2
Temporal inter-comparison of gradient measure-
ments Centre for
Ecology and Hydrology (CEH), Hungarian Forest Research Insti-
tute (FRI), German Federal Agricultural Research Institute (FAL-
D) and Swiss Federal Research Station for Agroecology and Agri-
culture (FAL-CH). FAL-CH gradient data comences on 30 May
2000. Tick labels mark 00:00 GMT. erally the fluxes from the different systems showed a similar
structure and response to the management activities on the
field. The larger flux values (e.g. FAL-D on 1 and 2 June Biogeosciences, 6, 819–834, 2009 www.biogeosciences.net/6/819/2009/ www.biogeosciences.net/6/819/2009/ H 3.4
Inter-comparison of gradient measurements 53.4.1
Comparisons of χ(1 m) and Fz(zo) against FAL-
CH C. Milford et al.: Ammonia fluxes over intensively managed grassland Table 4. Summary of regression results of χ(1 m) of the individual systems versus the value for the mean gradient (χ(1 m)mg) expressed
in µg m−3. The regression is given first as a simple linear function (as shown in Fig. 4): χ(1 m)individ=cχ(1 m)mg+b and secondly as
log10[χ(1 m)individ]=e log10 [χ(1 m)mg]+d. Data from 3, 8, 9 and 10 June are excluded. Table 4. Summary of regression results of χ(1 m) of the individual systems versus the value for the mean gradient (χ(1 m)mg) expressed
in µg m−3. The regression is given first as a simple linear function (as shown in Fig. 4): χ(1 m)individ=cχ(1 m)mg+b and secondly as
log10[χ(1 m)individ]=e log10 [χ(1 m)mg]+d. Data from 3, 8, 9 and 10 June are excluded. Linear results
c (slope)
c 95% confidence limits
b (intercept)
b 95% confidence limits
n
r2
χ(1 m)FAL−D
0.99
[0.98, 1.01]
1.28
[1.19, 1.37]
1525
0.94
χ(1 m)CEH
0.82
[0.81, 0.83]
−0.21
[−0.27, −0.15]
1256
0.93
χ(1 m)FRI
1.15
[1.13, 1.17]
−0.70
[−0.81, −0.59]
1097
0.95
χ(1 m)FAL−CH
0.92
[0.91, 0.94]
−0.32
[−0.44, −0.20]
990
0.94
Log Transformed results
e (slope)
e 95% confidence limits
d (intercept)
d 95% confidence limits
n
r2
χ(1 m)FAL−D
0.89
[0.88, 0.90]
0.18
[0.17, 0.19]
1525
0.93
χ(1 m)CEH
0.98
[0.96, 1.00]
−0.12
[−0.13, −0.10]
1256
0.86
χ(1 m)FRI
1.25
[1.22, 1.28]
−0.20
[−0.22, −0.18]
1097
0.86
χ(1 m)FAL−CH
1.15
[1.11, 1.19]
−0.20
[−0.23, −0.17]
990
0.75 Fig. 4. Regression of χ(1 m) from each system against mean gradi-
ent concentration, χ(1 m)mg, for (a) German Federal Agricultural
Research Institute (FAL-D), (b) Centre for Ecology and Hydrology
(CEH), (c) Hungarian Forest Research Institute (FRI) and (d) Swiss
Federal Research Station for Agroecology and Agriculture (FAL-
CH). Data from 3, 8, 9 and 10 June 2000 are not included. 3.3
Assessment of advection corrections Estimates of the difference in the vertical flux due to advec-
tion (the advection error, 1Fz,adv) were derived from both
measurements and modelling by Loubet et al. (2009). The
modelled estimates of 1Fz,adv were applied to correct the
flux measurements (Fz(1 m)) in order to provide an estimate
of the fluxes at the canopy surface (Fz (zo)). Examples of
the magnitude of the advection errors in relation to the flux
measurements for particular periods are given in Loubet et
al. (2009). The modelled advection errors during the pre-cut
period due to the farm 610 m from Site 1 estimated by Loubet
et al. (2009), ranged between 0 to 27 ng m−2 s−1. However, Biogeosciences, 6, 819–834, 2009 www.biogeosciences.net/6/819/2009/ 826 C. Milford et al.: Ammonia fluxes over intensively managed grassland 3.4.2
Regressions of χ(1 m) and Fz(zo) against mean
gradient estimate After the data processing procedures were conducted as de-
tailed in Sect. 2.4 the “mean gradient estimate” was calcu-
lated for Fz(zo) and χ(1 m) (Fz(zo)mg and χ(1 m)mg), this is
the arithmetic mean of all available individual measurements
remaining in the dataset. These data include the corrections
for advection. A comparison of χ(1 m)mg versus the indi-
vidual systems was conducted (Fig. 4a–d). The regression
results are presented in Table 4, these include results cal-
culated from the simple linear function and also using log
transformed data. Data from 3, 8, 9 and 10 June 2000 were not included
in the regression because on these days there was signifi-
cant disagreement between the systems with FAL-D and FRI
giving higher estimates compared with the CEH and FAL-
CH estimates. To include the FAL-D and FRI estimates for
these days in the regression would bias the regression to-
wards FAL-D and FRI and might give a false impression of
the overall dataset. With data from 3, 8, 9 and 10 June removed, Fig. 4a–d
demonstrate that there was close agreement of χ(1 m) be-
tween each individual system and the mean estimate across
the full concentration range. As indicated by the tempo-
ral graphs, FAL-D and FRI showed slightly higher concen-
trations than the best estimate, whilst FAL-CH and CEH
showed slightly lower. The r2 value for all the linear regres-
sions was high (>0.93) (Table 4) which gives confidence in
the 4 systems and the values of χ(1 m)mg. Fig. 4. Regression of χ(1 m) from each system against mean gradi-
ent concentration, χ(1 m)mg, for (a) German Federal Agricultural
Research Institute (FAL-D), (b) Centre for Ecology and Hydrology
(CEH), (c) Hungarian Forest Research Institute (FRI) and (d) Swiss
Federal Research Station for Agroecology and Agriculture (FAL-
CH). Data from 3, 8, 9 and 10 June 2000 are not included. of concentrations. A similar effect was seen in some peri-
ods for the FRI AMANDA data of χ(1 m) and Fz (zo) (data
not shown). CEH AMANDA χ(1 m) and Fz (zo), underes-
timated the FAL-CH mini-WEDD values, but did not show
the variation in agreement demonstrated by FAL-D and FRI. of concentrations. A similar effect was seen in some peri-
ods for the FRI AMANDA data of χ(1 m) and Fz (zo) (data
not shown). C. Milford et al.: Ammonia fluxes over intensively managed grassland 827 Fig. 5. Regression of NH3 flux from each system against mean
gradient flux for (a) German Federal Agricultural Research Insti-
tute (FAL-D), (b) Centre for Ecology and Hydrology (CEH), (c)
Hungarian Forest Research Institute (FRI) and (d) Swiss Federal
Research Station for Agroecology and Agriculture (FAL-CH). Data
from 3, 8, 9 and 10 June 2000 are not included. χ(1m) (µg m-3)
0
5
10
15
20
25
30
35
21 may
23 may
25 may
27 may
29 may
31 may
02 jun
04 jun
06 jun
08 jun
10 jun
12 jun
14 jun
16 jun
Fz(z0) (ng m-2 s-1)
-500
0
500
1000
1500
2000
2500
3000
3500
a)
b)
χ(1m)ag
χ(1m)mg
Fz(z0)ag
Fz(z0)mg
Figure 6. Mean gradient estimate of a) χ(1m)mg and b) net NH3 flux (Fz(zo)mg) showing
response to management activities. The alternative gradient estimate is also shown (χ(1m)ag
and Fz(zo)ag ) on 3, 8, 9 and 10 June 2000, these are days with high instrument uncertainty (see
text). Vertical lines indicate cutting (dashed line), removal of the grass from the field (dotted
line) and NH4NO3 fertilization (solid line). Fig. 6. Mean gradient estimate of (a) χ(1 m)mg and (b) net NH3
flux (Fz(zo)mg) showing response to management activities. The
alternative gradient estimate is also shown (χ(1 m)ag and Fz(zo)ag
on 3, 8, 9 and 10 June 2000, these are days with high instrument
uncertainty (see text). Vertical lines indicate cutting (dashed line),
removal of the grass from the field (dotted line) and NH4NO3 fer-
tilization (solid line). Fig. 5. Regression of NH3 flux from each system against mean
gradient flux for (a) German Federal Agricultural Research Insti-
tute (FAL-D), (b) Centre for Ecology and Hydrology (CEH), (c)
Hungarian Forest Research Institute (FRI) and (d) Swiss Federal
Research Station for Agroecology and Agriculture (FAL-CH). Data
from 3, 8, 9 and 10 June 2000 are not included. χ(1m) (µg m-3)
0
5
10
15
20
25
30
35
21 may
23 may
25 may
27 may
29 may
31 may
02 jun
04 jun
06 jun
08 jun
10 jun
12 jun
14 jun
16 jun
Fz(z0) (ng m-2 s-1)
-500
0
500
1000
1500
2000
2500
3000
3500
a)
b)
χ(1m)ag
χ(1m)mg
Fz(z0)ag
Fz(z0)mg
Figure 6. Mean gradient estimate of a) χ(1m)mg and b) net NH3 flux (Fz(zo)mg) showing
response to management activities. C. Milford et al.: Ammonia fluxes over intensively managed grassland Therefore, this alternative estimate was
also provided to other end-users of the data. Such a log transformation for the fluxes data is less suited,
due to the existence of negative fluxes. The most nega-
tive flux, recorded for FAL-D was −315 ng m−2 s−1 and
a transformation of (log10[Fz(zo)individ + 320]=e log10[Fz
(zo)mg + 320] + d) was tested. However, this did not succeed
in normalizing the data, while the variance was again larger
at smaller values (due to scatter at smaller fluxes). For this
reason, only the linear relationships are shown for the fluxes. Such a log transformation for the fluxes data is less suited,
due to the existence of negative fluxes. The most nega-
tive flux, recorded for FAL-D was −315 ng m−2 s−1 and
a transformation of (log10[Fz(zo)individ + 320]=e log10[Fz
(zo)mg + 320] + d) was tested. However, this did not succeed
in normalizing the data, while the variance was again larger
at smaller values (due to scatter at smaller fluxes). For this
reason, only the linear relationships are shown for the fluxes. The regression of Fz(zo)mg versus Fz(zo) from the indi-
vidual systems (Fig. 5a–d, Table 5) demonstrates that once
the four uncertain days (3, 8, 9 and 10 June) were removed
from the regression then FAL-D tended to underestimate the
flux compared with the mean gradient estimate, as did CEH. By contrast, FRI and FAl-CH both overestimated the mean
gradient flux by about 10% compared with the mean gradi-
ent estimate. C. Milford et al.: Ammonia fluxes over intensively managed grassland The alternative gradient estimate is also shown (χ(1m)ag
and Fz(zo)ag ) on 3, 8, 9 and 10 June 2000, these are days with high instrument uncertainty (see
text). Vertical lines indicate cutting (dashed line), removal of the grass from the field (dotted
line) and NH4NO3 fertilization (solid line). Fig. 6. Mean gradient estimate of (a) χ(1 m)mg and (b) net NH3
flux (Fz(zo)mg) showing response to management activities. The
alternative gradient estimate is also shown (χ(1 m)ag and Fz(zo)ag
on 3, 8, 9 and 10 June 2000, these are days with high instrument
uncertainty (see text). Vertical lines indicate cutting (dashed line),
removal of the grass from the field (dotted line) and NH4NO3 fer-
tilization (solid line). χ(1m) (µg m-3)
0
5
10
15
20
25
30
35
21 may
23 may
25 may
27 may
29 may
31 may
02 jun
04 jun
06 jun
08 jun
10 jun
12 jun
14 jun
16 jun
Fz(z0) (ng m-2 s-1)
-500
0
500
1000
1500
2000
2500
3000
3500
a)
b)
χ(1m)ag
χ(1m)mg
Fz(z0)ag
Fz(z0)mg Fig. 5. Regression of NH3 flux from each system against mean
gradient flux for (a) German Federal Agricultural Research Insti-
tute (FAL-D), (b) Centre for Ecology and Hydrology (CEH), (c)
Hungarian Forest Research Institute (FRI) and (d) Swiss Federal
Research Station for Agroecology and Agriculture (FAL-CH). Data
from 3, 8, 9 and 10 June 2000 are not included. Figure 6. Mean gradient estimate of a) χ(1m)mg and b) net NH3 flux (Fz(zo)mg) showing
response to management activities. The alternative gradient estimate is also shown (χ(1m)ag
and Fz(zo)ag ) on 3, 8, 9 and 10 June 2000, these are days with high instrument uncertainty (see
text). Vertical lines indicate cutting (dashed line), removal of the grass from the field (dotted
line) and NH4NO3 fertilization (solid line). Fig. 6. Mean gradient estimate of (a) χ(1 m)mg and (b) net NH3
flux (Fz(zo)mg) showing response to management activities. The
alternative gradient estimate is also shown (χ(1 m)ag and Fz(zo)ag
on 3, 8, 9 and 10 June 2000, these are days with high instrument
uncertainty (see text). Vertical lines indicate cutting (dashed line),
removal of the grass from the field (dotted line) and NH4NO3 fer-
tilization (solid line). similar for the full range of concentrations, and therefore this
is more appropriate for the comparison of the data than the
log transformed data. operating correctly. 3.4.2
Regressions of χ(1 m) and Fz(zo) against mean
gradient estimate CEH AMANDA χ(1 m) and Fz (zo), underes-
timated the FAL-CH mini-WEDD values, but did not show
the variation in agreement demonstrated by FAL-D and FRI. The regression results for the log transformed data show
lower values of r2 with respect to the linear results. This
is due to increased scatter of small concentrations near the
detection limit in the log transformed dataset, resulting in a
greater variance in the dataset for low concentrations. By
contrast, in the simple linear plot (Fig. 4) the variance in y is Biogeosciences, 6, 819–834, 2009 www.biogeosciences.net/6/819/2009/ 3.5.2
Post-cutting, pre-fertilizing The accumulated flux was −0.03, 0.50 and 3.41 kg N ha−1
for the pre-cutting, post cutting and post fertilizing periods,
respectively. This accumulated flux up to 10 days after the
fertilization represented 3.2% of the N applied, or an equiva-
lent of 3.6% if the post-cutting emissions were included. Immediately after cutting (29 May 2000, 06:00 GMT) the
NH3 flux switched to emission. The emission had a
diurnal pattern with very small fluxes during night-time
and emission fluxes increasing during the daytime; daily
peak emission values were 135 to 700 ng m−2 s−1. The
mean flux during the cutting period was 100 ng m−2 s−1 (all
data) or 138 ng m−2 s−1 (only data which passes the mi-
cromet criteria). These values are equivalent to 71 and
98 g N ha−1 day−1, respectively. 3.5.1
Pre-cutting Prior to cutting of the grass, the flux was predominantly de-
position to the surface (see Fig. 2 for typical diurnal course). The mean flux of the pre-cutting period was −5.8 ng m−2 s−1
if all data were included and −6.0 ng m−2 s−1 if only data
which passed the micrometeorological criteria were in-
cluded. Any emission which was observed was generally
small, the maximum emission observed over the period was
42 ng m−2 s−1. The intensive grassland was generally acting
as a sink for NH3 during this period. There was a rapid increase in NH3 flux observed following
the fertilization with N (5 June 2000, 06:00 GMT), with val-
ues peaking at 3820 ng m−2 s−1. During the first two nights
after fertilization (5 June and 6 June) there were mean noc-
turnal emissions of 1050 ng m−2 s−1 and 150 ng m−2 s−1, re-
spectively. The mean flux over the whole post-fertilizing pe-
riod was 474 ng m−2 s−1 (all data) or 559 ng m−2 s−1 (only
data which passed the micrometeorological criteria). These
values are equivalent to 337 and 398 g N ha−1 day−1, respec-
tively. The emission flux decreased on 11 June 2000, but was
still up to 565 ng m−2 s−1 13 days after cutting. 3.5
NH3 concentration and flux in relation to manage-
ment activities c (slope)
c 95% confidence limits
b (intercept)
b 95% confidence limits
n
r2
Fz (zo)FAL−D
0.89
[0.88, 0.90]
33.45
[28.68, 38.22]
1525
0.95
Fz (zo)CEH
0.68
[0.67, 0.69]
−8.86
[−10.75, −6.97]
1256
0.96
Fz (zo)FRI
1.08
[1.07, 1.09]
−5.36
[−9.16, −1.55]
1097
0.98
Fz (zo)FAL−CH
1.10
[1.09, 1.11]
−5.58
[−11.16, 0.00]
990
0.98 Table 6. Variations in 15 min measurements of NH3 concentration at 1 m from the mean gradient, χ(1 m)mg, throughout different measure-
ment periods, before and after micromet filtering. Period
Dataset
Mean
µg m−3
Stdev
µg m−3
Median
µg m−3
Min
µg m−3
Max
µg m−3
n
Data
Coverage (%)
Pre-cutting
All data
3.22
2.06
2.64
0.29
15.00
705
93.6
Pre-cutting
After filtering
3.25
2.03
2.64
0.35
10.81
592
78.6
Post-cutting/
Pre-fertilizing
All data
4.32
2.15
3.96
0.58
12.32
675
98.7
Post-cutting/
Pre-fertilizing
After filtering
4.50
2.46
3.86
0.58
12.32
393
57.5
Post-fertilizing
All data
8.38
5.55
6.78
0.92
31.52
970
99.7
Post-fertilizing
After filtering
9.21
5.76
7.38
0.92
31.52
667
68.6
Pre-cutting: 21 May, 10:00 GMT–29 May, 06:00 GMT; Post-cutting/Pre-fertilizing: 29 May, 06:00 GMT–5 June, 06:00 GMT; Post fertilizing: 5 June, 06:00 GMT–15 June,
12:00 GMT Table 6. Variations in 15 min measurements of NH3 concentration at 1 m from the mean gradient, χ(1 m)mg, throughout different measure-
ment periods, before and after micromet filtering. Pre-cutting: 21 May, 10:00 GMT–29 May, 06:00 GMT; Post-cutting/Pre-fertilizing: 29 May, 06:00 GMT–5 June, 06:00 GMT; Post fertilizing: 5 June, 06:00 GMT–15 June,
12:00 GMT 3.5
NH3 concentration and flux in relation to manage-
ment activities The resulting mean gradient concentrations of NH3 at 1 m
(χ(1 m)mg) and flux (Fz(zo)mg) for the whole period (Fig. 6)
demonstrate clearly the effect of the management activities
(cutting and fertilizing) on the concentration and flux. In
addition to the mean gradient estimate, Fig. 6 also shows
the alternative gradient estimate for χ(1 m) and Fz(zo) on
3, 8, 9 and 10 June. Statistics for the mean gradient esti-
mate of χ(1 m) and Fz(zo) were calculated for the three pe-
riods: i) pre-cutting; ii) post-cutting/pre-fertilizing and iii)
post-fertilizing (Tables 6 and 7). These data have been cor-
rected for both advection and storage errors. The regression of Fz(zo)mg versus Fz(zo) from the indi-
vidual systems (Fig. 5a–d, Table 5) demonstrates that once
the four uncertain days (3, 8, 9 and 10 June) were removed
from the regression then FAL-D tended to underestimate the
flux compared with the mean gradient estimate, as did CEH. By contrast, FRI and FAl-CH both overestimated the mean
gradient flux by about 10% compared with the mean gradi-
ent estimate. The effect of the data filtering for micrometeorological
restrictions (Sect. 2.4, step vii) is illustrated, with slightly
larger χ(1 m) and Fz(zo) from the filtered dataset. This re-
flects a bias in the filtered dataset, that more data were ex-
cluded from night-time conditions, when χ(1 m) and Fz(zo)
were smallest. As a result of the disagreement between systems on the
3, 8, 9 and 10 June an “alternative gradient estimate” for χ
(1 m) and Fz(zo) was proposed for these days. The alterna-
tive gradient estimate (subscript ag) consisted of the mean of
the two systems (CEH and FAL-CH). It was not considered
that there was sufficient justification to remove the high mea-
surements from the mean dataset. However, it was suspected
that on these days the two high systems might not have been Biogeosciences, 6, 819–834, 2009 www.biogeosciences.net/6/819/2009/ C. Milford et al.: Ammonia fluxes over intensively managed grassland 828 Table 5. Summary of linear regression results of Fz(zo) of the individual systems versus Fz(zo)mg expressed in ng m−2 s1, given as
Fz(zo)individ=c Fz (zo)mg+b. The estimates are derived from the data shown in Fig. 5. Table 5. Summary of linear regression results of Fz(zo) of the individual systems versus Fz(zo)mg expressed in ng m−2 s1, given as
Fz(zo)individ=c Fz (zo)mg+b. The estimates are derived from the data shown in Fig. 5. 3.5.4
Inter-instrument differences for χ(1 m) and Fz(zo)
for main management periods It is relevant to consider the statistics of χ(1 m) and Fz(zo)
between the different ammonia analyzers for the main man-
agement periods. With longer-averaging periods the uncer-
tainties between the different systems decreased (Table 8). Biogeosciences, 6, 819–834, 2009 www.biogeosciences.net/6/819/2009/ C. Milford et al.: Ammonia fluxes over intensively managed grassland 829 Table 7. Variations in 15 min measurements of mean gradient net NH3 flux (Fz(zo)mg) throughout different measurement periods, before
and after micromet filtering. Period
Dataset
Mean
Stdev
Median
Min
Max
n
Data
ng m−2 s−1
ng m−2 s−1
ng m−2 s−1
ng m−2 s−1
ng m−2 s−1
Coverage (%)
Pre-cutting
All data
−5.8
13.1
−6.0
−64.2
42
705
93.6
Pre-cutting
After filtering
−6.0
13.9
−6.7
−64.2
42
592
78.6
Post-cutting/ Pre-fertilizing
All data
99.8
131.7
52.4
−54.3
703
675
98.7
Post-cutting/ Pre-fertilizing
After filtering
138.2
143.7
97.6
−49.2
703
393
57.5
Post-fertilizing
All data
474.0
611.0
191.7
−2.2
3821
970
99.7
Post-fertilizing
After filtering
559.4
603.5
304.5
1.4
3821
667
68.6
Pre-cutting: 21 May, 10:00 GMT–29 May, 06:00 GMT; Post-cutting/Pre-fertilizing: 29 May, 06:00 GMT–5 June, 06:00 GMT; Post fertilizing: 5 June 06:00 GMT–15 June,
12:00 GMT Table 7. Variations in 15 min measurements of mean gradient net NH3 flux (Fz(zo)mg) throughout different measurement periods, before
and after micromet filtering. Table 7. Variations in 15 min measurements of mean gradient net NH3 flux (Fz(zo)mg) throughout different measurement periods, before
and after micromet filtering. Pre-cutting: 21 May, 10:00 GMT–29 May, 06:00 GMT; Post-cutting/Pre-fertilizing: 29 May, 06:00 GMT–5 June, 06:00 GMT; Post fertilizing: 5 June 06:00 GMT–15 June,
12:00 GMT May, 06:00 GMT; Post-cutting/Pre-fertilizing: 29 May, 06:00 GMT–5 June, 06:00 GMT; Post fertilizing: 5 June 06:00 GMT–15 June, Table 8. Standard errors (SE) and % standard errors in the concen-
tration and flux estimates between the 4 ammonia sampling systems
according to the different management periods (for the full dataset). Table 8. Standard errors (SE) and % standard errors in the concen-
tration and flux estimates between the 4 ammonia sampling systems
according to the different management periods (for the full dataset). Table 8. Standard errors (SE) and % standard errors in the concen-
tration and flux estimates between the 4 ammonia sampling systems
according to the different management periods (for the full dataset). Fig. 7. 3.5.4
Inter-instrument differences for χ(1 m) and Fz(zo)
for main management periods Time-course of the uncertainty in mean Fz(zo) and χ(1 m)
presenting 1 hourly running medians of the 15 min estimates, shown
together with the magnitude of advection and storage errors: (a)
Percentage and absolute Standard Error (SE) in the measured am-
monia flux, (b) % SE in χ(1 m), (c) absolute storage error (Fz,sto)
and Fz,sto as a % of Fz(1 m), (d) absolute advection error (Fz,adv),
sum of the modelled advection errors at z=1 m due to the experi-
mental field and farm and Fz,adv as a % of Fz(1 m). Vertical lines
indicate cutting (dashed line), removal of the grass from the field
(dotted line) and NH4NO3 fertilization (solid line). Variable
Pre-cutting
Post-cutting
Post-fertilizing
Mean χ(1 m)
3.22
4.32
8.38
SE in χ(1 m)
0.55
0.81
1.24
% SE in χ(1 m)
17.0
18.7
14.8
Mean Fz(zo)
−5.8
99.8
474.1
SE in Fz(zo)
4.4
33.1
93.7
% SE in Fz(zo)
76.2
33.2
19.8 For the four different measurement systems, the relative stan-
dard error in χ(1 m) was similar for the three measurement
periods at 15–20%. By contrast, the relative standard error in
Fz(zo) varied substantially between the three measurement
periods. This was as expected, with better agreement be-
tween the different ammonia analyzers being found for the
periods with larger fluxes. 3.6
Time-course of uncertainties in the mean estimates
of χ(1 m) and Fz(zo) 7d) were a similar mag-
nitude to those for storage, with most values being less than
10% of Fz. Although larger absolute advection errors were
observed post cutting and post fertilizing, the values as a per-
centage of the measured flux were smaller for these periods
than for the pre cutting period. teractions with other atmospheric components (Sutton et al.,
2009a). The availability of replicate instruments in the present ex-
periment also highlight the need for caution when interpret-
ing results from the more usual situation of a single ammo-
nia flux detection system. Table 8 shows that the different
instruments used here agreed to within 20% (standard error)
for ammonia concentrations in the three management peri-
ods. By contrast, while the flux by the three systems had a
standard error of 20% for the post-fertilization period (when
fluxes were largest and easiest to detect), the uncertainties
were larger for the pre-fertilization periods. This indicated
an uncertainty of 33% for the post-cut period and 76% for the
pre-cut period. As demonstrated by Fig. 2, the uncertainties
can be even larger for 15 min measurements (even leading to
uncertainties in flux direction). As a result, great caution is
needed when interpreting such NH3 flux data when only one
system is available. In this situation, it is thus essential to
perform regular calibration and also zero-concentration tests
(switching off air supply and checking the zero of the ana-
lyzer) and zero-flux tests (e.g. by bringing inlets to a com-
mon height and demonstrating that the measured flux is zero,
Sutton et al., 1993a). Intercomparisons of measured ammo-
nia fluxes with process models must similarly recognize the
substantial uncertainty in the measured fluxes. 3.6
Time-course of uncertainties in the mean estimates
of χ(1 m) and Fz(zo) The availability of up to four parallel measurements of the
ammonia concentrations and fluxes enabled an assessment of
the uncertainty in the mean estimates of Fz(zo) and χ(1 m). The standard error (SE) of the mean 15 min values was cal-
culated as σn−1/√n, where σn−1 is the sample standard de-
viation and n is number of estimates available for a given
15 min period. Hence the magnitude of the SE depended on
both the level of agreement of the denuders and the number
of denuders operating at a given time. The time course of the
SE is presented for Fz(zo) and χ(1 m) (Fig. 7a and b). The
SE of the 15 min values in the pre-cut period with substan-
tial bi-directional exchange was typically around 60%, with
absolute SE values of 10 to 20 ng m−2 s−1 (Fig. 7a). Follow-
ing cutting, the errors differed diurnally with daytime SE in
the flux typically 40%. The % SE was smallest following Fig. 7. Time-course of the uncertainty in mean Fz(zo) and χ(1 m)
presenting 1 hourly running medians of the 15 min estimates, shown
together with the magnitude of advection and storage errors: (a)
Percentage and absolute Standard Error (SE) in the measured am-
monia flux, (b) % SE in χ(1 m), (c) absolute storage error (Fz,sto)
and Fz,sto as a % of Fz(1 m), (d) absolute advection error (Fz,adv),
sum of the modelled advection errors at z=1 m due to the experi-
mental field and farm and Fz,adv as a % of Fz(1 m). Vertical lines
indicate cutting (dashed line), removal of the grass from the field
(dotted line) and NH4NO3 fertilization (solid line). Biogeosciences, 6, 819–834, 2009 www.biogeosciences.net/6/819/2009/ C. Milford et al.: Ammonia fluxes over intensively managed grassland 830 fertilization with daytime values typically 15%, increasing
to typically 30% from 10 June. The overall median uncer-
tainty in the 15 min estimates of χ(1 m) was 18%, with val-
ues mostly in the range 5 to 50% (Fig. 7b). Regarding the
storage and advection calculations, for much of the period
these were rather small, but they were important in specific
instances. The storage errors were generally less than 10% of
the measured flux, although individual values occurred in the
range 25–125% during periods of rapidly changing χ(1 m)
(Fig. 7c). The advection errors (Fig. 4.1
Inter-comparison of gradient measurements Overall, the inter-comparison of gradient measurements was
encouraging, with three of the flux measurements being
within 11% of the mean estimate and the other within 32%
(Table 5). There was also close agreement achieved across
the concentration range, except for some days where over-
estimation of concentration is suspected to have occurred in
some of the systems, possibly due to high operating temper-
atures and inaccuracies in the measurement which could be
exacerbated in the large concentration range, where the cal-
ibration is no longer as robust. The inter-comparison high-
lighted the need for regular calibration of flux gradient sys-
tems and regular quality standard checks. As concluded by
Harrison and Kitto (1990), operator differences can induce
the same amount of variation in NH3 measurements as dif-
ferent measuring techniques and, although techniques such
as AMANDA and the WEDD have been shown to be reli-
able in measuring NH3, operators have to be vigilant in their
running of these systems. A reliable clean deionised water
supply, regular changing of pump tubing and regulation of
instrument operating temperature are all essential to main-
tain the reliability of these systems. 4.2
Influence of management activities on NH3 flux The present measurements support previous findings of en-
hanced emissions following grass cutting (Sutton et al.,
1997, 2001a; Milford et al., 2001), with a mean flux of
98 g N ha−1 day−1 after cutting compared with measure-
ments of predominantly deposition before cutting. These
emissions appear to be a consequence of altered N processing
in the remaining sward rather than the lying cut grass (Sutton
et al., 1997; David et al., 2009; Mattsson et al., 2008). The
modelling of Sutton et al. (2001) and Riedo et al. (2002) ex-
plained this on the basis of a larger ammonia compensation
point of the remaining plant (Riedo et al., 2002) and these in-
teractions are further investigated by Burkhardt et al. (2009),
Personne et al., (2009) and Sutton et al. (2009a). As expected, emissions were also enhanced following fer-
tilization, with a mean flux of 398 g N ha−1 day−1. These
fluxes are somewhat larger in magnitude to those observed
earlier (Sutton et al., 1997; Milford et al., 2001). Expressed
as the % emission of fertilizer N applied, in the present case
3.2% of the N (applied as calcium ammonium nitrate) was
lost as ammonia within 10 days of application. The emis-
sion factor for ammonium nitrate fertilizer applied to grass-
lands in the UK is 1.6% of the applied N (van der Weer-
den and Jarvis, 1997). By comparison using AMANDA flux
measurement, Milford et al. (2001) also estimated that 1.6%
of fertilizer N (as ammonium nitrate) was lost as ammonia
from a Scottish grassland. Strictly, the value from Milford
et al. (2001) is not comparable with the present study, since Having four independent systems did result in a robust
final dataset with an extremely high data coverage of NH3
concentrations and fluxes of 98%. The main restriction on
estimates was a filter according to strict micrometeorologi-
cal criteria, which reduced the flux data coverage to 69% if
these filter criteria were applied. With typically 50–70% data
coverage of concentration and 30–50% for flux being more
common in other NH3 studies (e.g., Erisman et al., 1998;
Sutton et al., 2001b; Horvath et al., 2005) and with the con-
tinuous record of uncertainty from the replicate systems, it is
clear that this dataset is unique in coverage and robustness. Biogeosciences, 6, 819–834, 2009 5
Conclusions An inter-comparison of four NH3 gradient measurement sys-
tems was conducted over intensively managed grassland dur-
ing a four week period in May–June 2000 as part of the
GRAMINAE Integrated Experiment. The inter-comparison
was encouraging, with three of the flux measurements be-
ing within 11% of the mean and the other within 32%. The inter-comparison highlighted the need for regular cali-
bration of flux gradient systems and regular checks against
quality standards. Operating four systems resulted in ex-
ceptionally high data coverage of measured ammonia fluxes
(98%) and provided a continuous record of the uncertainty
of the data. The measurements supported previous findings
of enhanced emissions from grass cutting with a mean daily
flux of 98 g N ha−1 day−1 after cutting compared to measure-
ments of predominantly deposition before cutting. Emissions
were also enhanced following fertilization, with a mean flux
of 398 g N ha−1 day−1. The results of this intercomparison
provide a robust dataset for the evaluation of the processes
controlling ammonia exchange, while indicating the need for
caution in interpreting unreplicated ammonia flux measure-
ments. It is clear that measurement of ambient ammonia
concentration and flux still remains a challenge and future
research should focus on continuing to improve ammonia
measurement techniques and the reliability of flux measure-
ments. Dabney, S. M. and Bouldin, D. R., Fluxes of ammonia over an al-
falfa field, Agron. J., 77, 572–578, 1985. David, M., Roche, R., Mattsson, M., Sutton, M. A., D¨ammgen, U.,
Schjoerring, J. K., and Cellier, P.: The effects of management on
ammonia fluxes over a cut grassland as measured by use of dy-
namic chambers, Biogeosciences Discuss., 6, 1599–1623, 2009,
http://www.biogeosciences-discuss.net/6/1599/2009/. Denmead, O. T., Simpson, J. R., and Freney, J. R.: Ammonia flux
into the atmosphere from a grazed pasture, Science, 185, 609–
610, 1974. Denmead, O. T., Freney, J. R., and Simpson, J. R.: A closed am-
monia cycle within a plant canopy, Soil. Biol. Biochem., 8, 161–
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Influence of management activities on NH3 flux As such it provides an important resource for interpretation
of ammonia fluxes in relation to bioassays, models and in- Biogeosciences, 6, 819–834, 2009 www.biogeosciences.net/6/819/2009/ 831 C. Milford et al.: Ammonia fluxes over intensively managed grassland their estimate referred to total emissions (including from
grass cutting). Hence if the present losses following cutting
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trogen, further emphasizing the larger values of this study. The larger emissions measured here are possibly a result of
the high temperatures which were observed, particularly in
the latter period of the campaign. The mean air temperature
at 1 m was 17.6◦C during 5–15 June 2000, although daily
maximums reached 39◦C (Nemitz et al., 2009b). The poten-
tial for NH3 emission increases with increasing temperature
due to the dependence of solubility of NH3 on temperature,
which affects the stomatal compensation point (Farquhar et
al., 1980) as well as the NH3 gaseous concentration at the
soil and litter surface (Nemitz et al., 2001b, 2004). Acknowledgements. The authors gratefully acknowledge funding
from the European Commission under the GRAMINAE project
and from a wide range of national funding agencies, in particular
the UK Defra (AEQ Division). Final synthesis of these results
was conducted under the EC NitroEurope Integrated Project. The
authors also gratefully acknowledge support from the Spanish
Ministry of Education and Science through the Juan de la Cierva
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Permeability of the peroxisomal membrane: lessons from the glyoxylate cycle
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Frontiers in physiology
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Edited by: y
Vladimir I. Titorenko, Concordia
University, Canada
Reviewed by:
J. Kalervo Hiltunen, University of
Oulu, Finland
Vladimir I. Titorenko, Concordia
University, Canada
*Correspondence:
Andreas Hartig, Department of
Biochemistry and Cell Biology, Max
F. Perutz Laboratories, University of
Vienna, Dr. Bohrgasse 9,
Vienna A-1030, Austria
e-mail: andreas.hartig@univie.ac.at Reviewed by:
J. Kalervo Hiltunen, University of
Oulu, Finland
Vladimir I. Titorenko, Concordia
University, Canada *Correspondence:
Andreas Hartig, Department of
Biochemistry and Cell Biology, Max
F. Perutz Laboratories, University of
Vienna, Dr. Bohrgasse 9,
Vienna A-1030, Austria
e-mail: andreas.hartig@univie.ac.at REVIEW ARTICLE
published: 14 August 2013
doi: 10.3389/fphys.2013.00204 published: 14 August 2013
doi: 10.3389/fphys.2013.00204 INTRODUCTION The single membrane separating the peroxisomal matrix from
the surrounding was shown to be permeable only for small
molecules such as tri- and dicarboxylates and amino acids
(Antonenkov et al., 2009). Specific transporters have not yet
been identified but features compatible with pore-like structures
were demonstrated (Verleur and Wanders, 1993; Antonenkov and
Hiltunen, 2006). Peroxisomes are defined as organelles encasing the metabolism
of H2O2. This highly reactive molecule is generated by several
oxidative reactions and degraded inside the organelle by abun-
dant amounts of catalase. In addition, peroxisomes fulfill a
number of important metabolic functions for eukaryotic cells
requiring an active communication between the peroxisomal
lumen and the cytosol or other organelles. Major functions
include the β-oxidation of fatty acids, parts of the glyoxy-
late cycle and parts of the photorespiration (Hu et al., 2012;
Waterham and Wanders, 2012). For β-oxidation fatty acids
have to enter peroxisomes, and the resulting acetyl-CoA is
distributed throughout the cells. Photorespiration in plants
involves mitochondria, chloroplasts and peroxisomes and there-
fore requires extensive metabolite exchange. A key intermediate
shared by the photorespiration and the glyoxylate cycle is gly-
oxylate, usually generated and metabolized inside peroxisomes. The glyoxylate cycle was originally considered a metabolic pro-
cess localized to peroxisomes (Breidenbach and Beevers, 1967). This localization was rationalized as a means to increase the
efficiency of the flux of intermediates. However, the finding
that parts of the whole cycle are extra-peroxisomal in the yeast
Saccharomcyes cerevisiae and in the plant Arabidopsis thaliana
indicated, that a functional glyoxylate cycle requires the trans-
fer of various metabolites across the peroxisomal membrane,
too (Minard and McAlister-Henn, 1991; Courtois-Verniquet
and Douce, 1993; Taylor et al., 1996; Kunze et al., 2002;
Pracharoenwattana et al., 2007). The nature of the molecules
crossing the peroxisomal membrane varies with the organism
under study. In this review we will address the questions how and where
glyoxylate is generated and how metabolites of the two most
prominent pathways partially localized to peroxisomes, the gly-
oxylate cycle and the photorespiration, are thought to cross the
membrane on their way in and out of the peroxisomes. Markus Kunze 1 and Andreas Hartig 2* 1 Department of Pathobiology of the Nervous System, Center for Brain Research, Medical University of Vienna, Vienna, Austria
2 Department of Biochemistry and Cell Biology, Max F. Perutz Laboratories, University of Vienna, Vienna, Austria Glyoxylate serves as intermediate in various metabolic pathways, although high
concentrations of this metabolite are toxic to the cell. In many organisms glyoxylate
is fed into the glyoxylate cycle. Enzymes participating in this metabolism are located
on both sides of the peroxisomal membrane. The permeability of this membrane
for small metabolites paves the way for exchange of intermediates between proteins
catalyzing consecutive reactions. A model, in which soluble enzymes accumulate in
close proximity to both ends of pore-like structures forming a transmembrane metabolon
could explain the rapid and targeted exchange of intermediates. The metabolites passing
the membrane differ between the three model organisms Saccharomyces cerevisiae,
Arabidopsis thaliana, and Candida albicans, which reflects the ease of evolutionary
adaptation processes whenever specific transporter proteins are not involved. The atypical
permeability properties of the peroxisomal membrane together with a flexible structural
arrangement ensuring the swift and selective transport across the membrane might
represent the molecular basis for the functional versatility of peroxisomes. Keywords: glyoxylate, glyoxylate cycle, peroxisomes, pore forming protein, membrane permeability, metabolite
transfer, metabolon, photorespiration Keywords: glyoxylate, glyoxylate cycle, peroxisomes, pore forming protein, membrane permeability, metabolite
transfer, metabolon, photorespiration REVIEW ARTICLE
published: 14 August 2013
doi: 10.3389/fphys.2013.00204 REVIEW ARTICLE
published: 14 August 2013
doi: 10.3389/fphys.2013.00204 PEROXISOMES AND GLYOXYLATE METABOLISM Inside peroxisomes the accumulation of glyoxylate is prevented by
conversion into glycine making use of a transaminase reaction or
by condensation with acetyl-CoA into malate catalyzed by malate
synthase (MLS), one of the key enzymes of the glyoxylate path-
way (for reviews see Wanders and Waterham, 2006; Theodoulou
and Eastmond, 2012). Glyoxylate can be produced from different
precursor molecules (Figure 1A). Cleavage of isocitrate generates
glyoxylate and succinate in the glyoxylate cycle (Figure 1B). The
oxidation of glycolate to glyoxylate is catalyzed by glycolate
oxidase being part of the photorespiratory process generating
H2O2 within peroxisomes (Figure 1C). Another possible source
of glyoxylate is the degradation of purines in those organisms
metabolizing the intermediate uric acid (Figure 1D). Moreover,
in mammals glyoxylate may represent a degradation product of
hydroxyproline originally derived from collagen (Salido et al.,
2012) or alternatively, may be the result of a conversion from August 2013 | Volume 4 | Article 204 | 1 www.frontiersin.org Permeability of the peroxisomal membrane Kunze and Hartig FIGURE 1 | Glyoxylate generation and consumption. (A) Glyoxylate is
generated from different precursor molecules and converted into stable
metabolites for further utilization. The proteins involved are ureidoglycolate
hydrolase (UGH), isocitrate lyase (ICL), glycolate oxidase (GO), malate
synthase (MLS), glutamine:glyoxylate aminotransferase (GGAT). (B) In the
glyoxylate cycle two acetyl-CoA are condensed to succinate and 2
CoA (not shown). The proteins involved are citrate synthase (CIT),
aconitase (ACO), isocitrate lyase (ICL), malate synthase (MLS), malate
dehydrogenase (MDH). (C) The generation and consumption of glyoxylate
in the photorespiratory process. The stoichiometry of the reaction
sequence to obtain three molecules of 3-phospho-glycerate from two
oxygenation products of ribulose-1,5-bisphosphate is indicated by the FIGURE 1 | Continued
serine-hydroxymethyltransferase (SHMT), serine:glyoxylate
aminotransferase (SGAT), hydroxypyruvate reductase (HPR), glycerate kinase
(GLYK). (D) The reactions in the purine degradation pathway leading to
glyoxylate. The proteins involved are urate oxidase (UO), allantoin synthase
(HIU-hydrolase + OHCU decarboxylase) (AS), allantoinase (alantoin
amidohydrolase) (AL), allantoate amidohydrolase (AAH), ureidoglycine
aminohydrolase (UGlyAH), ureidoglycolate amidohydrolase (UAH). glycine catalyzed by D-amino acid oxidase generating H2O2
(Ohide et al., 2011). The glyoxylate cycle allows the formation of 4-carbon units
from 2-carbon units (Figure 1B). The resulting succinate serves
to replenish the tricarboxylic acid (TCA) cycle representing
the major collector and distributor of small carbon units. Alternatively, succinate or its follow-up product oxaloacetate
serves as precursor for many biosynthetic processes. PEROXISOMES AND GLYOXYLATE METABOLISM This biosyn-
thetic pathway is absent in all animals except nematodes
(Kondrashov et al., 2006). Acetyl-CoA fed into the glyoxylate
cycle can be derived from different sources, such as β-oxidation
of fatty acids, degradation of amino acids or in case of micro-
bial organisms from external carbon sources such as ethanol or
acetate. The glyoxylate cycle shares a series of three enzymatic
activities with the TCA-cycle, namely malate dehydrogenase
(MDH), citrate synthase (CIT), and aconitase (ACO) activity. The
two unique activities, isocitrate lyase (ICL) and malate synthase
(MLS) generate and consume the name-giving metabolite gly-
oxylate. The cleavage of isocitrate bypasses the decarboxylation
reactions and the synthase reaction leads to the net-condensation
of acetyl-CoA units. In many organisms these activities can be
carried out by two or more isoenzymes with different localization
signals and different expression patterns catalyzing the respective
reactions. Photorespiration is required in all photosynthetic organisms
and serves as carbon recovery system (Maurino and Peterhansel,
2010). Oxygenation of ribulose-1,5-bisphosphate by ribulose-
bisphosphate-carboxylase-oxygenase (RuBisCO) competes with
CO2 fixation upon high partial oxygen pressure leading to the
formation of 2-phosphoglycolate and 3-phosphoglycerate in the
chloroplast (Figure 1C). 3-phosphoglycerate is channeled into
the Calvin cycle and 2-phosphoglycolate is recycled into 3-
phosphoglycerate in a series of reactions involving peroxisomes
and mitochondria. In peroxisomes glycolate is oxidized to gly-
oxylate generating H2O2, which is immediately decomposed
by catalase. Glyoxylate is transaminated to glycine using either
serine or glutamine as nitrogen source and further transported
to the mitochondria. Here, two glycines are converted into one
serine which is now returned into peroxisomes. The peroxi-
somal serine:glyoxylate aminotransferase and hydroxypyruvate
reductase convert serine into glycerate which is transferred to
chloroplasts, where it enters the Calvin cycle in the form of
3-phosphoglycerate. In summary, two glycolate molecules are
consumed to produce glycerate and CO2 and the generation of
two H2O2 is confined to peroxisomes. FIGURE 1 | Glyoxylate generation and consumption. (A) Glyoxylate is
generated from different precursor molecules and converted into stable
metabolites for further utilization. The proteins involved are ureidoglycolate
hydrolase (UGH), isocitrate lyase (ICL), glycolate oxidase (GO), malate
synthase (MLS), glutamine:glyoxylate aminotransferase (GGAT). (B) In the
glyoxylate cycle two acetyl-CoA are condensed to succinate and 2
CoA (not shown). The proteins involved are citrate synthase (CIT),
aconitase (ACO), isocitrate lyase (ICL), malate synthase (MLS), malate
dehydrogenase (MDH). (C) The generation and consumption of glyoxylate
in the photorespiratory process. PEROXISOMES AND GLYOXYLATE METABOLISM The stoichiometry of the reaction
sequence to obtain three molecules of 3-phospho-glycerate from two
oxygenation products of ribulose-1,5-bisphosphate is indicated by the
numbers below the molecules (1×, 2×). The proteins involved are
ribulose-bisphosphate-carboxylase-oxygenase (RuBisCO), phosphoglycolate
phosphatase (PGLP), glycolate oxidase (GO), glutamine:glyoxylate
aminotransferase (GGAT), glycine-decarboxylase (GlyDC),
(C
ti
d) FIGURE 1 | Glyoxylate generation and consumption. (A) Glyoxylate is
generated from different precursor molecules and converted into stable
metabolites for further utilization. The proteins involved are ureidoglycolate
hydrolase (UGH), isocitrate lyase (ICL), glycolate oxidase (GO), malate
synthase (MLS), glutamine:glyoxylate aminotransferase (GGAT). (B) In the
glyoxylate cycle two acetyl-CoA are condensed to succinate and 2
CoA (not shown). The proteins involved are citrate synthase (CIT),
aconitase (ACO), isocitrate lyase (ICL), malate synthase (MLS), malate
dehydrogenase (MDH). (C) The generation and consumption of glyoxylate
in the photorespiratory process. The stoichiometry of the reaction
sequence to obtain three molecules of 3-phospho-glycerate from two
oxygenation products of ribulose-1,5-bisphosphate is indicated by the
numbers below the molecules (1×, 2×). The proteins involved are
ribulose-bisphosphate-carboxylase-oxygenase (RuBisCO), phosphoglycolate
phosphatase (PGLP), glycolate oxidase (GO), glutamine:glyoxylate
aminotransferase (GGAT), glycine-decarboxylase (GlyDC),
(C
ti
d) The degradation pathway of purines leads to uric acid, which
in many organisms is further metabolized within peroxisomes
to allantoine and allantoic acid (Figure 1D). In some animals (Continued) (Continued) August 2013 | Volume 4 | Article 204 | 2 Frontiers in Physiology | Integrative Physiology Permeability of the peroxisomal membrane Kunze and Hartig the mitochondrial activity (Regev-Rudzki et al., 2005) attributing
Aco1p to the TCA- and the glyoxylate cycle, whereas Aco2p may
be important for fermentation (Van den Berg et al., 1998). the allantoicase activity leads to the cleavage of allantoic acid
into urea and glyoxylate (Hayashi et al., 2000). In plants, some
fungi and bacteria allantoic acid is further processed within the
endoplasmic reticulum into ureidoglycine and ureidoglycolate,
both giving rise to glyoxylate upon degradation (Werner and
Witte, 2011). The glyoxylate derived from purine degradation is
supposedly converted into glycine or condensed with acetyl-CoA
catalyzed by a MLS activity providing the versatile metabolite
malate. In essence, the heterocyclic core structure is converted
into a nitrogen containing product and glyoxylate. The latter is
recycled into biosynthetic processes in all organisms, the nitro-
gen containing product is either excreted (animals) or recycled
(plants). Even more complex is the situation in A. thaliana. ENZYMES ALLOCATED TO THE GLYOXYLATE CYCLE Due to the fact that three of the five enzymatic activities con-
tributing to the glyoxylate cycle are shared with the TCA cycle
and additional enzymatic activities have been described an assign-
ment of individual proteins to the glyoxylate cycle is not trivial. Some of the encoded proteins can be excluded due to an inap-
propriate expression pattern, but for some of the proteins only
the phenotype of cells lacking the respective activity is a con-
vincing argument for the function in the glyoxylate cycle. As
the situation in the yeast S. cerevisiae has been analysed in
detail previously (Kunze et al., 2006), we will only summarize
the most important information and then concentrate on more
recent results obtained in the thale cress A. thaliana as the plant
model organism and in the human opportunistic fungus Candida
albicans. The corresponding proteins are listed in Table 1. The expression pattern of enzymes contributing to the gly-
oxylate cycle is very characteristic, namely high expression after
imbibition during seed germination followed by strong reduction
upon postgerminative growth. Isoenzymes that are not expressed
in the phase of germination can be excluded from a contribution
to the glyoxylate cycle, such as CYS1 (Pracharoenwattana et al.,
2005). During this developmental phase both, the β-oxidation
of fatty acids and the glyoxylate cycle are equally needed and
therefore the expression profiles of the corresponding genes are
similar (Eastmond and Graham, 2001; Rylott et al., 2001). A
detailed and precise cluster analysis of expression profiles during
seedling development in soybean remained inconclusive since the
expression pattern of the two key enzymes ICL and MLS did not
fall into the same cluster and expression differences of the iso-
forms of CIT and MDH were not distinguishable (Gonzalez and
Vodkin, 2007). In the genome of S. cerevisiae the key enzymes ICL and MLS
are encoded by two genes each. Based on the high expression
levels when cells grow on ethanol, acetate or fatty acids and
the inability of cells lacking these proteins to grow on these
carbon sources Icl1p and Mls1p have been attributed to the gly-
oxylate cycle (Fernandez et al., 1992; Hartig et al., 1992). The
two others, Icl2p and Dal7p contribute to the mitochondrial
propionate metabolism and the purine degradation, respectively
(Hartig et al., 1992; Luttik et al., 2000). PEROXISOMES AND GLYOXYLATE METABOLISM Although
only one gene codes for ICL (Eastmond et al., 2000) and
one for MLS (Cornah et al., 2004), eight genes encode NAD+
dependent MDHs, five genes code for CITs and three genes code
for ACOs (summarized in Table 1). The localization, expres-
sion pattern and phenotype of the corresponding T-insertion
mutants may help to discern the respective function of each
individual protein. Nevertheless, the attribution of a func-
tion in the glyoxylate cycle appears difficult. Three of the
five CITs, CYS1-3 harbor peroxisomal targeting information
whereas CYS4 and CYS5 include mitochondrial leader peptides
(Pracharoenwattana et al., 2005). Similarly, among the MDH pro-
teins two are peroxisomal, two mitochondrial, one is observed in
chloroplasts and three in the cytosol (Pracharoenwattana et al.,
2007) [The Arabidopsis Information Resource (TAIR), http://
www.arabidopsis.org/, Huala et al., 2001]. www.frontiersin.org ENZYMES ALLOCATED TO THE GLYOXYLATE CYCLE However, this assay shows differences in the re-routing
of acetate between seedlings from wild-type plants and plants,
which are blocked in the glyoxylate cycle due to lack of ICL or
MLS, although the differences appear small in the absence of MLS
(Cornah et al., 2004). Experiments to discern the individual roles
of the three cytosolic MDH were not yet carried out. Similarly,
among the three ACO proteins a function in the glyoxylate cycle
has not been assigned to anyone of them (Peyret et al., 1995; Arnaud et al., 2007). Altogether, in A. thaliana the assignment of
individual ACOs, CITs, and MDHs to the glyoxylate cycle remains
open. p
The situation in the yeast C. albicans is different. Each one
of the enzymes ICL, MLS, and CIT is encoded by a single gene
(Piekarska et al., 2008). In this organism the same enzyme Cit1p
seems to contribute to the mitochondrial TCA-cycle and the gly-
oxylate cycle such as demonstrated for Candida tropicalis (Ueda
et al., 1997). However, it cannot be excluded that a cytosolic
form is derived from alternative translation as two variants are
described (C4YLG7 and Q59ZZ5), but none of the described
variants contains a peroxisomal targeting signal type 1 (PTS1;
C-terminus KYIELVKNINKA). ACO activity is encoded by two
genes, MDH activity by three genes (Jones et al., 2004). In each
case one of the variants has a closer similarity to the respective
glyoxylate cycle enzyme in S. cerevisiae, but experimental evidence
for their role is missing. ENZYMES ALLOCATED TO THE GLYOXYLATE CYCLE From the three genes
coding for MDH, a mitochondrial (Mdh1p; McAlister-Henn and
Thompson, 1987), a cytosolic (Mdh2p; Minard and McAlister-
Henn, 1991), and a peroxisomal (Mdh3p; Steffan and McAlister-
Henn, 1992) gene product are derived. Only the cytosolic and
the mitochondrial variants are expressed when cells grow on
C2-carbon sources (ethanol/acetate) and the cytosolic enzyme
was required for growth under these conditions (Minard and
McAlister-Henn, 1991; McCammon, 1996). This experimental
evidence indicates that the cytosolic Mdh2p participates in the
glyoxylate cycle. An unambiguous attribution of a function in the glyoxylate
cycle to a distinct protein could be based on the characteristics of
cells deficient in the glyoxylate cycle function. A. thaliana seeds
lacking ICL are able to germinate, but their seedling establish-
ment is severely impaired in the absence of light or carbohydrates
offered as alternative carbon source (Eastmond et al., 2000). In
other words, gluconeogenesis is compromised but lipid respira-
tion is still active in these mutants. A. thaliana seeds lacking MLS,
the other unique enzyme of the glyoxylate cycle, display a simi-
lar defect in seedling establishment in the absence of light or
carbohydrates. Regarding the compensation of the establishment
defect by light the seeds respond differently. In the absence of
MLS activity a lower light dose is required to promote seedling
establishment than in the absence of ICL activity (Cornah et al.,
2004). The latter phenotype led to the suggestion that in the
absence of MLS glyoxylate, which is produced by ICL can enter
gluconeogenesis by hijacking enzyme activities from the photo-
respiratory pathway. Similarly, among the three CIT proteins encoded within the
yeast genome only Cit2p is considered to contribute to the gly-
oxylate cycle, because of its expression pattern on various carbon
sources and because of the mitochondrial location of the other
two enzymes Cit1p and Cit3p. Interestingly, Cit2p is essential
for growth on ethanol or acetate only, when an alternative route
probably involving one of the two mitochondrial activities is
blocked (Van Roermund et al., 1995). Finally, one gene encoding
ACO (ACO1; Gangloff et al., 1990) gives rise to the cytosolic and Phenotypes of plants lacking various isoforms of either CYS
or MDH are less revealing to discern a specific function. During
seedling establishment both peroxisomal MDHs (pMDH1 and
pMDH2) and peroxisomal CITs (CYS2 and CYS3) are expressed. ENZYMES ALLOCATED TO THE GLYOXYLATE CYCLE August 2013 | Volume 4 | Article 204 | 3 www.frontiersin.org www.frontiersin.org Permeability of the peroxisomal membrane Kunze and Hartig Seeds lacking either one of the peroxisomal activities present with
disturbed fatty acid degradation (Pracharoenwattana et al., 2005,
2007). Thus, a contribution of these proteins to the glyoxylate
cycle cannot be delineated from the block in seedling establish-
ment obtained in these mutant plants, because the lack of fatty
acid degradation elicits a similar block in seedling establishment. Conversely, no noticeable defect in establishment was observed
for seedlings from plants lacking mitochondrial MDH excluding
an essential role of these MDHs in either β-oxidation of fatty acids
or the glyoxylate cycle (Tomaz et al., 2010). When the metabolic
distribution of exogenously added acetate within seedlings is
used as indicator for the functionality of the glyoxylate cycle, no
differences between seedlings lacking the peroxisomal MDH and
wild type seedlings could be observed (Pracharoenwattana et al.,
2007). However, this assay shows differences in the re-routing
of acetate between seedlings from wild-type plants and plants,
which are blocked in the glyoxylate cycle due to lack of ICL or
MLS, although the differences appear small in the absence of MLS
(Cornah et al., 2004). Experiments to discern the individual roles
of the three cytosolic MDH were not yet carried out. Similarly,
among the three ACO proteins a function in the glyoxylate cycle
has not been assigned to anyone of them (Peyret et al., 1995; Seeds lacking either one of the peroxisomal activities present with
disturbed fatty acid degradation (Pracharoenwattana et al., 2005,
2007). Thus, a contribution of these proteins to the glyoxylate
cycle cannot be delineated from the block in seedling establish-
ment obtained in these mutant plants, because the lack of fatty
acid degradation elicits a similar block in seedling establishment. Conversely, no noticeable defect in establishment was observed
for seedlings from plants lacking mitochondrial MDH excluding
an essential role of these MDHs in either β-oxidation of fatty acids
or the glyoxylate cycle (Tomaz et al., 2010). When the metabolic
distribution of exogenously added acetate within seedlings is
used as indicator for the functionality of the glyoxylate cycle, no
differences between seedlings lacking the peroxisomal MDH and
wild type seedlings could be observed (Pracharoenwattana et al.,
2007). PERMEABILITY OF THE PEROXISOMAL MEMBRANE To seal certain reactions in a compartment and to restrict the
generation of reactive molecules cells seem to spare no effort to
translocate the corresponding proteins and metabolic precursors
into distinct compartments. The localization of various oxidases
to peroxisomes is a typical example for an energy spending
activity preventing contamination of the cytosol by H2O2. In
turn, the membrane delimiting a compartment thought to pro-
tect the cellular interior from detrimental effects by small, highly
reactive molecules could be expected to be impermeable for
such substances. However, early studies provided evidence for
a permeability of the peroxisomal membrane for small solutes
such as urate or amino acids and for density gradient material
such as sucrose (De Duve and Baudhuin, 1966; Van Veldhoven
et al., 1987). For cofactors and larger substrates required for
peroxisomal enzymes such as NAD or acetyl-CoA the peroxiso-
mal membrane was shown to act as barrier similar to the inner
mitochondrial membrane (Van Roermund et al., 1995). Shuttle
systems were proposed to functionally connect the peroxisomal
lumen with the cytosol exchanging substrates and keeping the
cofactors in the reduced or in the oxidized state as required
(Elgersma et al., 1995; Van Roermund et al., 1995; Antonenkov
and Hiltunen, 2006; Visser et al., 2007). g
g y
y
y
In A. thaliana the key enzymes ICL and MLS are considered
peroxisomal containing a C-terminal PTS1 [EGTSLVVAKSRM
for ICL and IVAHYPINVSRL for MLS, Arabidopsis subcellular
database (SUBA), (Heazlewood et al., 2007)]. In contrast, ACOs
lack peroxisomal targeting information and were allocated to the
cytosol and the mitochondria (Arnaud et al., 2007). The extra-
peroxisomal location of ACO activity was confirmed in castor
bean and potato (Courtois-Verniquet and Douce, 1993). Which
of the other gene products, namely of MDH and of CIT, are con-
tributing to the glyoxylate cycle is unclear. Among the MDHs it
can be assumed that one or more of the three cytosolic isoforms
participates in the glyoxylate cycle as mutant plants lacking either
the peroxisomal (Pracharoenwattana et al., 2007) or the mito-
chondrial (Tomaz et al., 2010) isoenzymes show no signs of a
glyoxylate cycle defect. g y
y
y
In C. albicans both key enzymes of the glyoxylate cycle, ICL and
MLS were found in peroxisomes irrespective of the carbon source
used (Piekarska et al., 2008), rendering this microorganism an
alternative yeast model with key enzyme distribution similar to A. thaliana. SUBCELLULAR LOCALIZATION OF THE ENZYMES OF THE GLYOXYLATE
CYCLE Upon growth on ethanol or acetate as sole carbon source
with all other glyoxylate cycle enzymes in the cytosol the extra-
peroxisomal fraction of Cit2p might suffice to generate four-
carbon units through the glyoxylate cycle. of intermediates might facilitate diffusion controlled transfer,
which appears unlikely for highly reactive compounds such as
glyoxylate. PERMEABILITY OF THE PEROXISOMAL MEMBRANE However, the exclusive presence of one citrate synthase
gene (CIT1) and the necessity of a peroxisomal and mitochon-
drial shuttle mechanism for acetyl-CoA for growth on oleic acid
indicates that peroxisomal CIT is not available, but a cytosolic
isoform might well be. The similarity of the MDH proteins
to the homologs from S. cerevisiae suggests that the cytosolic
Mdh2p contributes to the glyoxylate cycle as well. Finally, the
cytosolic ACO Aco1p (Jones et al., 2004) might be supple-
mented by a minor peroxisomal subfraction that is caused by
a weak PTS1 [HGSALNFIKSKY, http://mendel.imp.ac.at/pts1/,
Neuberger et al., 2003]. A concept for the peroxisomal membrane permeability
reconciling conflicting data was put forward by Hiltunen and co-
workers (Antonenkov et al., 2004a,b). Pore-like structures permit
the free exchange of small solutes (MW < 300D) across an other-
wise impermeable membrane inhibiting the transfer of molecules
such as acetyl-CoA, ATP, or NAD. Accordingly, pore-forming
activities were reported in peroxisomes isolated from plants,
mammalian tissue and yeast (Reumann et al., 1995; Antonenkov
et al., 2005; Grunau et al., 2009), but the molecular nature of the
channel proteins remained largely unknown. In contrast, specific
transporters for adenine nucleotides and NAD were identified
corroborating the impermeability of the peroxisomal membrane
for such bulky molecules (Palmieri et al., 2001; Bernhardt et al.,
2012; for reviews see Antonenkov and Hiltunen, 2012; Hu et al.,
2012). Briefly summarized, the localization of proteins participat-
ing in the glyoxylate cycle on different sides of the peroxisomal
membrane in all three model organisms requires the transport of
intermediates across the lipid barrier. )
The localization of enzymatic activities of the glyoxylate cycle
on different sides of the peroxisomal membrane requires an
efficient transport of intermediates across the lipid barrier (Kunze
et al., 2006). When acetyl-CoA is generated inside the peroxisomal
matrix—usually via β-oxidation of fatty acids—it remains con-
fined to peroxisomes probably because of its size (Van Roermund
et al., 1995; Antonenkov and Hiltunen, 2006). CIT catalyzes the
condensation of acetyl-CoA with oxaloacetate into citrate, which
in turn is exported and serves as substrate for extra-peroxisomal
ACO. The resulting isocitrate is imported into peroxisomes in
those organisms in which the corresponding cleavage activity,
ICL resides inside peroxisomes (A. thaliana, C. albicans). Thereby,
succinate, the net product of the cycle is released within peroxi-
somes and requires an additional export mechanism. Conversely,
glyoxylate is directly handed over to the second acetyl-CoA SUBCELLULAR LOCALIZATION OF THE ENZYMES OF THE GLYOXYLATE
CYCLE In S. cerevisiae the enzymes contributing to the glyoxylate cycle
are distributed between the peroxisomal matrix and the cytosol. Table 1 | Enzymatic activities required for the glyoxylate cycle. Enzyme
Saccharomyces cerevisiae
Candida albicans
Arabidopsis thaliana
Protein
Gene
Targeting
Protein
Gene
Targeting
Protein
Gene
Targeting
information
information
information
Isocitrate lyase
ICL1
YER065C
Cytosolic
ICL1
CaO19.14134
Peroxisomal
ICL
At3g21720
Peroxisomal
ICL2
YPR006C
Mitochondrial
Malate synthase
MLS1
YNL117W
Peroxisomal
MLS1
CaO19.12296
Peroxisomal
MLS
At5g03860
Peroxisomal
DAL7
YIR031C
PTS1
Malate
MDH1
YKL085W
Mitochondrial
MDH1
CaO19.12072
MITO
pMDH1
At2g22780
Peroxisomal
dehydrogenase
MDH2
YOL126C
Cytosolic
MDH2
CaO19.7481
pMDH2/MDHG1
At5g09660
Peroxisomal
MDH3
YDL078C
Peroxisomal
MDH3
CaO19.1278
PTS1
mDH3/MDHM1
At1g53240
Mitochondrial
mDH4/MDHM2
At3g15020
Mitochondrial
MDH5/MDHC1
At1g04410
MDH6/MDHC2
At5g43330
MDH7/MDHC3
AT5G56720
cMDH8/MDHP1
At3g47520
Chloroplast
Citrate synthase
CIT1
YNR001C
Mitochondrial
CIT1
CaO19.11871
Mitochondrial
CSY1
At3g58740
PTS2
CIT2
YCR005C
Peroxisomal
Q59ZZ5
CSY2
At3g58750
Peroxisomal
CIT3
YPR001W
Mitochondrial
CSY3
At2g42790
Peroxisomal
CSY4
At2g44350
Mitochondrial
CSY5
At3g60100
Mitochondrial
Aconitase
ACO1
YLR304C
Mitochondrial
ACO1
CaO19.13742
MITO
ACO1
At4g35830
Mitochondrial
ACO2
YJL200C
Mitochondrial
ACO2
CaOrf19.6632
MITO
ACO2
At4g26970
Mitochondrial
ACO3
At2g05710
Mitochondrial
Proteins, corresponding genes, accession number, and targeting information are shown for enzymes carrying out activities required for the glyoxylate cycle
in S. cerevisiae, C. albicans, and A. thaliana. PTS and MITO indicates predicted targeting information (general prediction: http://wolfpsort.org/, PTS1-predictor:
http://mendel.imp.ac.at/pts1/, Mito-predictor: http://ihg.gsf.de/ihg/mitoprot.html). Cytosolic, peroxisomal, mitochondrial, and chloroplast indicates experimentally
verified localization. Bold underlined are proteins experimentally proven to contribute to the glyoxylate cycle. Table 1 | Enzymatic activities required for the glyoxylate cycle. Proteins, corresponding genes, accession number, and targeting information are shown for enzymes carrying out activities required for the glyoxylate cycle
in S. cerevisiae, C. albicans, and A. thaliana. PTS and MITO indicates predicted targeting information (general prediction: http://wolfpsort.org/, PTS1-predictor:
http://mendel.imp.ac.at/pts1/, Mito-predictor: http://ihg.gsf.de/ihg/mitoprot.html). Cytosolic, peroxisomal, mitochondrial, and chloroplast indicates experimentally
verified localization. Bold underlined are proteins experimentally proven to contribute to the glyoxylate cycle. August 2013 | Volume 4 | Article 204 | 4 Frontiers in Physiology | Integrative Physiology Permeability of the peroxisomal membrane Kunze and Hartig Icl1p, Mdh2p, and Aco1p were described in the cytosol (Minard
and McAlister-Henn, 1991; Taylor et al., 1996; Regev-Rudzki
et al., 2005), and Cit2p was found in the peroxisomal fraction
(Lewin et al., 1990). Mls1p is either targeted to peroxisomes
when cells utilize oleic acid or distributed across the cytosol
when cells utilize ethanol (McCammon et al., 1990; Kunze et al.,
2002). TRANSFER OF METABOLITES Usually, the concentration of free intermediates occurring in
metabolic pathways is relatively low, because a local accumu-
lation of participating enzymes, e.g., within an organelle, or a
physical interaction between successive enzymes allows a chan-
neling of intermediates. In extreme cases, large protein complexes
such as the fatty acid synthase transfer small molecules from one
active center to the next. If a membrane separates consecutive
steps an efficient metabolic pathway requires a direct shuttling
of intermediates either through a transporter protein or through
a proteinaceous channel linking enzymes on both sides of the
membrane. Alternatively, a comparably high net concentration August 2013 | Volume 4 | Article 204 | 5 www.frontiersin.org www.frontiersin.org Permeability of the peroxisomal membrane Kunze and Hartig Permeability of the peroxisomal membrane
FIGURE 2 | Metabolites of the glyoxylate cycle crossing the
peroxisomal membrane. The peroxisomal membrane facilitates the
transfer of small metabolites. The transport of glyoxylate cycle
intermediates and of C2-units is shown for S. cerevisiae (A), A. thaliana (B), and C. albicans (C). Hypothetical pore-forming proteins
permitting the export of intermediates are colored orange, hypothetical
pore-forming proteins permitting the import of intermediates are
colored green. Broken lines are drawn to close the glyoxyate cycle. Intermediates that cross the peroxisomal membrane are indicated
bold. Px, peroxisomal side of the membrane, Cyt, cytosolic side of
the membrane. consuming enzyme, MLS, which is a peroxisomal constituent in
all organisms. Some organisms such as S. cerevisiae do not harbor a peroxiso-
mal ICL (Taylor et al., 1996), but instead generate the products of
this reaction in the cytosol. The disadvantage that a small reactive
molecule such as glyoxylate needs to be translocated across a
membrane to reach MLS might be balanced by the advantage
that succinate is already in the cytosol, which is one step closer to
its usual final destination, mitochondria. The interesting obser-
vation that the relevant MDH activity for the glyoxylate cycle is
exerted by cytosolic isoforms in the yeast S. cerevisiae and suppos-
edly also in A. thaliana calls for an additional export of malate and
the subsequent import of oxaloacetate to close the cycle (Minard
and McAlister-Henn, 1991; Pracharoenwattana et al., 2007). All in all a series of transport steps is required to complete a full
round of the glyoxylate cycle. If ICL is extra-peroxisomal, e.g., in
the yeast S. TRANSFER OF METABOLITES cerevisiae, citrate and malate are exported, and gly-
oxylate and oxaloacetate are imported (Figure 2A). If ICL resides
inside peroxisomes citrate, malate and succinate are exported,
whereas isocitrate and oxaloacetate are imported (Figure 2B), a
situation occurring in the plant A. thaliana. A similar flux of inter-
mediates occurs in C. albicans, although citrate is generated in the
cytosol and does not occur in peroxisomes (Figure 2C). The export of malate and import of oxaloacetate as part of the
glyoxylate cycle mimics a redox-shuttle system consisting of a per-
oxisomal and a cytosolic MDH activity that mediates a net-export
of reduction equivalents generated by the β-oxidation of fatty
acids to the cytosol. Thus, peroxisomal malate generated by MLS
might be fueled into the same path to the cytosol as malate gen-
erated by the re-oxidation of NADH (peroxisomal ScMdh3p or
AtpMDH1/pMDH2). Similarly, the transport of isocitrate might
be part of a shuttle mechanism coupling the import of isocitrate
and the export of α-oxo-glutarate with the reduction of NADP+
inside peroxisomes. Such a hypothetical net-exchange is based on
a cytosolic isocitrate dehydrogenase (ScIdp2p) and a peroxisomal
isoform (ScIdp3p) converting isocitrate into α-oxo-glutarate and
CO2 and vice versa. The export of citrate is not restricted to the glyoxylate cycle
but also presents a mode of export for C2-units that are generated
as acetyl-CoA by fatty acid β-oxidation within peroxisomes. In
this case the export of citrate is ultimately balanced by the import
of oxaloacetate, the precursor for CIT. The export of citrate is
considered the only export pathway for C2-units in plants since
lack of the peroxisomal CITs, CYS2 and CYS3, blocks β-oxidation
of fatty acids in A. thaliana (Pracharoenwattana et al., 2005),
whereas in the yeast S. cerevisiae an additional export system for
acetyl-units exists. For the latter, a peroxisomal form of carnitine
acetyl-transferase (Cat2p; Elgersma et al., 1995) generates acetyl-
carnitine that can be translocated across the membrane and
rebuilt into acetyl-CoA either in the cytosol or within mitochon-
dria employing either one of three carnitine acetyltransferases,
Cat2p, Yat1p, or Yat2p (Swiegers et al., 2001). In S. Frontiers in Physiology | Integrative Physiology TRANSFER OF METABOLITES Furthermore,
a glycolate/glycerate transporter required for photorespiration
(PLGG1) has been identified in the chloroplast membrane (Pick
et al., 2013), but no homologous proteins were found in plant
peroxisomes. In contrast, strains of baker’s yeast, S. cerevisiae, have
been selected for efficient growth in the presence of ethanol. Consequently, this organism most efficiently utilizes acetyl-CoA
generated from this carbon source in the cytosol, which is
apparently supported by the potential to relocate the complete
glyoxylate cycle to the cytosol. Despite overwhelming evidence for the free exchange of small
solutes the molecular nature of pore-forming activities in plant
and yeast peroxisomes remained unknown (Reumann et al., 1997,
1998; Grunau et al., 2009). Moreover, the yeast peroxisomal mem-
brane pores were shown to conduct solutes of the glyoxylate cycle
(Antonenkov et al., 2009; Grunau et al., 2009). So far, only in
mammalian peroxisomes a protein, Pxmp2, was identified that
exhibits channel-forming capacities (Rokka et al., 2009). The bipartite localization of enzymes provokes unusual
intricacies for a straightforward metabolic pathway consisting of
five enzymatic activities. Evolutionary optimization and pressure
to increase efficiency demand a physiological advantage to com-
pensate for this complexity. We suggest that the distribution of
the glyoxylate cycle enzymes on different sides of the peroxisomal
membrane might be due to the combination of (i) the unavoid-
able provision of its substrate, acetyl-CoA, inside peroxisomes by
fatty acid β-oxidation (Kunze et al., 2006) and (ii) the incom-
patibility of some of its enzymes, namely ACO (Verniquet et al.,
1991) and to a certain extent also ICL (Yanik and Donaldson,
2005), with the oxidative milieu within peroxisomes including
high H2O2 concentrations. Thus, during evolution neither com-
partmentation of the complete pathway within peroxisomes nor
the transfer of all enzymes into the cytosol appeared as feasible
alternative. Provided that the transport of intermediates is essential for
the metabolic activity, the lack of such pores should result in
non-functional peroxisomes. Therefore, the corresponding genes
should have turned up in various genetic screens searching for
mutants with dysfunctional peroxisomes. However, more than
one gene could encode redundant functions escaping the detec-
tion of these genes in screens. Likewise, such porins should
be rather abundant constituents of the peroxisomal membrane
and as such should have been identified in various proteomic
approaches. TRANSFER OF METABOLITES In this organism Mls1p
localization differs between cells grown in medium containing
ethanol as sole carbon source, when acetyl-CoA is generated pri-
marily in the cytosol, and cells utilizing oleic acid generating
acetyl-CoA within peroxisomes (Kunze et al., 2002, 2006). y
p
In other organisms such as the plant A. thaliana ICL and MLS
are peroxisomal. In this case an efficient coupling of the glyoxy-
late cycle to β-oxidation of fatty acids is essential, because both,
energy and biomass production for germination and seedling
outgrowth rests on the β-oxidation of fatty acid. The resulting
acetyl-CoA is used to generate energy via the TCA-cycle and
oxidative phosphorylation and to feed the glyoxylate cycle to
cover the needs for biosynthetic processes. For the opportunistic
fungus C. albicans carbohydrates and fatty acids are the prevalent
carbon sources in its natural habitat, the mammalian gut. Thus,
acetyl-CoA is expected to be obtained primarily inside mitochon-
dria following glycolysis or inside peroxisomes via β-oxidation
under such conditions. Even when these cells were grown in the
presence of ethanol or acetate both key enzymes (ICL and MLS)
are located in peroxisomes (Piekarska et al., 2008). Needless to
mention, that neither C. albicans nor plants ever face ethanol
or acetate as carbon source in their natural environment. This
supports the hypothesis that the prevailing source of acetyl-CoA
under natural conditions determines the localization of glyoxylate
cycle key enzymes. For both model systems neither evolutionary
pressure nor man-made selection forced the organism to change
the intracellular location of parts of the glyoxylate cycle. The transfer of the various small intermediates is compatible
either with specific transporter proteins or with atypical per-
meability properties of the membrane. The mitochondrial and
chloroplast membranes contain numerous transporter proteins
specific for small organic compounds, but corresponding pro-
teins were not identified in peroxisomes. Few examples may
illustrate this. In mitochondria of S. cerevisiae a carnitine acyl-
carnitine carrier protein Crc1p and a citrate-oxoglutarate carrier
were identified, but no peroxisomal paralog was discovered yet
(Van Roermund et al., 1999; Castegna et al., 2010). Similarly,
a succinate-fumarate transporter was identified in the inner
mitochondrial membranes of S. cerevisiae and A. thaliana
[Sfc1p/Acr1p (Bojunga et al., 1998) and AtMSFC1 (Catoni et al.,
2003), respectively], but no homologous protein was identified
to mediate plant peroxisomal succinate export. TRANSFER OF METABOLITES The most abundant yeast peroxisomal membrane
protein reported is Pex11p, but its localization at the outer surface
renders a function in solute transport rather unlikely (Erdmann
and Blobel, 1995; Van Roermund et al., 2000; Opalinski et al.,
2011). However, the tight coupling of peroxisomal acetyl-CoA
generation and its fueling into the glyxoylate cycle that appears
optimal for growth of A. thaliana and C. albicans in their natu-
ral habitats might restrain the incorporation of extra-peroxisomal
acetyl-CoA into the glyoxylate cycle. When such organisms uti-
lize the less physiological carbon source acetate as carbon source
they satisfy all their energetic and biosynthetic needs from acetyl-
CoA that is primarily generated in the cytosol. In this case
the peroxisomal membrane could act as barrier that separates TRANSFER OF METABOLITES cerevisiae the
β-oxidation of fatty acids requires only one mode of export and
thus the lack of either the peroxisomal CIT Cit2p or the car-
nitine acetyltransferase Cat2p localized to the peroxisomes and
the mitochondria is tolerated for growth on oleic acid as car-
bon source, but upon deletion of both genes (CIT2CAT2) FIGURE 2 | Metabolites of the glyoxylate cycle crossing the
peroxisomal membrane. The peroxisomal membrane facilitates the
transfer of small metabolites. The transport of glyoxylate cycle
intermediates and of C2-units is shown for S. cerevisiae (A), A. thaliana (B), and C. albicans (C). Hypothetical pore-forming proteins
permitting the export of intermediates are colored orange, hypothetical
pore-forming proteins permitting the import of intermediates are
colored green. Broken lines are drawn to close the glyoxyate cycle. Intermediates that cross the peroxisomal membrane are indicated
bold. Px, peroxisomal side of the membrane, Cyt, cytosolic side of
the membrane. FIGURE 2 | Metabolites of the glyoxylate cycle crossing the
peroxisomal membrane. The peroxisomal membrane facilitates the
transfer of small metabolites. The transport of glyoxylate cycle
intermediates and of C2-units is shown for S. cerevisiae (A), A. thaliana (B), and C. albicans (C). Hypothetical pore-forming proteins
permitting the export of intermediates are colored orange, hypothetical
pore-forming proteins permitting the import of intermediates are
colored green. Broken lines are drawn to close the glyoxyate cycle. Intermediates that cross the peroxisomal membrane are indicated
bold. Px, peroxisomal side of the membrane, Cyt, cytosolic side of
the membrane. August 2013 | Volume 4 | Article 204 | 6 Frontiers in Physiology | Integrative Physiology Permeability of the peroxisomal membrane Kunze and Hartig cells were not able to utilize oleate (Van Roermund et al., 1999). Moreover, under these conditions the cytosolic re-conversion
of acetyl-carnitine into acetyl-CoA is also essential, as mutant
cells lacking CIT2 and either one of the other carnitine acetyl-
transferases (YAT1 or YAT2) were unable to consume oleic acid
(Swiegers et al., 2001). In C. albicans a peroxisomal condensation
of acetyl-CoA with oxaloacetate producing citrate is not possible
due to the lack of a peroxisomal CIT. Therefore, the export of
acetyl-units depends on the carnitine form and consequently, in
the absence of the peroxisomal isoform of Cat2p cells were unable
to grow on oleic acid (Strijbis et al., 2010). observations in the yeast S. cerevisiae. August 2013 | Volume 4 | Article 204 | 7 BIPARTITE ENZYME DISTRIBUTION OF GLYOXYLATE CYCLE
ENZYMES: SUBCELLULAR DISTRIBUTION FOLLOWS
GENERATION OF SUBSTRATES This
phentotype of C. albicans cells might be due to their inability
to feed cytosolic acetyl-CoA into the glyoxylate cycle, because
MLS is enclosed by the peroxisomal membrane. This notion is
supported by the observation, that the ability to utilize acetate
efficiently can be restored in these cells (FOX2), when the
compartmentation of peroxisomal enzymes is prevented by the
deletion of the PTS1-receptor PEX5 (FOX2PEX5) (Piekarska
et al., 2008). Thus, in organisms optimized for an efficient con-
sumption of fatty acids the redirection of cytosolic acetyl-CoA
units obtained from other sources such as acetate into per-
oxisomes is a critical step for its fueling into the glyoxylate
cycle. FIGURE 3 | Metabolite transport processes for the glyoxylate cycle and
the photorespiratory process are considered to be similar. In plant FIGURE 3 | Metabolite transport processes for the glyoxylate cycle and
the photorespiratory process are considered to be similar. In plant
peroxisomes glyoxylate is a key metabolite of the glyoxylate cycle and the
photorespiration, the latter involving mitochondria and chloroplasts. Hypothetical pore-forming proteins permitting the export are colored orange,
hypothetical pore-forming proteins permitting the import are colored green. The broken lines with arrow heads indicate reactions of the glyoxylate cycle
and the conversion of two molecules of glycine into serine inside
mitochondria without further details. Reactions of the photorespiration
process are encircled. peroxisomes glyoxylate is a key metabolite of the glyoxylate cycle and the
photorespiration, the latter involving mitochondria and chloroplasts. Hypothetical pore-forming proteins permitting the export are colored orange,
hypothetical pore-forming proteins permitting the import are colored green. The broken lines with arrow heads indicate reactions of the glyoxylate cycle
and the conversion of two molecules of glycine into serine inside
mitochondria without further details. Reactions of the photorespiration
process are encircled. protein the close apposition of chloroplasts and peroxisomes is
lost, glyoxylate accumulates (Schumann et al., 2007). Under normal conditions the glyoxylate cycle and the
photorespiration do not overlap although they occur in the
same organelle and share glyoxylate as key intermediate. This
separation is due to the developmental program, which shows
highest glyoxylate key enzyme activities during seed germina-
tion [2–4 days postimbibition (Eastmond and Graham, 2001)],
whereas the enzymes of the photorespiration are induced by
light (Bertoni and Becker, 1993) upon cotyledon development. BIPARTITE ENZYME DISTRIBUTION OF GLYOXYLATE CYCLE
ENZYMES: SUBCELLULAR DISTRIBUTION FOLLOWS
GENERATION OF SUBSTRATES The hypothesis that substrate availability was the driving force
for changes in enzyme localization, has been proposed based on August 2013 | Volume 4 | Article 204 | 7 www.frontiersin.org Permeability of the peroxisomal membrane Kunze and Hartig FIGURE 3 | Metabolite transport processes for the glyoxylate cycle and
the photorespiratory process are considered to be similar. In plant
peroxisomes glyoxylate is a key metabolite of the glyoxylate cycle and the
photorespiration, the latter involving mitochondria and chloroplasts. Hypothetical pore-forming proteins permitting the export are colored orange,
hypothetical pore-forming proteins permitting the import are colored green. The broken lines with arrow heads indicate reactions of the glyoxylate cycle
and the conversion of two molecules of glycine into serine inside
mitochondria without further details. Reactions of the photorespiration
process are encircled. cytosolic acetyl-CoA from those peroxisomal enzymes of the gly-
oxylate cycle that utilize it, namely MLS in both organisms and
CIT in A. thaliana. Under these conditions the mitochondrial
energy production from cytosolic acetyl-CoA is not expected to
be limited, but the biosynthetic capacity of the glyoxylate cycle is
restricted by the transfer of acetyl-CoA into peroxisomes involv-
ing a specific import mechanism for this intermediate. Such a
limitation is avoided in S. cerevisiae, where the glyoxylate cycle
can be relocated to the cytosol. y
Interestingly, A. thaliana and C. albicans can grow under
conditions when acetate is utilized as sole carbon source (Hooks
et al., 2004; Zhou and Lorenz, 2008), but they are dependent on
specific peroxisomal functions. In A. thaliana a genetic screen for
mutants that are unable to utilize exogenous acetate identified two
genes encoding peroxisomal proteins (Hooks et al., 2004). The
peroxisomal transporter protein COMATOSE known to trans-
port fatty acids (Hooks et al., 2007) and the intraperoxisomal
short chain acyl-CoA/acetyl-CoA synthase (At3g16910, AAE7)
(Turner et al., 2005; Shockey and Browse, 2011) are required
for the integration of acetate into organic compounds (Allen
et al., 2011). Interestingly, neither C. albicans nor S. cerevisiae cells
require functional peroxisomes for the utilization of acetate, as
cells harboring a deletion of a PEX-gene can grow on medium
solely containing this carbon source (Piekarska et al., 2008). However, upon selective interruption of fatty acid β-oxidation
by the ablation of the enzyme exerting the second step (Fox2p),
C. albicans cells cannot utilize acetate any more, but S. cerevisae
cells can do so (Hiltunen et al., 1992; Piekarska et al., 2008). BIPARTITE ENZYME DISTRIBUTION OF GLYOXYLATE CYCLE
ENZYMES: SUBCELLULAR DISTRIBUTION FOLLOWS
GENERATION OF SUBSTRATES Upon this development the change of glyoxylate cycle containing
peroxisomes (“glyoxysomes,” see Pracharoenwattana and Smith,
2008) into photorespiratory active peroxisomes is considered
prototypical for the exchange of enzymatic equipment of such
organelles (Titus and Becker, 1985; Behrends et al., 1990) and is
even accompanied by the highly surprising removal of ICL from
“glyoxsomes” (Lingard et al., 2009). However, employing artifi-
cial experimental conditions by exposing outgrowing seedlings
to light these developmental programs become blended, and so
does the enzymatic equipment. This is demonstrated by the com-
parison of plants in which the glyoxylate cycle is interrupted
either by ICL deficiency or MLS deficiency. The observed growth
defect of these plants in the dark can be completely overcome
by exposing seedlings lacking MLS to light, but seedlings lacking
ICL can only partially recover (Cornah et al., 2004). The sug-
gestion that the addition of photorespiratory equipment allows
the re-direction of glyoxylate from MLS to serine/glutamine-
glyoxylate aminotransferase is supported by the re-organization
of the incorporation pattern of radioactively labeled acetyl-CoA
into different water soluble intermediates. Thereby, wild type
plants and plants lacking MLS display a more similar phenotype
than plants lacking ICL. Frontiers in Physiology | Integrative Physiology CONCLUSIONS AND PERSPECTIVES The peroxisomal matrix is surrounded by a single membrane that
allows the enclosure of a variety of highly reactive and even toxic
compounds. However, in vitro the membrane appears permeable
to small molecules and neither a chemical nor an electrochemical
gradient has been detected, which represents a major difference
to the mitochondrial inner membrane or the thylakoid mem-
brane. The biophysical properties of the peroxisomal membrane
demands pore-like structures in addition to the few transmem-
brane proteins with transporter function for larger molecules. The frequent exchange of intermediates between peroxisomes
and the intracellular environment requires directed transport
processes with high selectivity, which appears incompatible with
pore-like exchange modules. However, a model in which soluble
enzymes accumulate in proximity to both ends of such pore-like
structures forming a transmembrane metabolon would explain
rapid and selective exchange based on a local increase in interme-
diate concentration. The observation that the enzymes of the gly-
oxylate cycle are distributed across both sides of the peroxisomal
membrane would be prototypical for such transport processes,
because an efficient transfer of small organic metabolites across
the peroxisomal membrane is essential for this pathway. The
high variability in the nature of small organic molecules trans-
ported across the peroxisomal membranes between S. cerevisiae,
C. albicans, and A. thaliana may reflect the ease of adaptation pro-
cesses whenever specific transporter proteins are not involved. Similar considerations are applicable to the photorespiration,
although the direct exchange of metabolites with other organelles
renders the latter apparently more complex. A transport system
of such high versatility generates an organelle of high plasticity
and allows rapid adjustments to environmental changes and
to developmental programs generally considered an important
feature of peroxisomes. According to our model in S. cerevisiae the loosely associated
protein complex outside peroxisomes would consist of ACO, ICL,
and MDH, on the inside of MLS and CIT, and the channel(s)
would allow transferring citrate and malate from the matrix to
the cytosol and/or glyoxylate and oxaloacetate from the cytosol
to the peroxisomal matrix. When the peroxisomal Mdh3p has
no access to the complex formed it is excluded from partici-
pation in the glyoxylate cycle. In plants, five metabolites have
to cross the membrane. ACO and MDH acting outside peroxi-
somes require the export of citrate and malate and the import
of isocitrate and oxaloacetate for further processing. MODEL FOR THE FLUX OF INTERMEDIATES ACROSS A
MEMBRANE the cellular metabolism. Since according to our model ICL would
loosely associate with CIT and MLS it is reasonable to suggest that
the simultaneous export of succinate using the same channel unit
represents an additional drive for the cleavage reaction. The localization of enzymes catalyzing successive metabolic
reactions on opposite sides of the peroxisomal membrane and
the permeability of this membrane for small molecules raises
the question how fast small metabolites can cross the barrier
membrane. Such a transfer of intermediates might be accom-
plished either (i) by an unspecific pore or (ii) by a series of
specific transporter or facilitator proteins or (iii) by the direct
coupling of specific enzymes on both sides of an unspecific pore
forming a transmembrane metabolon. A simple diffusion con-
trolled mechanism involving only a pore-like structure appears
unlikely, because after reaching the other side of the mem-
brane small metabolites could easily diffuse and the concentra-
tion might become (quite) low. This may happen, permitting
the adjustment of concentrations between the cytosol and the
peroxisomal matrix. However, such a model appears less likely
for instable, highly reactive or even toxic intermediates such
as glyoxylate. Alternatively, specific transporters or facilitators
might close the gap between successive enzymatic reactions and
guarantee specificity of transport, however these proteins have
not been found so far. An interesting alternative might be a
transmembrane metabolon comprised of transiently accumulat-
ing metabolic enzymes such as the glyoxylate cycle enzymes on
both sides of an unspecific pore. We propose that in this extended
form of the classical metabolon (Srere, 1987) pore-like channels
participate in the assembly of supramolecular complexes, thereby
linking proteins on both sides of the membrane enabling the swift
transfer or exchange of metabolites. Such structural arrangement
could enhance efficiency and ensure sufficient flux of interme-
diates across the peroxisomal membrane. Importantly, the tran-
siently formed transmembrane metabolon would generate only
local concentration peaks of intermediates at the entry sides of
the pores. In our model metabolically active proteins transiently
acquire a pore leading to a rapid transfer of the corresponding
metabolites across a short distance. In this respect the obser-
vation seems interesting that polyethylene glycol (PEG), which
stabilizes peroxisomal membrane integrity during the isolation
procedure (Antonenkov et al., 2004a) is also known as stabilizing
agent for supramolecular protein complexes such as metabolons
(Beeckmans and Kanarek, 1981; Barnes and Weitzman, 1986). METABOLIC FLUXES IN RELATED METABOLIC PATHWAYS Photorespiration represents another example for a metabolic pro-
cess in which glyoxylate is formed and intermediates traverse the
peroxisomal membrane. Considering only the transport reactions
between chloroplasts and peroxisomes two molecules of glyco-
late are transported into peroxisomes and one molecule glycerate
is exported. The exchange with the mitochondria includes the
export of two molecules of glycines and the import of one
molecule serine (Figure 3). Already 40 years ago peroxisomes,
mitochondria, and chloroplasts were often seen in close vicinity
in the electron microscope (Frederick and Newcomb, 1969). A
direct apposition of the peroxisomal membrane and the outer
membranes of chloroplasts (Schumann et al., 2007) supports
the hypothesis of direct exchange of metabolites thereby avoid-
ing diffusion of intermediates into the cytosol. In leaves of A. thaliana, in which due to a mutation in a peroxisomal membrane August 2013 | Volume 4 | Article 204 | 8 Frontiers in Physiology | Integrative Physiology Permeability of the peroxisomal membrane Kunze and Hartig www.frontiersin.org MODEL FOR THE FLUX OF INTERMEDIATES ACROSS A
MEMBRANE Such a model would explain how a variety of substrates could
cross the membrane without specific transporter molecules. The
selectivity would be maintained via the transient association of
proteins on both sides of the pore-like channels providing control
and efficiency. Furthermore, the peroxisomal membrane would per se not
represent a principal barrier for glyoxylate cycle intermediates
or any other small metabolite, but controls the velocity of the
continuous flux. In addition, this model could also explain how
shuttles may function. A local concentration of enzymes on
both sides of a membrane-spanning pore could facilitate the
shuttling of reduction equivalents in [isocitrate/α-oxo-glutarate
(Van Roermund et al., 1998)] or out [malate/oxaloacetate (Van
Roermund et al., 1995), lactate/pyruvate (Baumgart et al., 1996),
G3P/DHAP (Gee et al., 1974)] of the peroxisomal matrix (for
review see Antonenkov and Hiltunen, 2006). The exchange of
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Corrected: Author correction
ARTICLE
OPEN
Automated generation and ensemble-learned matching of
X-ray absorption spectra
Chen Zheng1, Kiran Mathew2, Chi Chen1, Yiming Chen1, Hanmei Tang1, Alan Dozier3, Joshua J. Kas4, Fernando D. Vila4, John J. Rehr4,
Louis F. J. Piper5,6, Kristin A. Persson2 and Shyue Ping Ong 1
X-ray absorption spectroscopy (XAS) is a widely used materials characterization technique to determine oxidation states,
coordination environment, and other local atomic structure information. Analysis of XAS relies on comparison of measured spectra
to reliable reference spectra. However, existing databases of XAS spectra are highly limited both in terms of the number of
reference spectra available as well as the breadth of chemistry coverage. In this work, we report the development of XASdb, a large
database of computed reference XAS, and an Ensemble-Learned Spectra IdEntification (ELSIE) algorithm for the matching of
spectra. XASdb currently hosts more than 800,000 K-edge X-ray absorption near-edge spectra (XANES) for over 40,000 materials
from the open-science Materials Project database. We discuss a high-throughput automation framework for FEFF calculations, built
on robust, rigorously benchmarked parameters. FEFF is a computer program uses a real-space Green’s function approach to
calculate X-ray absorption spectra. We will demonstrate that the ELSIE algorithm, which combines 33 weak “learners” comprising a
set of preprocessing steps and a similarity metric, can achieve up to 84.2% accuracy in identifying the correct oxidation state and
coordination environment of a test set of 19 K-edge XANES spectra encompassing a diverse range of chemistries and crystal
structures. The XASdb with the ELSIE algorithm has been integrated into a web application in the Materials Project, providing an
important new public resource for the analysis of XAS to all materials researchers. Finally, the ELSIE algorithm itself has been made
available as part of veidt, an open source machine-learning library for materials science.
npj Computational Materials (2018)4:12 ; doi:10.1038/s41524-018-0067-x
INTRODUCTION
X-ray absorption spectroscopy (XAS) is a widely used technique in
the study of the properties, physical states, and local environments of materials.1–3 When incident X-ray photons with energy
greater than the binding energy are absorbed by an atom, a corelevel electron is removed from its quantum level. In XAS, the
absorption coefficient, μ(E) is measured as a function of X-ray
energy E. Detailed descriptions of X-ray absorption theory and
equation have been included in many excellent books and review
papers.4,5
The X-ray absorption fine structure (XAFS) is typically divided in
to two regimes: X-ray absorption near-edge structure (XANES) and
extended X-ray absorption fine structure (EXAFS).6 The XANES is a
fingerprint of the oxidation states and coordination chemistries of
the absorbing atom. Quantitative XANES analyses are typically
difficult and are usually conducted in combination with principle
component analysis or least-squares fitting. The EXAFS provides
local atomic structure information, which can be extracted via
coupling with theoretically calculated XAFS spectra using wellestablished software packages.7 One of the main challenges of
interpreting XANES and EXAFS lies in obtaining reference spectra
to fit the unknown spectra; measuring XAFS spectroscopy
experimentally is laborious and time-consuming, requiring X-ray
beams of finely tunable energy that are accessible only through
synchrotron radiation facilities.5 To the authors’ knowledge, open
reference databases usually contain at most hundreds of XAS
spectra. For example, the electron energy-loss spectroscopy (EELS)
database8 initiated in the 1990s contains 271 spectra, but only 21
of which are XAS spectra and 17 of which are K-edge spectra. EELS
is theoretically equivalent to X-ray absorption9 under common
acquisition conditions, but is of lower quality in terms of signal to
noise ratio and energy resolution. Most XAS data are available only
via publications in the literature, which cannot be extracted easily
for comparison.
In recent years, theoretical calculations of XAFS have become
more accurate and accessible due to the successful development
of ab initio codes, such as the FEFF program,10,11 as well as
advances in computing power. In this work, we will discuss the
development of a high-throughput framework to generate a
reference XAS database (XASdb) for all materials in the Materials
Project12 database. This framework combines the power of the
Python Materials Genomics (pymatgen) materials analysis library13
with the FireWorks workflow management software14 to carry out
hundreds of thousands of XAFS calculations using the FEFF9
code.10 This framework has been implemented in the Atomate
package.15 More importantly, we have developed a novel
automated XANES spectra matching algorithm that leverages
1
Department of NanoEngineering, University of California San Diego, 9500 Gilman Drive, Mail Code 0448, La Jolla, CA 92093-0448, USA; 2Department of Materials Science,
University of California Berkeley, Berkeley, CA 94720, USA; 3Division of Applied Research and Technology, National Institute for Occupational Safety and Health, Centers for
Disease Control, Cincinnati, OH 45226, USA; 4Department of Physics, University of Washington, Seattle, WA 98195, USA; 5Department of Physics, Applied Physics and Astronomy,
Binghamton University, Binghamton, NY 13902, USA and 6Materials Science & Engineering, Binghamton University, Binghamton, NY 13902, USA
Correspondence: Kristin A. Persson (kapersson@lbl.gov) or Shyue Ping Ong (ongsp@eng.ucsd.edu)
These authors contributed equally: Chen Zheng, Kiran Mathew, Chi Chen
Received: 30 September 2017 Revised: 8 February 2018 Accepted: 12 February 2018
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1234567890():,;
ensemble learning techniques to identify similar XANES spectra
from our computed reference XASdb. We believe the combination
of the XASdb with these machine-learned spectra matching tools
will be an invaluable resource to the materials research community by greatly enhancing the efficiency at which experimental
XAS spectra can be analyzed. It should be noted that this work
primarily focuses on common K-edge XANES spectra; higher edge
XANES and EXAFS computations and analysis are currently
ongoing and will be discussed in future publications.
RESULTS AND DISCUSSION
We have selected the latest version (v9) of the popular FEFF
program as our software of choice in this work. FEFF is a program
for ab initio multiple-scattering calculations of XAFS and various
other spectra for clusters of atoms. This choice is motivated by
three factors: (i) FEFF-computed spectra has been shown to yield
excellent agreement with experimentally measured spectra in a
broad range of studies;16–18 (ii) FEFF calculations are relatively
inexpensive compared to other approaches for computing XAS
spectra (e.g., a typical FEFF calculation takes < 1 h on a single
node, while multi-day, multi-core calculations are necessary for
DFT-based spectra calculations); and (iii) FEFF requires minimal
adjustable parameters. These three advantages make FEFF an
ideal candidate for automation to generate XAS spectra across a
broad range of chemistries. A key step in any automation
framework is benchmarking of computational parameters for
convergence and accuracy. The benchmarking dataset and criteria
are detailed in the Methods section. The Pearson correlation
coefficient, as given by the following expression, is used as the
benchmarking criterion.
PD
i¼1 Xi X Yi Y
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ffi;
SPearson ðX; Y Þ ¼ r
(1)
2 PD
2
PD
i¼1 Xi X
i¼1 Yi Y
where Xi and Yi represent the absorption coefficients of two
spectra on the same energy grid. The value of Spearson can range
from −1 to 1, with a value of 1 being a perfect match. Used in this
context, the Pearson correlation coefficient is a similarity metric,
i.e., it measures the degree of similarity between two spectra.
We have tested the convergence of the FEFF calculated spectra
with respect to four parameters: the radius of the cluster
considered in the full multiple-scattering (FMS) calculation (selfconsistent field (SCF) rfms1), the total number of multiplescattering paths considered (FMS rfms), the exchange-correlation
potential (EXCHANGE), and the treatment of the core (COREHOLE)
(see Methods for a detailed description of the FEFF input file).
The SCF rfms1 was varied from 2 to 8 Å, and the spectrum at the
highest value (8 Å) was set as the reference for each material.
Figure 1 shows the computed Pearson correlation coefficients
between spectra computed at lower rfms1 and the reference. We
find that the computed spectra are converged (Spearson > 0.95) at
around rfms1 = 6 Å for all material, though the Al K-edge for
aluminum nitride is converged only for rfms1 = 6.5 Å. Given that
the computational cost increases substantially for rfms1 > 7 Å (see
Supplementary Fig. 1), we have chosen rfms1=7 Å as the default
setting for SCF in the high-throughput XANES computations.
The rfms field in the FMS card was varied from 3.0 to 11.0 Å at
1.0 Å intervals, and the spectrum at the highest value (11 Å) is set
as the reference for each material. We find that the computed
spectra are converged (Spearson > 0.95) around rfms = 9 Å for all
materials (see Supplementary Fig. 2(a)). Since the computational
cost increases substantially for rfms >9 Å (see Supplementary Fig.
2(b)), we have chosen rfms=9 Å as the default setting for FMS in
the high-throughput XANES computations.
In FEFF9, two approximations of the core-hole potentials have
been implemented, i.e., a fully screened potential based on the
npj Computational Materials (2018) 12
Fig. 1 Benchmarking results of rfms1 parameter in the SCF card for
K-edge XANES of various materials. The rfms1 parameter specifies
the radius of the cluster considered for the full multiple scattering
during self-consistent potential calculations. Pearson correlation
coefficients were calculated between spectra calculated at different
rfms1 and the reference calculated at rfms1=8.0 Å
final-state rule (FSR) and a linear random-phase-approximation
(RPA) screening. Systematic reviews of these two approaches have
been done by Rehr et al.19 We evaluated the performance of all
three core-hole options in FEFF9 on the computed K-edge XANES.
As shown in Supplementary Fig. 3(a), spectra obtained using both
the FSR and RPA are in much better agreement with experimental
results than ones without core-hole treatment. The spectra
computed without a core-hole treatment lack the edge enhancement observed in the experiments. In general, spectra obtained
using FSR and RPA are similar (Supplementary Fig. 3(b)). We have
chosen RPA screening as the default setting for the highthroughput XANES computations as the FSR might breakdown
for the L-shell metals.20
Similar evaluations of the EXCHANGE card options reveal that
the default Hedin-Lundquist model is the best option (see
Supplementary Fig. 4).
Sensitivity of computed XAS spectra to lattice parameters
The FEFF code uses a self-consistent DFT calculation of the Fermi
energy based on the real-space Green’s function (RSGF) approach
with muffin-tin potentials for a given lattice structure. Comparing
to the full-potential calculations, we find that the FEFF calculation
of the densities of states is typically in fairly good agreement with
DFT for many materials. In the Materials Project, the PerdewBerke-Ernzerhof (PBE)21 generalized gradient approximation functional was used as the default for all relaxation calculations. As it is
well known that PBE leads to systematic errors of up to 5% in the
lattice parameters (with a tendency to overestimate),22–25 we
tested the sensitivity of computed XANES spectra to ±5% changes
in the lattice parameters. The results are shown in Fig. 2.
We find that the Fermi energy level of the spectrum is sensitive
to the lattice parameter variation (Fig. 2a). The Fermi energy level
shifts towards lower energy as the lattice parameter increases,
while the spacing of the spectral features contracts at the same
time. An example for Na K-edge of Na2O is shown in Fig. 2b, and
additional examples are available in Supplementary Fig. 5.
A portion of the Fermi energy shift can be attributed to the
artifacts of the FEFF’s potential approximation model (see
Supplementary Fig. 8). Nevertheless, the shape of the spectra
remains unchanged. While different corrections to eliminate the
artificial component of the dependence have been reported,26
these approaches are not amenable to a high-throughput
approach. Here, we note that due to the approximations used in
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Fig. 2 a Relationship between the Fermi energy level of K-edge XANES and a lattice parameter changes. Fermi energy levels of the unstrained
structures are used as references. b Visualization of Na K-edge XANES spectra in Na2O (mp-2352) calculated with different applied strain values
FEFF, we need to calibrate the Fermi level with experimental
spectra. Therefore, a pure energy shift only translates to an energy
calibration value in the post processing.
In summary, the PBE-relaxed structures from the Materials
Project can be used as the input for high-throughput XANES
calculations, even though there are other functionals27,28 that may
provide better lattice parameters estimates.29–32
Workflow and database
Using the high-throughput parameters outlined above, we
developed a high-throughput workflow for FEFF XAS calculations
within the open source computational materials science workflow
package Atomate.15 Atomate provides a high-level interface to
compose workflows using the widely used open source materials
science software such as Pymatgen,13 FireWorks,14 and Custodian.
The proposed default FEFF9 parameters have been implemented
as “input sets” in Pymatgen,13 which ensures reproducible and
automated generation of standardized input files for any material.
The compounds used in the high-throughput spectra generation
were obtained from the Materials Project database.12 For each
compound, the K-edge XANES spectrum was computed with each
symmetrically unique site in the structure as the absorbing atom.
All computed spectra, as well as accompanying meta-data (e.g.,
input structure, absorbing atom, materials project id, etc.), are
stored in a MongoDB database for on-demand querying and
retrieval of data. So far, K-edge XANES spectra have been
computed for more than 40,000 unique materials in the Materials
Project database, which amounts to over 800,000 K-edge spectra.
This is by far the largest repository of XANES spectra in the world,
and is growing rapidly. Future plans include the calculation of
XANES for L, M, and N shells as well as EXAFS spectra.
Spectra matching using ensemble learning
To extract the most utility and power from the XASdb, we have
developed a novel Ensemble-Learned Spectra IdEntification
(ELSIE) algorithm that allows for rapid identification of matching
spectra for any experimental XAS spectra. The main goal of
spectral matching is to obtain a list of compounds (the “hit list”)
whose spectra are most similar to that of the target spectrum. The
success and failure of matching is defined by the characteristics of
the spectrum. In the case of XANES spectra, the relevant
information to be extracted is the coordination environment
and oxidation state of the absorbing atom. As multiple materials
can have atoms in the same oxidation state and coordination
environment, we define the matching to be successful if the
correct coordination environment and oxidation state are within
the top entries.
The ELSIE algorithm uses the ensemble method to improve the
robustness of XAS identification. In ensemble learning, the core
concept is the combination of multiple weak learners to achieve
superior performance. It relies on the assumption that each weak
learner is better than a random guess, and each weak learner
captures different aspects of the problem. At the core of the
algorithm is the process of building individual weak learners.
Taking inspiration from the spectra matching algorithms for
Raman spectroscopy33 and other spectra,34,35 we broke down the
problem of matching XAS spectra into two main steps, namely
preprocessing and similarity computations. We define each weak
learner to be a combination of a preprocessor (a specific series of
preprocessing steps) with a similarity metric. Figure 3 provides an
overview of the ELSIE algorithm (see Methods section for the
details on the construction of the ELSIE algorithm).
We evaluated the ELSIE algorithm using 13 XANES spectra from
EELSDb (Supplementary Table 1), supplemented by six highquality experimental XANES spectra of V2O5, V2O3, VO2, LiNiO2,
LiCoO2, and NiO from previous studies.36,37 The inclusion of this
latter dataset is motivated by our desire to improve the diversity
of the test data, especially with regards to transition metal species.
The first step is to narrow down the candidate computed
reference spectra by the absorption element (A). Though this
information is usually known a priori, the characteristic XAS
absorption edge energy follows a power law with the atomic
number,5,6 which leads to clearly separated energy ranges. Hence,
we can identify the absorption element with 100% accuracy by
comparing the energy range of the target spectrum to tabulated
X-ray absorption edge data.38
Once the absorbing element A is identified, the computed
spectra of all materials within the same chemical system are
queried from the XASdb. For example, for the Al K-edge of Al2O3,
we include the Al K-edge spectra of all Al and AlxOy materials as
reference spectra. We excluded compounds with energy above
hull (Ehull) larger than 100 meV/atom since they are not likely to be
stable.39 For C K-edge XANES of the diamond structure (Fd3m), we
relaxed the constraint to 200 meV/atom as the corresponding
entry (mp-66, diamond) has an Ehull of 136 meV/atom. It should be
noted that though the individual absorption spectrum for each
symmetrically distinct site was computed for all crystal structures
in the Materials Project database, the reference spectra used for
comparison with the target spectra are constructed by summing
these individual spectra taking into account the site multiplicities.
To evaluate the overall performance of ELSIE, we looked at
three key metrics: (i) whether the correct structure is within the
top 5 ranked computed spectra, (ii) whether the top ranked entry
has the absorbing species in the correct oxidation state, and (iii)
whether the top ranked entry has the absorbing species in the
correct coordination environment, i.e., coordination number and
geometry. Where the exact structural information is not available
(e.g., in the experimental spectra from EELSdb), it is assumed that
those spectra correspond to the ground state structures in the
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Identify
Absorption
Species
Database
Learner 1
Learner N
Peak Shifting /
Alignment
Peak Shifting /
Alignment
Spectra Norm.
(Optional)
Spectra Norm.
(Optional)
Feature Trans.
Feature Trans.
Intensity Norm.
Intensity Norm.
Similarity
Measure
Similarity
Measure
Rank 1
Rank N
Combined
Rank
Prob. Each
Spectrum
Fig. 3 Workflow schema of the Ensemble-Learned Spectra IdEntification (ELSIE) algorithm. The algorithm consists of two steps. In the first
step, the absorption species is identified and used to narrow down the candidate computed reference spectra. In the second step, the spectral
matching ensemble yields a rank-ordered list of computational spectra according to similarity with respect to the target spectrum
Materials Project database with the same chemical composition. It
should also be noted that some reference materials may have the
same element in multiple oxidation states and coordination
environments. Therefore, the application of metrics (ii) and (iii)
merely indicates whether at least one of the distinct sites in the
top entry have the correct oxidation state and coordination
environment. The results are summarized in Table 1.
npj Computational Materials (2018) 12
Of the 19 test spectra, we find that the correct structure is
within the top 5 ranked structures for 11 systems, i.e., only 57.9%
accuracy. However, the correct oxidation state and coordination
environment are in the top entry for 16 and 15 systems, i.e.,
accuracies of 84.2% and 78.9%, respectively. The best coefficient α
is found to be 0.01. Given that XANES is a technique primarily used
to extract oxidation state and coordination environment
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Table 1.
Performance of ELSIE algorithm on 19 test spectra
Formula Space
group
Absorbing
species
Correct
structure
within
top 5
rank?
Correct
oxidation
state in
top
entries?
Correct
coordination
environment
in top entries?
SiO2
P32 21
Si
No
Yes
Yes
Si
AlPO4
Fd3m
I4
Si
Al
Yes
No
Yes
Yes
Yes
Yes
Yes
SiC
F43m
Si
No
Yes
Al2O3
R3c
Al
Yes
Yes
Yes
Al
Fm3m
Al
Yes
Yes
Yes
Na2O
Fm3m
Na
Yes
No
No
C
Fd3m
C
No
Yes
No
B2O3
P32 21
B
Yes
No
No
Si3N4
Si3N4
P31c
Si
P63 =m Si
Yes
Yes
Yes
Yes
Yes
Yes
AlN
P63 mc
Al
Yes
Yes
Yes
NaCl
Fm3m
Na
Yes
Yes
Yes
V2O5
Pmmn
V
No
Yes
No
VO2
P21 =c
V
No
Yes
Yes
Yes
V2O3
R3c
V
No
Yes
LiNiO2
R3m
Ni
No
No
Yes
NiO
LiCoO2
Fm3m
R3m
Ni
Co
Yes
Yes
Yes
Yes
Yes
Yes
information, these results are a major validation of the effectiveness of the ELSIE matching algorithm.
To emphasize the effectiveness of the ensemble approach, we
also performed the same benchmark using a single learner
utilizing just the sigmoid squashing function and cosine similarity
measure on spectra that have been pre-normalized with respect
to summed intensity. The ELSIE algorithm outperforms the single
learner approach by 15.8% in identifying both the correct
oxidation state and coordination environment.
We will now illustrate the performance of our spectral matching
algorithm with a few case studies on diverse chemistries. For all
spectra, we have confined our comparison to the energy range
from −10 to 45 eV from the absorption edge, which is the region
typically referred to as XANES.
Case study 1: main group metals
Figure 4a, b shows the ELSIE spectral matching results of the Al Kedge XANES of α-Al2O3 and Na K-edge XANES of NaCl,
respectively. For both target spectra, the correct oxidation states
and coordination environments are found in the top candidates.
Furthermore, we may observe that our proposed peak shifting
approach is effective in aligning the target and reference spectra.
Figure 4c shows a notable case—the Na K-edge of Na2O—
where the ELSIE algorithm fails. Here, the ELSIE algorithm returns
elemental Na as the top ranked result, as opposed to Na2O. The
main reason for this failure is that the FEFF-computed spectra is
not in good agreement with experimental spectra (see Supplementary Fig. 7 for this and a few other examples). Possible
solutions include the use of real-space full-potential multiplescattering theory or other first principle approaches.40 For Na2O in
particular, we find that the experimental Na K-edge XANES of
Na2O is more similar to the computed Na K-edge XANES of
Na2CO3 (Supplementary Fig. 7(c)), which may indicate possible
contamination by the atmosphere in experiments.
Case study 2: transition metal oxides
Figure 5 shows the ELSIE spectra matching results of the Ni K-edge
XANES in NiO, Co K-edge XANES in LiCoO2. From Fig. 5a, we note
that although the computed peak positions and amplitude are not
in great quantitative agreement with the experimental measured
spectra, the ground state NiO entry is nevertheless returned as the
top ranked candidate. In particular, the small Ni 1s-3d peak at
8332 eV in the experimental Ni K-edge XANES of NiO is not
present in the FEFF calculated spectra. There is, however, a small
peak at 8337 eV in the FEFF calculated spectra, which we believe is
the Ni 1s-3d peak. The inaccuracy in the position of the peak may
be due to the muffin-tin approximation used in FEFF.
For LiCoO2 (Fig. 5b), the ground state structure of LiCoO2 (R3m)
is among the top five entries. All Co3+ ions in the top entry (Li
(CoO2)2) are in octahedral coordination, i.e., the same coordination
environment of Co3+ ions in LiCoO2 (R3m). We may, therefore,
conclude that the ELSIE algorithm performs satisfactorily in both
instances.
Figure 5c shows the ELSIE spectra matching results for the V Kedge of V2O5 (Pmmn). The ELSIE algorithm fails to retrieve the
correct square-pyramidal coordination environment of V5+ in V2O5
(Pmmn). Indeed, vanadium ions in the top five matches returned
by the ELSIE algorithm are in octahedral coordination. Here, the
relative similarity of the V K-edge spectra for the different V
oxidation states and coordination environments seems to be the
key issue. Further structural refinement based on EXAFS simulations, therefore, becomes critical, which will be available in the
XASdb in the near future.
In conclusion, we have demonstrated the development of a
large database for XAS using high-throughput FEFF calculations.
Parameter benchmark results indicate that the overall quality of
the FEFF9 calculations with default input parameters is in
quantitative agreement with experiments, which is adequate for
comparison purposes. We developed a novel spectra-matching
algorithm—the ELSIE algorithm—that enables the rapid matching
of computed reference spectra to any target spectra. The
ensemble learning approach far outperforms any single approach
based on a pre-defined set of preprocessing and similarity metric;
outstanding ~84 and ~79% accuracies in identifying the correct
oxidation state and coordination environment are demonstrated
based on a diverse test set comprising 19 experimental XANES
spectra. The XASdb with the ELSIE algorithm has been integrated
into a web application in the Materials Project, providing an
important new public resource for the analysis of XAS to all
materials researchers, and the ELSIE algorithm itself has been
made available as part of veidt, an open source machine-learning
library for materials science.
METHODS
Benchmarking details
Robust, well-defined datasets are necessary for any benchmarking
exercise. We have used the existing high-quality K-edge XAS spectra
available in the open EELS Data Base (EELSDb)8 as reference data, and
matched them with the corresponding materials in the Materials Project12
using the Materials API41 and pymatgen.13 For materials in the EELSDb
without structural information, ground state structures with identical
chemical compositions in the Materials Project were used. For spectra in
EELSDb taken using the same materials, we selected one and adopted it in
our study. Supplementary Table 1 summarizes the 13 unique materials
used in this work.
FEFF
The FEFF software calculates X-ray absorption spectra using the RSGF
formulation of the multiple-scattering theory.11 The X-ray absorption μ is
written in terms of the imaginary part of the one-particle Green’s function
G(r,r′; E), which incorporate both the inelastic losses and other quasiparticle
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(a)
(b)
(c)
Fig. 4 Results of the similarity ranking returned by the ELSIE matching algorithm on a Al K-edge XANES of α-Al2O3 entry; b Na K-edge XANES
of NaCl; and c Na K-edge of Na2O. Detailed information about the retrieved compounds can be found in the Materials Project website, a Al2O3
(Pbcn, mp-1938), Al2O3 (Pna21, mp-2254), Al2O3 (R3c, mp-1143), and Al2O3 (C2/m, mp-7048), b NaCl (Fm3m, mp-22862), Na (Im3m, mp-127), Na
(P63/mmc, mp-10172) and Na (I43d, mp-567772), and c Na (Im3m, mp-127), Na (P63/mmc, mp-10172), Na2O (Fm3m, mp-2352), and Na (I43d,
mp-567772), in decreasing similarity order
(a)
(b)
(c)
Fig. 5 Results of the similarity ranking returned by the ELSIE matching algorithm on a Ni K-edge XANES of NiO; b Co K-edge XANES of LiCoO2;
and c V K-edge of V2O5. Detailed information about the retrieved compounds can be found in the Materials Project website, a NiO (Fm3m, mp19009), NiO2 (P63/m1, mp-543096), NiO2 (R3m, mp-25593) and NiO (Fm3m, mp-715434), b Li(CoO2)2 (P2/m, mp-553952), Li6CoO4 (P42/nmc, mp18925), CoO2 (P3m1, mp-714976) and LiCoO2 (R3m, mp-24850), and c V2O5 (C2/c, mp-542844), VO2 (Pnnm, mp-714880), V6O13 (Cmcm, mp715617) and V9O17 (P1, mp-716723), in decreasing similarity order
npj Computational Materials (2018) 12
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effects. In terms of G(r,r′; E), μ is given by:
1
b rGðr; r 0 ; E Þ2
b r 0 c θΓ ðE EF Þ;
(2)
μ ¼ Im c2
π
where θΓ is a broadened step function at the Fermi energy EF. This yields a
unified treatment of EXAFS and XANES. The treatment of X-ray absorption
can then be separated into atomic and scattering parts, i.e., G(r,r′; E) =GC(r,r
′; E)+GSC(r,r′; E). The exact result of GSC(r,r′; E) is given by the full matrix
inverse, or equivalently, a sum over all multiple-scattering paths.42 For the
XANES calculation, FEFF implements the FMS technique, which includes
the contributions from all orders of scattering within a cluster containing
the absorber and scatterers. The FEFF code also incorporates a GW-based
self-energy based on the Hedin-Lundqvist plasmon-pole model, which
includes effects of electron–electron interactions such as mean-free paths
and self-energy shifts. This method has been well tested and is usually a
good approximation for EXAFS and reasonable for XANES. FEFF includes a
screened corehole and gives results for excitonic enhancements comparable to GW/Bethe-Salpeter equation (BSE) calculations in many materials.
FEFF can also incorporate Debye-Waller factors using correlated-Debye or
more advanced models. Further details on the FEFF code and its
theoretical foundations can be found in ref. 11 for interested readers.
In the FEFF input file, parameters are specified in control “cards”. The
following parameters in FEFF were tested for convergence.
i. Self-consistent field (SCF): The rfms1 field in the SCF card specifies
the radius of the cluster considered in the FMS calculation. The
higher the rfms1 is, the greater the number of atoms is included in
calculation.
ii. Full multiple scattering (FMS): The rfms field in the FMS card
determines the total number of multiple-scattering paths considered in the XANES calculation. Default values are used for the other
five optional fields in the FMS card.
iii. EXCHANGE: The EXCHANGE card specifies the exchange-correlation
potential model used for XANES calculation. No shift was applied to
the Fermi energy level in this work, i.e., the second and third fields of
the EXCHANGE card were kept being 0.
iv. COREHOLE: The COREHOLE card is used to specify the treatment of
the core during XAS calculations. ‘‘Core hole’’ is the hole in the
orbital formed by the excitation of a single electron from that
orbital.5 In FEFF9 code, a combination of BSE and time-dependent
density functional theory (TDDFT) is used to improve the
approximation of the core hole interactions.10,20
ELSIE algorithm construction
We adopted the concept of ensemble method to index the most similar
spectra from the database with respect to a target spectrum. Each weak
learner has a unique combination of a few spectral preprocessing
techniques and one similarity metric, we will describe the preprocessing
approaches and similarity metrics in turn.
Each preprocessor comprises a series of steps, designed to emphasize or
weaken certain characteristics of the experimental and computed spectra.
A preprocessor is generated as follows:
1. Peak shifting and quantization: This step is necessary to all
preprocessors. Because of the differences in energy sampling
intervals and energy ranges, linear interpolation was used to
convert each spectrum to a vector of 200 intensity values with
identical energy grid. The reference spectra are shifted such that the
onset of absorption, which is well-defined by the photoelectric
effect, is aligned with that of the target spectra. This onset is
determined by ascertaining the lowest incident energy at which the
computed absorption intensity reaches 6% of the peak intensity.
2. Pre-normalization: We included an optional pre-normalization step
to rescale the intensity to a similar range. Given the spectrum X with
Xi represents the ith intensity, four normalization approaches are
adopted:43
Xi
Xinorm ¼ P :
Xi
(3)
Xi
Xinorm ¼ pffiffiffiffiffiffiffiffiffiffiffi
P 2:
Xi
(4)
Xinorm ¼
Xi Xmin
:
Xmax Xmin
(5)
Xinorm ¼ ðXi μÞ=σ,where μ ¼
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
X
ðXi μÞ2 =n:
σ¼
P
7
Xi =n and
(6)
3. Feature transformation: Several feature transformation functions
were implemented in the third step, which include the square root
and sigmoid squashing functions. The sigmoid squashed spectrum
is calculated
using X 0 ¼ 1cos2 ðπX Þ. The squared root squashing uses
pffiffiffi
X 0 ¼ X , where X′ is the squashed new spectrum. This technique
has shown to improve the response sensitivity with respect to
different spectral features.44 The feature transformation functions
also include taking the first or second order derivative of spectrum,
or weighted the spectra with the first and second order derivatives.
This step is necessary to make distinct weak learners.
4. Normalization: This last step is for all preprocessors. The spectra are
all
PDnormalized such that the sum of intensities is equal to 1, i.e.
i¼1 Xi ¼1.
Both the computed and target spectra are processed using the same
series of steps for each preprocessor.
The preprocessed target and computed spectra are then compared in a
pairwise manner using a similarity metric. Only bin-to-bin similarity metrics
are used in the ELSIE algorithm development as they are less
computationally demanding for high-throughput datasets.45 Four commonly used similarity metrics in the literatures are used in the ELSIE
algorithm:
1. Pearson correlation: as defined in the Benchmarking section.
2. Euclidean similarity: In the D-dimensional spectral feature space, the
Euclidean distance between two spectra X and Y is given by the
following equation:
rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
XD
(7)
dEuc ¼
jX Yi j2 :
i¼1 i
The spectral similarity measure can be derived from the distance
calculated using the following expression:
SEuc ðX; Y Þ ¼ 1
dEuc ðX; Y Þ
;
max
dEuc
(8)
max
is the absolute maximum expected Euclidean distance
where dEuc
between two probability mass functions.45
3. Cosine similarity: The cosine similarity measure is the normalized
inner product and measures the angle between two spectral
vectors.46 The cosine similarity between two spectra can be
calculated as:
PD
i¼1 Xi Yi
SCos ¼ qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
(9)
PD 2ffiqffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
PD 2ffi :
X
i¼1 i
i¼1 Yi
4. Ruzicka similarity: The Ruzicka45 similarity between two spectra is
given
PD by the following equation:
minðXi ; Yi Þ
SRuz ¼ PDi¼1
:
(10)
max
ðXi ; Yi Þ
i¼1
The combination of preprocessors and similarity metrics results in a total
of 168 learners that can potentially be used to construct the ELSIE
algorithm. To make an ensemble that outperforms individual learners, one
prerequisite is that each learner should have an error rate lower than
random guessing. We, therefore, filtered the 168 leaners to 33 and
adopted them in the ELSIE algorithm. The detailed filtering procedure can
be found in the Supplementary Information.
For each target spectrum, each learner (one preprocessor + one
similarity metric) outputs similarity scores for the reference spectra.
However, the quantitative scores for different similarity metrics cannot be
compared even for the same target spectrum. In the ELSIE algorithm, we
instead combine the reference spectra ranking from each learner to derive
an ensemble result. For a mixture of classifiers of various types, rankingbased combination methods have been shown to be more reliable.47
Based on the rankings, we compute the Borda count, defined as the
number of candidates that are ranked equal and below the specific
candidate. For example, the top spectrum among ten computed
candidates would receive a Borda count of 10, while the second ranked
spectrum has a Borda count of 9. For each target spectrum, the Borda
Published in partnership with the Shanghai Institute of Ceramics of the Chinese Academy of Sciences
npj Computational Materials (2018) 12
Automated generation and ensemble-learned matching...
C Zheng et al.
8
counts of the reference spectra under all learners are then summed to
arrive at a consensus ranking.48
Finally, the Borda ranks of all reference spectra are then combined with
a penalty term for the peak shift and converted to a probabilistic estimate
using the modified softmax function. The probability of a reference
spectrum Xk is indicated by P(Xk) where the superscript k indicates the k-th
spectrum, and is calculated as follows:
1. The Borda count of each reference (Rk) is normalized with respect to
Rk
This step is required to avoid the
the count sum: Rknorm ¼ P
Rk
exponential
overflow.
k
2. P X is then calculated by the following equation:
αjΔSk j
exp Rknorm exp δS
k
(11)
;
P X ¼P
k
exp Rknorm exp αjδΔSS j
where ΔSk could be calculated as ΔSk ¼Sk S:Sk is the peak shift amount
between the reference spectrum Xk and the target spectrum. S is the mean
peak shift of the reference spectra. δS is the standard deviation of Sk.
αjΔSk j
is therefore a term
Coefficient α is fitted to the test dataset. exp δS
that imposes a larger penalty on large peak shifts relative to smaller peak
shifts.
The algorithm itself has been highly optimized by leveraging on wellestablished numerical packages such as numpy and scipy.49,50 On a laptop
computer with Intel i5 2.6 GHz single CPU and 2 GB of RAM, the ELSIE
algorithm can perform a comparison between a target and candidate
spectrum in about 0.03 s. Typically, 20–30 spectra are selected for
comparison according to the rules that the computational reference
spectra should have identical absorption species, limited number of
elements and Ehull < 100 meV/atom. The overall time to perform a
complete ranking is, therefore, around 1 s, which allows for on-the-fly
matching of uploaded spectra.
Data availability
The computed spectra in the XASdb have been made available in the
Materials Project website. A new web application—the XASApp (https://
materialsproject.org/#apps/xas/)—has been developed which allows any
user to compare multiple X-ray absorption spectra and find matches within
the XASdb for an uploaded spectrum using the ELSIE algorithm.
The ELSIE algorithm has also been made publicly available as a part of
veidt, an open-source Python machine-learning library for materials science
developed by the Materials Virtual Lab that is available on the Python
Package Index and Github (https://github.com/materialsvirtuallab/veidt).
ACKNOWLEDGEMENTS
This work is supported by the National Science Foundation’s Cyberinfrastructure
Framework for 21st Century Science and Engineering (CIF21) program under Award
No. 1640899. The Materials Project, supported by the Department of Energy (DOE)
Basic Energy Sciences (BES) program, under Grant No. EDCBEE is gratefully
acknowledged for web dissemination and data infrastructure. The FEFF project is
supported primarily by DOE BES Grant DE-FG02-97ER45623. We also acknowledge
computational resources provided by Triton Shared Computing Cluster (TSCC) at the
University of California, San Diego, the National Energy Research Scientific
Computing Center (NERSC), and the Extreme Science and Engineering Discovery
Environment (XSEDE) supported by National Science Foundation under grant
number ACI-1053575. L.F.J.P. acknowledges support from the National Science
Foundation (DMREF-1627583).
AUTHOR CONTRIBUTIONS
C.Z., K.M., and C.C. performed the workflow design, code implementation, and
calculation analysis. Y.C., H.T., and A.D., J.J.K., F.D.V., and J.J.R. helped to the
simulations of XAS spectra. L.F.J.P. helped experimental XANES spectra analysis. K.P.
and S.P.O. is the primary investigators and supervised the workflow and code
development. All authors contributed to the writing and editing of the manuscript.
ADDITIONAL INFORMATION
Supplementary information accompanies the paper on the npj Computational
Materials website (https://doi.org/10.1038/s41524-018-0067-x).
Competing interests: The authors declare no competing interests
npj Computational Materials (2018) 12
Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims
in published maps and institutional affiliations.
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Open Access This article is licensed under a Creative Commons
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© The Author(s) 2018
Published in partnership with the Shanghai Institute of Ceramics of the Chinese Academy of Sciences
npj Computational Materials (2018) 12
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https://openalex.org/W2013887271
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https://zenodo.org/records/2323693/files/article.pdf
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German
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Über die basische Funktion des Methoxyls
|
Berichte der Deutschen Chemischen Gesellschaft
| 1,913
|
public-domain
| 6,520
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481. Hugo Kauffmann und Felix Kieser: lher die
basische Funktion des Methoxyls.
(Eiogegangen am 10. November 1913.) 1. Um in die basische und auxochrome Wirkungsweise des Me-
thoxyls weitere Einblicke zu gewinnen, haben wir, wie schon friiber
angegeben'), eine Anzahl m e t h o x y l i e r t e r D e r i v a t e des C h a l k o n s
und des T r i p h e n y l - c a r b i n o l s einer Untersuchung unterworkn. Pie Zahl der Methoxyle, die wir in die genannten Verbindungen ein-
fihrten, schwankte zwischen zwei und sechs. Der leitende Gedanke
war, zu priifen, wie die Funktionen einer Methoxylgruppe beeinflufit
werden, wenn sich an demselben Benzolring noch ein zweites oder
drittes Methoxyl befindet. B a e y e r und V i l l i g e r q haben beobachtet, daB beim Dibenzal-
aceton und Triphenyl-carbinol der basische EinfluD des Methoxyls am
stlrksten in para-Stellung und am schwachsten in meta-Stellung zum
Chromophor ist. Wir haben gefunden, daB diese Regel auch dann
noch Geltung besitzt, weun die Zahl der am gleichen Benzolring
sitzenden Methoxyle gr8Ber als eins ist. Dieser Regel liegt demnach
unzweifelhaft eine GesetxmEiBigkeit von allgemeiner Bedeutung zugrunde,
und es ist uns such gelungen, im Rahmen der Lehre von der Teil-
barkeit der Valenz Verstandnis fur diese GesetzmiiBigkeit zu erlangen. g
g
2. Wir haben, wie B a e y e r und Villiger, die Starke des basi-
schen Charakters der Verbindungen durch Titration der Eisessig-
SchwefelsEiure-Losung mit 75-prozentigem Alkohol gemessen. Urn uber
die GroBe des Beobachtungsfehlers AufschluB zu bekommen, hahen
wir jede einzelne Verbindung funfmal titriert und den mittleren Fehler
des Gesamtresultats berechnet. Dieser betrlgt durchschnittlich 1 ccrn
pro Millirnol und kommt also bei dem groIjen Unterschied der fur die
verschiedenen Substanzen erhaltenen Werte nicht in Betracht. Um
sicher zu sein, da13 sich die Substamen in der Eisessig-Schwefelsaure
nicht zersetzt haben, wurden diese Liisungen auf Eis gegossen, die g
g
2. Wir haben, wie B a e y e r und Villiger, die Starke des basi-
schen Charakters der Verbindungen durch Titration der Eisessig-
SchwefelsEiure-Losung mit 75-prozentigem Alkohol gemessen. Urn uber
die GroBe des Beobachtungsfehlers AufschluB zu bekommen, hahen
wir jede einzelne Verbindung funfmal titriert und den mittleren Fehler
des Gesamtresultats berechnet. Dieser betrlgt durchschnittlich 1 ccrn
pro Millirnol und kommt also bei dem groIjen Unterschied der fur die
verschiedenen Substanzen erhaltenen Werte nicht in Betracht. 3788 3788 Eisessig hellbraune Krystalle vom Schmp. 171O bildet. Das Xanthen
farbt konzentrierte Schwefelsaure purpurrot, also in ganz andrer Farbe
als das Carbinol. 0.1978 g Sbst.: 14.2 ccm N (220, 730 mm). 0.1978 g Sbst.: 14.2 ccm N (220, 730 mm). CssH1~NzO. Ber. N 7.74. Gef. N 7.79. 481. Hugo Kauffmann und Felix Kieser: lher die
basische Funktion des Methoxyls. (Eiogegangen am 10. November 1913.) B. 45, 781, 1333 [1912].
z, B. 35, 1194, 3013 [1902]. B. 45, 781, 1333 [1912]. 481. Hugo Kauffmann und Felix Kieser: lher die
basische Funktion des Methoxyls.
(Eiogegangen am 10. November 1913.) Um
sicher zu sein, da13 sich die Substamen in der Eisessig-Schwefelsaure
nicht zersetzt haben, wurden diese Liisungen auf Eis gegossen, die B. 45, 781, 1333 [1912]. z, B. 35, 1194, 3013 [1902]. z, B. 35, 1194, 3013 [1902]. Berichte d D. Chem. Gesellnohaft. Jahrg. XXXXVI. Berichte d D. Chem. Gesellnohaft. Jahrg. XXXXVI.
213 ausgcscbietlenen Verbindungen abgesaugt und ihre Schmelzpunkte be-
stimmt. Bis auf zwei Fllle, welche aber aus den Betrachtungen aus-
geschaltet wurden, stimmten die Schnielzpunkte mit denen der reinen
Y ubstanzen ti berein. 3. Eine Ubersicht uber die, auf eio Millimol berecbneten, gefun-
denen Zablen geben folgende Tabellen: 3. Eine Ubersicht uber die, auf eio Millimol berecbneten, gefun-
denen Zablen geben folgende Tabellen: C ha1 ko n e. C ha1 ko n e. C ha1 ko n e. Verbindung
2’. 4’- Di met hox ~-chel
kon
3’ 4’-
)D
4.2’.4’-Trimethoxp-chal kon
4.8’ 4’-
3
3.~-Diox~nietli~len-2’.4’-Dimathnxy-chalkon
>>
-3l.4’-
*
-41.5’-
>
2.4.2’.4’-Tetramethosy-chalkon
2.4.3’.4’-
D
3.4.Y.4’-
D
3 42.4’-
D
2.5 Y.4’-
>>
4.5 3l.4’-
>>
3.4.2’.4’.6’-
D
2..5.2’.4’.6’-
>>
2 4.?’.4’.G‘-Pentamethoxy-chalkon
Kalochromie-Farbe
citroocngelb
hellgelh
dunkel orange
kimchrot
dunkel braunrot
purpurrot
dunkel carmoisin
graiiatrot
tlnnkel grmatrot
dunkel kirschrot
dunkel rotbraun
purpur
kirschrot
dunkel kirschrot
rubinrot
)D
B
Basischer
Cherakter
ca. 9
ca. 4
84.7
47.9
93.6
74.4
62.4
110.2
68.C;
45.0
81.0
55.0
40.0
126.4
56.4
56 0 C ha1 ko n e. Diese Tabelle laBt die GesetzmaiSigLeit schon in voller Klarheit
hervortreten, obgleich sie noch einige Liicken enth6lt. Diese Liickra
kommen zum Teil dadurch herein, daB wir nicht alle der von uns
gewunschten \-erbindnngen in dem fur die Untersuchung erforderlichen
Grade von Reinheit herstellen konnten. So lassen namentlich die
Konderisationen des 2.5-Dimethoxy-acetophenons mit Aldebyden vie1
zu wunscben iibrig. Die GesetzmaBigkeit LuBert sich darin, da13 stets die o,p-Derivate,
d. h. die Resorcin-Derivate, s t a r k e r basisch als ihre Isomeren sind. A m geringsten basisch sind die Hydrochinon-Derivate, wiihrend die
Brenzcatechin-Derivate eine Mittelstellung einnehmen. Wir greifen
a19 Beispiel heraus : 2.4.2’.4’-Tetramethoxy-chalkon
1 10.2
3.4.2l.4’-
s
84.0 una
2.5.2l.4’-
*
55.0 Wiihrend hier Verbindungen aufgezahlt sind, bei welchen si& die zu
vergleichenden Metboxyle im A l d e h y d - R e s t befinden, sitaen sie im
folgenden Beispiel im Keton-R’est:
, - Wiihrend hier Verbindungen aufgezahlt sind, bei welchen si& die zu
vergleichenden Metboxyle im A l d e h y d - R e s t befinden, sitaen sie im
folgenden Beispiel im Keton-R’est:
, - 213 Berichte d D. Chem. Gesellnohaft. Jahrg. XXXXVI. 213 Piperonal-2’.4’-dimethoxy-acetophenon 93.6
D
-3‘.4’-
D
74.4
s
-2’.5’-
>
62.4 Wird in die Resorcin-Derivate noch ein weiteres Methoxyl und z w i r
in 6-Stellung eingefiihrt, so komrnen wir zu Phloroglucin-Deriraten,
die dieselben typischen Erscheinungcn zeigen : 2.4.2’.4‘.6’-Pent,smeth~~~y-chnlkon
126.4
3.4.2l.4’ 6’-
>
86.4
f.5.2’.4’.6’-
D
56 0 Wir mochten diesen allgeriieinen Ergebnissen noch einige besoii-
dere beifiigen. Wie vorauszusehen ist, sind die beiden Benzolringe
des Chalkons einander ungleichwertig, und dies druckt sich im bnsi-
schen Charakter der Methoryl-Derivate unverkennbar aus; cleun das
2.4.3‘.4’-Tetramethoxy-chalkon ergibt einen wesentlich andren TVert
81s das 3.4.2’.4’-Tetramethoxy-chalkon, - Wir hnben ferner mit dem
Benzal-3.4-dimethory-acetophenon verglichen
das Cionnninl-3.4-di-
methoxy- acetophenon. Die erstere Verbindung ergibt die Zahl
ca. 4, die zweite die Zahl 36. Man sieht hieraus, da13 ebenso wie
das Methoxyl auch die 1,’erliingerung der chromophoren Kette uiii
eine Athylen-Bindung den basischen Charakter zu steigern vermag. - Des weiteren schien es uns yon Interesse, den Benzol-Riug niit
dem Furan-Ring zii vergleichen. Zu diesem Zweck stellten wir das
Fural-3.4-dimethoxy-acetophenon her, das iins dann beim Titrieren
den Wert 66.5 ergab. Der Furanring ist also uicht our dem Benzol-
ring weit iiberlegen, sondern holt fast den Ring des Resorcin-diniethyl-
athers ein. 4. Die methoxylierten Triphenyl-carbinole gehorchen der Regel
in ebenso ausgepragter Weise. 4. Die methoxylierten Triphenyl-carbinole gehorchen der Regel
in ebenso ausgepragter Weise. 3791 Das Hauptergebnis ist ganz eklatant, denn das Resorcin-Derivat
mit der 2.4-Stellung der beiden Methoxyle iibertrifft seine lsomeren
ganz erheblich. Am nachsten kommt ibm, ebeoso wie bei den Chal-
konen, das Brenzcatechin-I)erirat, wahrend dns Hydrochinon-Derivat
sehr weit von ihrn absteht und nur einen ganx kleineo Wert gibt. Der groBe Gcgensatz zwischen Resorcio- und Hydrochinon-Derivat
springt bei den tetramethoxylierten
Verbindungen noch sehr vie1
Lervorstechender ins Auge, da das 2.4.2‘.4’-I’etramethoxy-fripheugI-
carbinol den Wert ca. 1200 gibt, wahrend das 2.5.2’.5’-Tetramethoxy-
tripheoyl-carbinol nur die Zabl 9 liefert. Bei den Hexamethosy-
Derivaten wachsen sich die Gegensiitze noch schroffer aus, denn das
2.4.2’.4’.2”.4’J-Hexarnethoxy-triphenyl-carbinol
verhalt sich bereits als
Farbstoff, uiibrend das 2.5.2’.5’.2”.5”-Hexamethoxy-triphenyl-carbinok
durch die Einfuhrung der Methoxyle seinen basischen Charakter n u r
unerheblich vergrol3ert. Man konnte geueigt seio, diese Iiefunde durch die hnnahine zu
cleuten, daD das Ringsystem des Kesorcin.dimethylathers i n hiherem
31aBe befahigt sei, die basischen Eigenschaften zu erwecken, als das
Ringsystem des Hydrochinon-dirnethyliitbers. Wiire diese Aulfassung
richtig, so muBteo jeue Resorcin-Derivate, aelche die Methoxyle n i c h t
in or~.lho-iJul.a-Stelluna zum Zentralkohlenstoffatom enthalten, sich gleich-
fall3 durcb stark basische Eigenschaften auszeicbnen. Urn diese rber-
legung zu verfolgen, haben wir auch das 3.5-Dimethoxy-triphenylcar-
binol in den Kreis unserer Beobachtungen gezogen. Diese Terbinduog
ist nur sehr schwach basisch und damit EHllt die obige Annahme. Hieraus folgt daun, daB nicht das Ringsystem als solches die Ent-
scheiduog hat, sondern da13 i n e r s t e r L i n i e d i e S t e l l u n g d e r
11 e t h ox y le z 11 m Ze n t ra 1 k o h 1 r n s t o f f nu scbl a g g e b e u d is t. Die stark basischen Resorcin-Derivate uod die Hydrochinon-Deri-
vate haben die ortlio-standigen Methoxyle gemeinsam. Sie unterscheiden
sich dadurch, darj bei den ersteren die iibrigen Methoxyle sich in
pra-Stellung, bei den letztereo in nwta-Steilung befinden. Die groBe
Differenz zwischen beideo Arten von Stoffen beruht also im wesent-
lichen wieder auf dem schou bei Monornethoxy-Derivateu hervortreten-
den Unterschied zwischen para- und metu-Stellung. 1st die Auffassung richtig, daB die Stellung der Jlethoxyle zuni
Xentralkohlenstoff den maSgebendeu Paktor bildet, dann rnurjten
I’hloroglucin-Derivate stlrker basisch sein als Resorcin-Derivate, denn
es tritt ja noch ein Methoxyl in ortho-Stellung, also in einer \\irk-
sarnen Stellung, hinzu. Trip h e n y 1 - c arbi n o I e. Trip h e n y 1 - c arbi n o I e. ._
__.__.-._____
Verbindung
~ Halochroniie-Farbe 1
~~~~~~~
2.4-Dimethoxy-triphenyl-carbinol
~
dunkel rot
3.4-
cirrmoisinrot
2.5-
n
’
dunkel grun
3.5-
i
schwach gelb
4.4.6-Trimethoxv-tri~henvl-carbinol
dunkel brauorot
. I
. *
2.3.4-
s
dunkel rot
2.4.2’.4’-Tetramethox~-triihenvl-carbinoI
dunkel blaurot
. . A
-
3.5.2j.5’-
>
i
dunkel grun
2.4.6.2’4’-Pentamethoxv-tri~henvl-carbinol dunkel blaurot
2.4.2’.4’.2”..i’’-Bex.?inethoxy- ~riph&yl-carbinol
x. n
2.5.2’.5’.2”.5”-
>\
indigoblan
57.0
43.0
ca. 9
K
K
ca. 11 3791 Diese Ansicht hat sich vollauf bestiitigt, dn
das 2.4.6-l‘rimethoxy-tripbenylcarbinol einen Wert von uber 350, das
2.4-Dimethox) -triphenylcarbinol nber our von ,-)7 lielert. Dali hierbeih ”3 *’ 3792
_. gerade der Eintritt der Methoxylgruppe in oif/tr,-Stellung aesentlicli
ist, ergibt sich durcb den Vergleich nlit dem ?..9.4-lrimetbosy-trj-
phenylcarbinol. Dieses hat dns dritte hlethoxyl in metn-Stellung aiif-
genommen und ist infolgedessen erheblich schwacher bnsisch als d n
Phloraglucin-T)erivat iind erreicht mit seinern”Wert von 53 kaum tlas
2.4-~imethos~-triphen~lcarhinol. Dieses mta-stiindige Methoxyl iar
also so gut wie wirkungslos. Auf Grund dieser Retracbtungeu ist z u schliefien, da8 Phloro-
glucin-Derivate gnoz hervorragend basisch sein werdeo, und wir baberi
uns daher beniuht, noch weitere Derivate darziistellen. Bis jetzt
konnten wir allerdings nur das 2.4.6.2’.4’-Pentarnethoxy- tripbenyl-
carbinol gewiuuen. Diese Verbindung besitzt in der Tat stark basi-
scben Charakter und deutliche Farbstoffeigenscbafteo und steht den1
2.4.2’.4’.2”.4”-Hexametho~~-triphenylcarbinol
ganz nnhe. Von beson-
derem lnteresse wire danach die Herstellung des ?.4.6.2’.4’.6’.2”.4”.6”
Enneamethoxy-triphenylcarbinols, das voraussichtlicb eine regelrechte
Farbbase ist. Wir versuchten zu diesem interessanten Korper mit
Hilfe des J o d - p h l o r o g l u c i n - trimethylathers’) zu gelangen, ver-
mochten aber diesen letzteren bis jetzt noch nicht mit Magoesium ztir
Renktion zu bringen. 5. Der tiefere Sinn der eben dargelegten Regelniafligkeiten w i d
Itlarer, wenn man die Sachlage vom Standpunlit der‘Lehre von drr
l e i l b a r k e i t d e r Valenz aus betrachtet. Der Benzolring 1aBt sich
auffassen als ein System ron drei Doppelbindungen, die einander kon-
jugiert sind, und diese Aulfsssung ermoglicht eine Losung der Frage,
die zunaclist nur an einem einzigen Henzolring, drr das Z?ntralkohlen-
stoffatom und ein para-standiges Aurochrom trage, besprochen
sei. 1’s ergibt sich dabei folgendes Pornielbild I: B
l
i
5
li
~ r - ,
-/
~. 1. Aux-4 ’ c,
I c‘c
11. 4 3.’-
i.l? c
::
2
8<
12
AUX B
l
i
5
li
~ r - ,
-/
~. 1. Aux-4 ’ c,
I c‘c
11. 4 3.’-
i.l? c
::
2
8<
12
AUX B
l
i
~ r - ,
~. 1. Aux-4 ’ c,
I c‘c
::
2 AUX Verfolgt nian die Kette vom Auxochrom zum Zentralkohlenstoff,
so erkennt man, da8 die Gruppierung: Aux, 4, 3, 3, I , C ein koii- I ) Dargestellt durch direktes Jodieren Ton Phlol.oglucin-trimetl~~l~tb
nittels Jod und Quecksilbwoxyd in alkoholischer Lhung. I ) Dargestellt durch direktes Jodieren Ton Phlol.oglucin-trimetl~~l~tbc
nittels Jod und Quecksilbwoxyd in alkoholischer Lhung. Man erhilt ihn
ails Ligroin i n langon, weiBen Nadeln vom Schmp. 1’210. 0.1918 g Sbst.: 0.1.534 g AgJ. CrH1,O1.T. Brr. J 43.17. Gef. J 43.23. I ) Dargestellt durch direktes Jodieren Ton Phlol.oglucin-trimetl~~l~tb
nittels Jod und Quecksilbwoxyd in alkoholischer Lhung. Man erhilt ihn
ails Ligroin i n langon, weiBen Nadeln vom Schmp. 1’210.
0.1918 g Sbst.: 0.1.534 g AgJ.
CrH1,O1.T. Brr. J 43.17. Gef. J 43.23. 1) Uber die Wirkungsweise der Auxochroiiie sielie: H. Kauffmann,
2) ElJenda S. 316.
Die Valenzlelirc, S. 500. 2) ElJenda S. 316. I) RaiiFErnnnn, Die Vnlenzlehre, S. 502 fF. 3791 Man erhilt ihn
ails Ligroin i n langon, weiBen Nadeln vom Schmp. 1’210. 3793 jugiertes System darstellt, das zwei Doppelbindungen enthllt. In
dieser Kette ist dnher ein ziemlich weitgehender Valenzausgleich mog-
licb, wodurcb am Ausochrom grol3e ') und am Zentralkohlenstoff nur
kleine Valenzbetriige zur Verfugung stehen. Die punktierten Liniea
versinnbildlichen diese Betrage. Bei einer Verbindung mit orttm-stsodigem Auxochrom ergibt sich
Sclienia 11. Die Kette vom Ausochrom zum Zentrnlkohlenstoff enthllt
cliesmal nur eine eiozige Doppelbindung und dieser Umstaod ist aus-
scblnggebend. E3 ist eine wicbtige Erfahrungstatsache der Lehre von
der 'I'eilbarkeit der Valenz, dab bei konjugierten Systemen die frei
verfiigbaren Valenzreste, welche an den beiden Enden des Systems
nuftreten, urn so gr6Ber smd, je ]anger die Kette der konjugierten
Doppelbindung ist '). Es sind also bei der orlho-stellung weniger
Valenrreste an1 Aiisocbrom uncl mehr am Zentralkohlenstoffutom dis-
pnnibel als hei der pua-Stellung, welche ia eine langere Kette kon-
jugierter Doppelbindungen besitxt. Rei der mefa-Stellung sind folgende Formelbilder zu diskutieren: Rei der mefa-Stellung sind folgende Formelbilder zu diskutieren: Aux
A U X Aux
A U X Man sieht sofort, daB in beiden Formeln zwischen Zentralkoblen-
stoffatom und Auxochrom keine Konjugation besteht. Sind die ver-
'rugbaren Valenzreste am Zentralkohlenstoff klein, so werden diejenigen
mi Auxochrom, wie die Rechnung zeigt, gleichfalls klein. Sind die
Valenzreste am Auxochrom grol3, so werden diejenigen am Zentral-
kohlenstoff ebenfalls grol3. 6. Diese Formelbilder lehren ohne weiteres, dn13 ein grundsiitz-
licher Unterschied zwischen der mela-Stellung und den andren Stellungeu
bestehen muB, und gerade darin, daB sie dies lehren, liegt ihre Be-
deutung. Diese Formelbilder sind daher fur alle weiteren Uberlegungen
nls unumgangliche Unterlage zu xvlhlen. Dnbei ist jedoch auf einen
besonderen Nebenum stand hiti zuweisen. Man kann bei para-stgndigen Auxochromen den K e k u l B - T hiele-
achen Beozolriog anzeicbnen wie man will, stets ziebt sich vom Zentral-
kohlenstoff zum Auxochrom eiue aus zmei Lhppelbindpugen bestehende 1 ) B. 46, 781 [1912].
Da unter Umstiinden mela-stindige Methoxyle
den basischen Charakter dennoch, wenn auch sehr wenip, steigern, SO miissen
im Molekul noch an andren Stellen disponible Valenzbetrjige vorhanden sein.
Diese BetrHge sind aber sehr vie1 kleiner und von uns vorlLuEig auBer Bc-
tracht gelassen. Sie miissen schon beim Triphenyl-carbinol selbst, das ja auch
schon basisch ist, vorhanden sein und k6nnen sogar beriicksichtigt werden.
Wenu air dies nicht taten, so geschah es nur, urn die Formeln und Uber-
legungen nicht durch Komplikationen uniibersichtlich zu machen. g
g
p
3) i,Valenzlehrea, S. 502 ff. konjugierte Kette. Ganz anders i n der ortho-Reihe. Schreibt nian
den Ring in andrer Weise: so bildet nicht mehr die Kette Aus, 2, 1, C, sondern Aus, 2, 3, 4,
5, 6, 1, C ein konjugiertes System. Diese Kette eotbalt sogar drei
Doppelbindungen und daher sollte der Vnlenzausgleich bier sehr stark
sein und sognr noch weiter gehen als beim Pam-Derivat. I)a nun
dies aber nicht der Fall ist, so ist der Schlul3 zu ziehen, daB, weun
der Valenzausgleich zwischen Ausochrom iind Chromophor IHngs
zweier verschiedener Ketten statttinden kann, er den h-urzeren Weg
bevorzugt. so bildet nicht mehr die Kette Aus, 2, 1, C, sondern Aus, 2, 3, 4,
5, 6, 1, C ein konjugiertes System. Diese Kette eotbalt sogar drei
Doppelbindungen und daher sollte der Vnlenzausgleich bier sehr stark
sein und sognr noch weiter gehen als beim Pam-Derivat. I)a nun
dies aber nicht der Fall ist, so ist der Schlul3 zu ziehen, daB, weun
der Valenzausgleich zwischen Ausochrom iind Chromophor IHngs
zweier verschiedener Ketten statttinden kann, er den h-urzeren Weg
bevorzugt. IXeser Schlul3folgeruog diirfte, menigstens soweit Benzolderivnte
in Betracht kommen, allgemeine Bedeutung zukommen. Denn vie
die lriphenj I-cnrbinole verhalten sich die Dibenzal-acetone u u d clip
Chalkone. 7. \\ie cler Vnlenz-Ausgleich die bnsischen lhktionen regelt, Isfit
sich auniichst bei den Triphenyl-carbinolen sehr anschaulich zeigen : AUS - CslT(\
Cs H j F C
c6 Ha Befindet sich das Ausochrom in einer wirksamen Steliung, so
\Iird der \ alenzbetrng am Zeotralkohlenstoff,
aelcher durcb die
punlitierte Linie ausgedrhckt ist, urn so kleiner, je stiirker das Auso-
chrorn ist. Zur Bindung eines vierten Atoms oder eines vierten Restes
an dns ZentralkoblenstoIFatom, etwn zur Bindung eines Chloratoms,
bleibt infolgedesseu UUI so weoiger Valenz zur Verfugung, je starker
das Ausochrom wirkt. J e mehr Ausochrome eiogefuhrt werden, desto
lockerer wird sich dieses vierte Atom verketten und in Form ekes
Ions abspalteo, d. h. desto ausgesprocheneren Salzcharnkter hat die
Verbindung, urn so weniger unterliegt sie der Hydrolyse. Dall sich dieses vierte Atom oder dieser vierte Reat io Form
eioes Ions abspaltet, IL13t sich noch naher begrunden. Dieser Kest
ist durch weniger als eioe ganze Valeoz an das Zentralkohlenstoff
gekettet, und infolgedessen bleibt an ihm ein Valenzbetrag disponibel,
der, wie die Rechoung') ergibt, ebeoso grol3 ist, wie die Summe der
disponiblen Vnlenzbetrlge an den Auxochromen. na im Rahmen der 3793 Valenzlehre, so wie sie von H. K a u I f m a n n formuliert und entwickelt
wurde, freie Valenzreste ausgeschlossen sind, so ist das Formelschema
dahin zu erganzen, da13 diese gleich groBen Betriige sich gegenseitig
absattigen. Damit gelangt man fur den Fall, daB niir ein Auxo-
chrom vorhanden ist, zu folgendem Bilde: AUX 5 cs HA \7
C6Hs-C
A n ,
c6 HJ in welchem An in iiblicher Weise ein Anion bedeutet. Die Uber-
tragung dieses Bildes auf auxochrom - reichere Verbindungen fuhrt
dann zu Formeln, die H. KauFfmann in einer friiheren Arbeit
bereits veroffentlicht hat'). Wesentlich fur das eben entwickelte Schema ist der Umstand,
daB es nur fur para- und ortho-standige Auxochrome moglich ist. Weil ein wela-stiindiges Auxochrom mit dem Chromophor keine kon-
jugierte Kette bildet, ist bei solchen metn-Derivaten, wie wieder aus
der Rechnung') ersichtlich ist, das angegebene Schema nusgesthlossen. Jede Einliihrung eines weiteren Auxochroms in ortho- und nament-
lich iu pra-Stellung bewirkt die weitere Absplitterung eines Valenz-
betrnges aus der Bindung zwischen Anion und Zentralkohlenstoff, in-
den1 das Auxochrom diesen Betrag beausprucht. Nach der Lehre
vun der Teilbarkeit der Valenz bedeutet aber eine Erhohung der
Valeuzzersplitterung nichts andres als eine Zunahme der Ionisierbar-
keit. Der mit An verbundene Rest hat demnach eine um SO griiBere
Neigung, nls Ion aufzutreteu, je grB13er die Anzahl der in ihm ent-
haltenen in or/ho- und para-Stellung befindlichen Auxochrome ist. Ein
hlnl3stab dieser Neigung ist der von uns gemessene bssische Charakter,
und clantit is/ ge:eigt, day die gegebeire Formrilierun,q tatsci'chlich ein an-
sckcoilieher ,qro]ihi.sclier Aic.sdntck d~.$ 7*on icy e.rpriittcutel1 zusammengc-
traqenen illaterials ist. S. Bei den Chalkonen liegen die Verhaltnisse analog, sowie man
dawu ausgeht, da13 bei ihneu wie beim Dibenzal-aceton die Salzbil- dung auf einem fibergang des Carbonyl-Snuerstoffes von der zwei-
wertigeo in die vierwertige Stufe beruht. Die SBnre addiert sich nn
den Carbonyl.Snuerstoff, jedocb nicht in der rein formalen Weibe, wie
dies gewiihnlich angenommen wird. Dieselben ~berlegungen wie oben
beini Triphenyl carbinol verlangen , daB der Saurereat gleichzeitig in)
Valenz-Ausgleich mit den Auxochroiuen steht und dieses Ergebnis fuhrt
xu folgender Formulierung I): A I I X - C ~ HI
CH = C B 2. C --
R
It
0
. .A n
H/ 2 ii s n m m e 11 fa s s tin g. I. I) Der Vollsthdigkeit halber sei beigefiigt, daO auch vom Saure-Wasser-
stoff Valenzlinien zum Ausochronl fiihren und ferner zum Anion.
Hierauf
ist in der hrbeit von H. Kauffmrnn in der Zeitsrhr. f. anorg. Chemie,
Clasaen-Festschrift, 81, 83 [ 19131, niher einqegangeo. Grnppe der Chalkone. Die untersuchten Chalkone wurden in der ublichen Weise durch
Kondensntion aus den eotsprechenden Aldehyden und K e t o n e n mit
Hilfe r o n Alkali hergestellt. Benzal -2.4- dime t h ox y -ace top hen on. 3793 Dcr basische Charakter halorhromer Verbindungen wirtl durch p a m -
stindige Methoryle kriittig, durch ordo-stkndige vie1 schwkher und durch
actn-sthdige unwesentlich gesteigert. Diesel Satz gilt aucli daiin, wenn
mehrere M e t h o x y l e ini gleichen Iiiiig sitzen und (laher sind Verbin-
dungen, .welclie die Methoxyle in Resorcin- oder Phloroglucin-Stellun~ tragen,
sehr stark basisch. 11. Diese Verhiltnisse sintl iiii Rahmen der Lehre von der Teilbarkeit
der Valenz leicht zu verstehen und ftihreii zu den Ton H. Kauffmann f i r
die halocbronien Verbindungen der Tripbenyl-carbinole, Dibenral-acetone und
Chalkone aufgestellten Formeln. 111. Wenn Auxochrom und Chromopbor zwei Wege far den \'alein-
Ausglricli haben, so bevorzugen sic den kiirzeren. 3.4.2l.4'- T c t r a m e t h ox y -c h D I k o 11. Das Konderisationsprodukt erliilt man. PUS Alkohol in schwach gelben
Krystfdlchen, die bei 89O schmelzen. In Xther uod Ligroin ist der Korper
so gut wie unloslich, leichter in Methyl- ond Athylnlkohol, schr leicht in
Cbloroforni, Aceton und Benzol ,
0.1811 g Sbst.: 0.4610 g (202, 0.1006 g HsO. 0.1811 g Sbst.: 0.4610 g (202, 0.1006 g HsO. g (
g
C19HwO5. .Ber. C 69.48, H 6.14. Gef. n 69.42,
n 6.23. 3.4- U i ox 7
III c t 111- 1 on- 'LI.4'- tl i m c t IJ o x y - c 11 n 1 k o n. Aus Alkohol in LlaDgclbcn Nidelchcn vom Schmp. 135O. Der KGrper
i d in Alkohol, Benrol, .\thcr rind Ligroiil schwcr, ' i n Accton untl Chloro-
form leicht 16slich. 0.1849 g Sbst.: 0.4687 g COs, 0.08i9 g H?O 0.1849 g Sbst.: 0.4687 g COs, 0.08i9 g H?O g
,
g
ClsHlr.05. Ber. C ti'3.20, H 5.17. Gel. x 69.13,
n 5 20. 2.4.2'.4'-Te t rani c t h ox y -c h a I k o ii. Aus Alkohol blnflgclbe Nuelchen vom Schmp. 128O. Leicht liislicli in
Benzol, Chloroform, Accton, Tctrnchlorkohlcnstoff, schwer in .xther und
J.igroiil. g
0.1825 g Sbst.: 0.464i g CO?, 0.1010 g H20 g
0.1825 g Sbst.: 0.464i g CO?, 0.1010 g H20 g
,
g
C19Hlo05. Ber. C 69.45, H 6.14. Gcf. D 69.44,
)) 6.19. 2.5.8'.4'-Tc t ram e thox y-c h rl k o n. Ails Methylalkohol in gliiizenden, gelben Bliittchen roiii Schmp. 90.5O. L6slichkeit wie beim vorhergchenden Isomeren Ails Methylalkohol in gliiizenden, gelben Bliittchen roiii Schmp. 90.5O. L6slichkeit wie beim vorhergchenden Isomeren. 6slichkeit wie beim vorhergchenden Isomeren. 0.1804 g Sbst.: 0.4592 g COs, 0.1000 g H10. g g
g
,
g
c i 9 1 1 2 ~ 0 5 . Ber. C 69.48, H 6.14. g
,
g
c i 9 1 1 2 ~ 0 5 . Ber. C 69.48, H 6.14. Get. n 69.42,
I) 6.20. 4.2'.4'-Trinietboxy-chalkon. hus i0-prozentigem Alkohol erhilt man es in blal3gelben Nadelchen vom
Sclrmp. 880. Die L6slichkeitsverb:iltnissc sind dieselben, s i e beim rorher-
gehenden Produkt. g
0.191T1 g Sbst.: 0.5051 g COr, 0.10.55 g HsO. g
0.191T1 g Sbst.: 0.5051 g COr, 0.10.55 g HsO. g
,
g
ClaHls04. Ber. C 72.4.5, H 6.08. Gef. x i2.36, ')
6.16. Benzal -2.4- dime t h ox y -ace top hen on. Aus Alkohol feine, blal3gelbe NBdelchen, die liei 7 8 O schmelzeii und in
Alkohol, Benzol und Chloroform leicht, in .ither und Ligruin schner 16s-
lieh sind. 0.1896 g Sbst.: 0.5279 R COP, 0.1016 g HzO. 0.1896 g Sbst.: 0.5279 R COP, 0.1016 g HzO. g
CI~Hlc03. Ber. C 76.08, H 6.02. GeF. D 75 93,
6.05. 3.4.3'.4'-Te t ram c t b o x y -chal kon. hus All;olrol i n feincn. hcllgclben Krpstallchen vom Schmp. 110". I.6slicli
Iceit wie oheir :iogegcben. hus All;olrol i n feincn. hcllgclben Krpstallchen vom Schmp. 110". I.6slicli
Iceit wie oheir :iogegcben. g g
0.1815 g Shjt.: 0.4609
CO?, 0.0999
H20. 0.1815 g Shjt.: 0.4609
CO?, 0.0999
H20. 0 1 : , H 2 0 0 5 . . Ber. C 69.48, H 6.14. ,
Gef. 69.26, * 6.16. 3.4. n i o x y m c t 11 y 1 c !I - 3l.4'- d i me th o x y-ch al k on. A u s Alkohol crhiilt man das Produkt in gclblichen, verfilzten Xadelclien
vom Schmp. 1440. Reziiglich seiner L6slichkeit siehe die aiidren Challtonc. 0.1636 g Sbst.: 0.4653 g (202, 0.0860 g H,O. A u s Alkohol crhiilt man das Produkt in gclblichen, verfilzten Xadelclien
vom Schmp. 1440. Reziiglich seiner L6slichkeit siehe die aiidren Challtonc. 0.1636 g Sbst.: 0.4653 g (202, 0.0860 g H,O. g (
g
,
CleHlcOj. Ber. C 69.20, 1-1 5.17. ,
Gef. s 69.15, )) 5.24. 2.5.3'.4'- Tc t ram e t h ox y - c ha1 k on. Aus Mctliylalkohol in hellgelben, kompakten Kry-stallen vom Schmp. S1O. Beziiglich Ihlichkeit in den gebriuchlicheten Lijsungsmitteln siehe oben. Aus Mctliylalkohol in hellgelben, kompakten Kry-stallen vom Schmp. S1O. Beziiglich Ihlichkeit in den gebriuchlicheten Lijsungsmitteln siehe oben. 0.1783 0" Slbst.: 0.4540 g COI, 0.0959 g H20. ,
g
Ber. C 69.46, 11 6.14. g
g
C19Hao0j. Ber. C 69.46, 11 6.14. g
C19Hao0j. Ber. C 69.46, 11 6.14. Gel. )) 69.48,
6.21. Das ?.5.?' 3'-Te t r a in e t ho x y-chal ko n konnten s i r bishcr auf keine
Wcise in krystallisierbareni Zustand erhalten. Das ?.5.?' 3'-Te t r a in e t ho x y-chal ko n konnten s i r bishcr auf keine
Wcise in krystallisierbareni Zustand erhalten. 2.4.3'.4'-T e t ram e t 11 ox y- ch n I k on. ..\us Alkohol in feinen, blnCgelben Niidrlclien ~ o r n Schnip. 115.5". 1 . 6 ~
lichkeit wie oben angegelien. ..\us Alkohol in feinen, blnCgelben Niidrlclien ~ o r n Schnip. 115.5". 1 . 6 ~
lichkeit wie oben angegelien. ..\us Alkohol in feinen, blnCgelben Niidrlclien ~ o r n Schnip. 115.5". 1 . 6
lichkeit wie oben angegelien. 0.1503 g S1)at.: 0.4585 g Cog, 0.0999 g HzO. g
g
g
ClsH2oO5. Ber. C 69.46, H 6.14. Ber. C 69.46, H 6.14. Gef. * 69.35,
6.20. 3 '. 4'- D i m e t h o x y - c h a I k on. Aus Methylalkohol in blaBgelben Blittchen vom Schrnp. 850. Der K6r-
per l6st sich leicht in Benzol, Chloroform, Aceton, Tetrachlorkolilenetoff, sehr
schwer in .ither und Ligroin. g
0.1887 g Sbst: 0.5251 g CO1, 0.1037 g HtO. g
0.1887 g Sbst: 0.5251 g CO1, 0.1037 g HtO. g
,
g
C,TH,GOS. Ber. C 76.08, H 6.02. Gel. D 75.89. )) 6.15. 4.3'.4'-Trime thox T - c h n l kon. 4.3'.4'-Trime thox T - c h n l kon. Ails Alkohol in gelblichen, deiben Nadeln vom Schmp. 90°. Iu seiner
LGslichkcit vcrhiilt es sich \vie dic andren Chalkone. C l a H l ~ O c . 0.1902 g Sht.: 0.5043 g CO?, 0.1052 g Hz0. Ber. C 72.45, €1 6.08. G f
73 30
6 19 Ails Alkohol in gelblichen, deiben Nadeln vom Schmp. 90°. Iu seiner
LGslichkcit vcrhiilt es sich \vie dic andren Chalkone. 0.1902 g Sht.: 0.5043 g CO?, 0.1052 g Hz0. Ails Alkohol in gelblichen, deiben Nadeln vom Schmp. 90°. Iu seiner LGslichkcit vcrhiilt es sich \vie dic andren Chalkone. 0.1902 g Sht.: 0.5043 g CO?, 0.1052 g Hz0. LGslichkcit vcrhiilt es sich \vie dic andren Chalkon
0.1902 g Sht.: 0.5043 g CO?, 0.1052 g Hz0. C l a H l ~ O c . g
g
Ber. C 72.45, €1 6.08. Gef. N 73.30,
D 6.19. 0.1SS8 g 5kt.: 0.4801 g '202, 0.1057 g B?O. 2,4,2’,4’,6’- I’ cn t a m e th o x y - c h r l I< o n. .4us Alkohol gclbliche Nidelchen vom Schmp. 127O
Die Substanz i b t
in .:ither nntl Ligroin so gut wic unliislieli, leicht dagegen in JJenzol, Chloroforni,
Acetoil und Eisessig. .4us Alkohol gclbliche Nidelchen vom Schmp. 127O
Die Substanz i b t
in .:ither nntl Ligroin so gut wic unliislieli, leicht dagegen in JJenzol, Chloroforni,
Acetoil und Eisessig. 0.1824 g Scst.: 0.4470 g COz, 0.1011 g 820. 0.1824 g Scst.: 0.4470 g COz, 0.1011 g 820. g
C20Hn106. Bcr. C 67.01, H 6.19. Gef. * 66.84, D 6.20. 3.4.2’.4‘.6’- Pen t a m e t h o x y- c h n Ikon. hns Alkohol i n schwach gelben, mikroskopischen Kadelchen Torn Schrnp. hns Alkohol i n schwach gelben, mikroskopischen Kadelchen Torn Schrnp. 1180. LBslich mie sein eben beschriebenes Isomeres. 0.181s g Sbst.: 0.4463 g COa. 0.1011 g HzO. g
g
Ca0HlaOs. Bcr. C 67.01, H 6.19. g
g
g
Ca0HlaOs. Bcr. C 67.01, H 6.19. Gef. D 66.95,
6.82. 3.4.2’.5’-Te t r a m e t h o x y -c h al k on. Aucli tliesc Substanz konntc crst nach langer Zeit in krystallisierbarem
Zustand erhalten werden. Aus Alkohol umkrystallisiert, erhalt nian sir: in
rreioeu, seideglanzenden Bkttchen vom Schmp. 128O. Der Kbrper xeigt keine
Halochroniie, ebenso ist seine vbllige Farblosigkeit auffallend. Seine Konsti-
tution als Chalkon liBt sich jedoch mit tler ;lnalysc gut rereinbaren. 0.1731 g Sbst.: 0.4440 g Cog, 0.093 g HaO. 0.1731 g Sbst.: 0.4440 g Cog, 0.093 g HaO. ClsHaoOs. Ber. C 69.48, H 6.14. Gef. D 69.32, x 6.?8. g
g
g
ClsHaoOs. Ber. C 69.48, H 6.14. ,
Gef. D 69.32, x 6.?8. ,
Gef. D 69.32, x 6.?8. F u r a 1-3.4 - cl i in e t h o x J - a ce t o p h o n on. F u r a 1-3.4 - cl i in e t h o x J - a ce t o p h o n on. .\us A l l t o l d blaBgelLe Nidelchen vom ‘Schmp. Slo. Beim Titrieren
mit 75-prozentigem Alkohol erbiilt man den Wert 66.5 f
1.1. .\us A l l t o l d blaBgelLe Nidelchen vom ‘Schmp. Slo. Beim Titrieren .\us A l l t o l d blaBgelLe Nidelchen vom ‘Schmp. Slo. Beim Titrieren
mit 75-prozentigem Alkohol erbiilt man den Wert 66.5 f
1.1. .\us A l l t o l d blaBgelLe Nidelchen vom ‘Schmp. Slo. Beim Titrieren
mit 75-prozentigem Alkohol erbiilt man den Wert 66.5 f
1.1. g
p
mit 75-prozentigem Alkohol erbiilt man den Wert 66.5 f
1.1. 0.1589 g Slsf.: 0.4s’21 g CO,, 0.0912 g HzO. 0.1589 g Slsf.: 0.4s’21 g CO,, 0.0912 g HzO. ClsHI+O,. Bor. C 69.74, H 5.46. g
g
,,
g
ClsHI+O,. Bor. C 69.74, H 5.46. g
,,
g
ClsHI+O,. Bor. C 69.74, H 5.46. Gef. D 69.65, * 5.40. C in n a rn a 1 - 3.4 - tl i m e t h o x y -ace t o 11 h e n o n. Xus Alkohol Eeinc, citronengelbe N8delchcn voin Schmp. Ill0. Bei cler Xus Alkohol Eeinc, citronengelbe N8delchcn voin Schmp. Ill0. Bei cler
Titrntion rnit 75prozentigem Alkohol erhilt man den Wert 36 0 f 0 5 Xus Alkohol Eeinc, citronengelbe N8delchcn voin Schmp. Ill0. Bei cler
Titrntion rnit 75prozentigem Alkohol erhilt man den Wert 36.0 f
0.5. 0.1683 g Sbst.: 0.4771 g COz, 0.0938 g Ht0. Cl,€1,,03. Ber. C 77.51, H 6.17. G F
77 31
6 “ Xus Alkohol Eeinc, citronengelbe N8delchcn voin Schmp. Ill0. Bei cler
Titrntion rnit 75prozentigem Alkohol erhilt man den Wert 36.0 f
0.5. 0.1683 g Sbst.: 0.4771 g COz, 0.0938 g Ht0. C
51 H 6 1 g
p
Titrntion rnit 75prozentigem Alkohol erhilt man den Wert 36.0 f
0.5. 0.1683 g Sbst.: 0.4771 g COz, 0.0938 g Ht0. g
g
,
g
Cl,€1,,03. Ber. C 77.51, H 6.17. g
,
g
Cl,€1,,03. Ber. C 77.51, H 6.17. GeF. )) 77.31,
6.“. 3.4.2'.5'-Te t r s m e t h o x y -chal kon. Das zuerst iilige Kondcnsationsprodukt wurde erst nach vierteljtihrigem
Stehen fcst. Es wurdc aus Alkohol urnkrystallisiert und zeigte d a m den
Schmp. 112". Auffillig ist an dieser Sobstanz die Farblosigkeit und das
Ausbleiben der Halochromie. Dic gut stirnmcnde hnalyse liiBt jedoch die an-
gegebcne Iconstit ution als wahrschcinlich crscheinen. Das zuerst iilige Kondcnsationsprodukt wurde erst nach vierteljtihrigem
Stehen fcst. Es wurdc aus Alkohol urnkrystallisiert und zeigte d a m den
Schmp. 112". Auffillig ist an dieser Sobstanz die Farblosigkeit und das
Ausbleiben der Halochromie. Dic gut stirnmcnde hnalyse liiBt jedoch die an-
gegebcne Iconstit ution als wahrschcinlich crscheinen. 0.1SS8 g 5kt.: 0.4801 g '202, 0.1057 g B?O. 0.1SS8 g 5kt.: 0.4801 g '202, 0.1057 g B?O. g
g
ClsHpoOj. Ber C 69.18, H 6.14. ,
Gef. )) 69.35, D 6.26. ,
Gef. )) 69.35, D 6.26. I ) B. 46, 2336 [1912].
'9 H. Meyer (hi. 8, 429 [18Si]) gibt fiir diesen Ester den Schmp. 810
:+\I:
wir haben in cl,ei,cins!iminung mit Eiilow (B. 36. 3902 [1902]) 430
!It~ol~act~
te t . 2.4 6- T r i m e t hox y - t ri p hen y lc nr b i n 01. l u s tlcm Keaktiousprodnkt YOU 3.6 g Brombcnzol, 10 g ahsolutem Ather
und 0.6 g Magnesinrnspiincn niit 3 g 5.-C.G-'Trimetho;rj.-benz0ptieii~n in 200 ccm
abaoluteni .\ther erhielt man iincli \-crtlunstcn tles .:tilers
und Uinkrystalli-
bieren aui Alkohol diescs Cnrbinol in feincn, weiGen Iirystiillchcn vom
Scbmp. 110-1
110. Iu seiner 1 iklichkeit rcrhiilt cs sicli \vie tlas obeu be-
rcliriebene Carbinol. 0.1628 g SbJSt.: 0.4487 g cot, 0.0913 g Hp@. 0.1628 g SbJSt.: 0.4487 g cot, 0.0913 g Hp@. g
,
g
p
CplH?zO). Rcr. C i5.39, H 6.33. ,
Gcf. D i.5.l'i,
w 6.25. 3 5 -D
i ni e tli O X y- t ri p 11 en y I cil r tJ i ti o 1. Zu der RUS 8 g Brou~benzol, 10 g absolutein iitlier untl 1.9 2 hlagncsium-
zpiinen hcrgestcllten Lijsung gibt nian eiiio Liisnnp voii 4 g 3..i-TI)ini~~tIroxy
I,enzoesiirir.c inethylcster") in 1 0 g absolutem .\ther. N:icIi Becndigung der
Kraktioii wird mit Wassei. nnti Essigsiiirc zcrsetzt untl die iitlierische LVaurig
niit Soda und Wasser gewnschen. N:~cli \.crtlunsten
d ~ , s .\tliers crhdt inan
t.inc liarte Krystallkrustc. Aus Alkohol umkrystallisiert, erhilr man den
G r p e r iii feirien, weiWen Niidelchen. (lie bei I 1 3 O scbinelzen. Er lu,t t.icli in
Ligroin selir schwer, lcicliter ill Allcoliol, stahr leicht i n Eenzul, Clilowlornr
und Aceton. 0.1521 6 Sbst.: 0.5250 g CO?, 0.lWI g 1120. 0.1521 6 Sbst.: 0.5250 g CO?, 0.lWI g 1120. 0.1521 6 Sbst.: 0.5250 g CO?, 0.lWI g 1120. g
g
Csl H20@3. Ber. C Td.71, H 6.30. ,
Gef. o iS.63. )) 6.27. 2,5, 2’, X, 6’- Pc n t a m e t h o s y- ch a 1 k o n. .\us Alkohol ill chii in en, gelhcn, glinzentlen Blittchcn vom Schlnp. 137”. Lbslichkeit aie oben. .\us Alkohol ill chii in en, gelhcn, glinzentlen Blittchcn vom Schlnp. 137”. Lbslichkeit aie oben. .\us Alkohol ill chii in en, gelhcn, glinzentlen Blittchcn vom Schlnp. 137”
Lbslichkeit aie oben. 0.1844 g Sbst.: 0.4.542 g Coal 0.1024 g H20. g
g
CzoH22Os. Ber. C 67.01, H 6.19. Gef. 67.18,
6.21. ?.42'.~'.2".4''-
Hex a m e t h o x y - t ri p h cn y Ic n r l ~ i
u J 1 ?.42'.~'.2".4''-
Hex a m e t h o x y - t ri p h cn y Ic n r l ~ i
u J 1 Dicsc: Substanz habeii wir sclion ausfiilii,lich bescliriet,en I )
untl t ragen
birr iiur noch die Analyse uach: y
0.1792 g Sbst.: 0.4178 g Cnz, 0.1030 6 HaO. y
0.1792 g Sbst.: 0.4178 g Cnz, 0.1030 6 HaO. y
0.1792 g Sbst.: 0.4178 g Cnz, 0.1030 6 HaO. H26Oi. Ber. C GS.15, H (i 41. Gcf. B 68.15,
)) 6.36. Die l~eiin entsprechmdeu )>Methan<< niit konzentriarter Scli\~elelsiirir~~
auf-
;i,etendt: Fiirbring ist, wie wir inzxisclien fi,ststellten, m i t einev Zersetzune
vc rk n i i p f t. 3801 3801 erhalt man das Carliinol in derbcn Krystallen, die aus Alkohol umkrysta1lisiei.t
bei 139O schmelzeii. Loslichkcit in den gebr8uchlichen I,6,ungsrnittelii
siehe oben. erhalt man das Carliinol in derbcn Krystallen, die aus Alkohol umkrysta1lisiei.t
bei 139O schmelzeii. Loslichkcit in den gebr8uchlichen I,6,ungsrnittelii
siehe oben. 0.1892 g Sbst.: 0.522 g co2, 0.10SG g HZO 0.1892 g Sbst.: 0.522 g co2, 0.10SG g HZO. g
,
g
C?? 8220,. Ber. C i5.39, H 6.33. g
C?? 8220,. Ber. C i5.39, H 6.33. Gef. 75.28, x 6.44. I) Zu dem Jod-resorcin-dimethylather, den wir schon beschriebeii halm
0.198% g Sbst.: 0.1772 g AgJ.
4B. 45, 2334 [191?]), haben wir noch die Analye nnchzntragen:
C,H9O2J. Ber. J 48.08. Gef. S 48.15. Gef. B 70.10,
6-41. Gef. B 70.10,
6-41. Das 3,3.4,2',4'-
Pent ame t h o x y- t r i p he n ylcar b i n o 1 haben wir ganz
analog dem ehen bescbriebencn Isomeren dargestellt. Das olige Reaktions-
produkt konnten wir jcdoch trotz vieler Versuche his jetzt noch nicht in kry-
stallisierbarem Zustande erhalten. 482. Hugo Eeuffmann:
Dae Geeetz der Desentralieation ohemiecher Funktionen. (Eingegangen am 10. November 1913.) 4.3.4-Trimethosv-triphenrIcarbiiiol. 4 g 2.3.4-Triniethoxy-benzoplienon
werdcn ~ I I
eincr L ~ S U I I , ~
aus 35 g Broni-
benzol und 0.6 g Mngnesiumspinen in 10 ccnt atwoluteni .'ither gegcben und
die iitheriache Lijsung drs Keaktionsproduktcs mit Wasser und Essigsiiure
zcrsetzt und mit Soda und Wnsser gcwasclien. Sacli Vertlampfen dcs .ithers I) Zu dem Jod-resorcin-dimethylather, den wir schon beschriebeii halm
0.198% g Sbst.: 0.1772 g AgJ.
4B. 45, 2334 [191?]), haben wir noch die Analye nnchzntragen: ?.4.6.2'.4'- P en t am et h o x y - t ri p h e n J 1 c n r h i n o 1. 3 g Jotl-resorcin-dimethplHther') werden mit 0.3 g aktivierten h l q -
oesiunispinen in 5 g absolutem .ither ZUT Reaktion gcbracht und hierauf 1.2 ;
2 4.6-Trimethoxy-benzophenon in 100 ccm absolutem .\her zugegeben. Nacb Beendigung der Reaktion zersetzt nian mit Wasser iind Essigsiure untl
wascbt die atheriscbe Lijsung rnit Sods und Wasser. Nacb Verdunsten tles
x thers erbiilt man es als Cnrbinol in beinshe farbloseri Krystallen, die nach
dem Urnkrystallisieren au5 Alkohol rein weiB werden und clan Sclimp. 120'. zeigen. Loslichkeit \vie oben angegeben. g
g g
O.lfiPO g Sbst.: 0.4678 g COY, 0.1043 g H20. O.lfiPO g Sbst.: 0.4678 g COY, 0.1043 g H20. g
CtaH26Oc. Ber. C 70.21, H 6.39. 482. Hugo Eeuffmann:
Dae Geeetz der Desentralieation ohemiecher Funktionen.
(Eingegangen am 10. November 1913.) Die vorangehende Arbeit beweist, da13 die von mir fiir die far-
'bigen l'riphen).l-carbinol-Salze
aufgestellte Formel sogar in feinere
Einzelheiten eindringt und daher sich auk neue als ein samtlicheu
ubrigen bekannten Pormeln iiberlegenes Schema darbietet. Die neue
Formel hat rnich auf ein wichtiges Grundgesetz aufmerksam gemncht,
das fur alle strukturchemischen Vorstellungen von grol3er Bedeutung
iut, und xuf dessen Bestehen ich daher irn Folgenden rnit kurzen
Worten hinmeisen rnochte. Ich scbicke voraus, daB bei den Eetrach-
tungen wieder die Anwendung des Nebenvalenz-Begriffes verrnieden
wird, da sonst das Gesetz weniger klnr hervortritt. I) Zu dem Jod-resorcin-dimethylather, den wir schon beschriebeii halm
0.198% g Sbst.: 0.1772 g AgJ. 4B. 45, 2334 [191?]), haben wir noch die Analye nnchzntragen:
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https://openalex.org/W2005205299
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https://europepmc.org/articles/pmc4251833?pdf=render
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English
| null |
Polyglutamine- and Temperature-Dependent Conformational Rigidity in Mutant Huntingtin Revealed by Immunoassays and Circular Dichroism Spectroscopy
|
PloS one
| 2,014
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cc-by
| 18,960
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RESEARCH ARTICLE OPEN ACCESS Citation: Fodale V, Kegulian NC, Verani M, Cariulo
C, Azzollini L, et al. (2014) Polyglutamine- and
Temperature-Dependent Conformational Rigidity in
Mutant Huntingtin Revealed by Immunoassays and
Circular Dichroism Spectroscopy. PLoS
ONE 9(12): e112262. doi:10.1371/journal.pone. 0112262 Editor: Patrick van der Wel, University of
Pittsburgh School of Medicine, United States of
America Valentina Fodale1, Natalie C. Kegulian2, Margherita Verani1, Cristina Cariulo1,
Lucia Azzollini1, Lara Petricca1, Manuel Daldin1, Roberto Boggio1,
Alessandro Padova1, Rainer Kuhn1¤, Robert Pacifici3, Douglas Macdonald3,
Ryan C. Schoenfeld3, Hyunsun Park3, J. Mario Isas2, Ralf Langen2*,
Andreas Weiss1*¤,
Andrea Caricasole1* Received: April 24, 2014
Accepted: October 6, 2014
Published: December 2, 2014 Received: April 24, 2014 Accepted: October 6, 2014 Published: December 2, 2014 1. IRBM Promidis, Pomezia, Rome, Italy, 2. Department of Biochemistry and Molecular Biology, Zilkha
Neurogenetic Institute, Keck School of Medicine, University of Southern California, Los Angeles, California,
United States of America, 3. CHDI Management/CHDI Foundation, Los Angeles, California, United States of
America Copyright: 2014 Fodale et al. This is an open-
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and repro-
duction in any medium, provided the original author
and source are credited. *langen@usc.edu (RL); andreas.weiss@evotec.com (AW); a.caricasole@irbm.it (AC) ¤ Current address: Evotec AG, Hamburg, Germany ¤ Current address: Evotec AG, Hamburg, Germany Data Availability: The authors confirm that all data
underlying the findings are fully available without
restriction. All relevant data are within the paper. Conformational Changes in Huntingtin Detected by Immunoassays and CD investigate the possibility of interrogating HTT protein conformation using these
assays. Methodology/Principal Findings: By performing TR-FRET measurements on the
same samples (purified recombinant proteins or lysates from cells expressing HTT
fragments or full length protein) at different temperatures, we have discovered a
temperature-dependent, reversible, polyglutamine-dependent conformational
change of wild type and expanded mutant HTT proteins. Circular dichroism
spectroscopy confirms the temperature and polyglutamine-dependent change in
HTTstructure, revealing an effect of polyglutamine length and of temperature on the
alpha-helical content of the protein. Conclusions/Significance: The temperature- and polyglutamine-dependent
effects observed with TR-FRET on HTT proteins represent a simple, scalable,
quantitative and sensitive assay to identify genetic and pharmacological modulators
of mutant HTT conformation, and potentially to assess the relevance of
conformational changes during onset and progression of Huntington’s disease. Abstract Funding: CHDI Foundation is a privately funded
nonprofit biomedical research organization exclu-
sively dedicated to discovering and developing
therapeutics that slow the progression of
Huntington’s disease. CHDI Foundation conducts
research in a number of different ways; for the
purposes of this manuscript, research was
conducted at IRBM Promidis and the University of
Southern California under a sponsored research
agreement. RL, MJI and NCK are also supported
by the Hereditary Disease Foundation. LP has
received funding from the European Community’s
Seventh Framework Programme FP7/2010 under
grant agreement no. 264508. The authors listed all
contributed to the conception, planning, and
direction of the research, including generation,
analysis, and interpretation of the data. Background: In Huntington’s disease, expansion of a CAG triplet repeat occurs in
exon 1 of the huntingtin gene (HTT), resulting in a protein bearing.35
polyglutamine residues whose N-terminal fragments display a high propensity to
misfold and aggregate. Recent data demonstrate that polyglutamine expansion
results in conformational changes in the huntingtin protein (HTT), which likely
influence its biological and biophysical properties. Developing assays to
characterize and measure these conformational changes in isolated proteins and
biological samples would advance the testing of novel therapeutic approaches
aimed at correcting mutant HTT misfolding. Time-resolved Fo¨rster energy transfer
(TR-FRET)-based assays represent high-throughput, homogeneous, sensitive
immunoassays widely employed for the quantification of proteins of interest. TR-
FRET is extremely sensitive to small distances and can therefore provide
conformational information based on detection of exposure and relative position of
epitopes present on the target protein as recognized by selective antibodies. We
have previously reported TR-FRET assays to quantify HTT proteins based on the
use of antibodies specific for different amino-terminal HTT epitopes. Here, we Competing Interests: The authors have declared
that no competing interests exist. RP, DM, RCS
and HP are employed by CHDI Management, Inc.,
as advisors to CHDI Foundation, Inc. VF, MV, CC,
LA, LP, MD, RB, AP, RK, AW, and AC are
employed by IRBM Promidis. NCK, JMI, and RL
are employed by the University of Southern
California. There are no patents, products in
development, or marketed products to declare. This declaration does not alter the authors9
adherence to all PLOS ONE policies regarding
sharing data and materials. 1 / 31 PLOS ONE | DOI:10.1371/journal.pone.0112262
December 2, 2014 Conformational Changes in Huntingtin Detected by Immunoassays and CD [13]. TR-FRET immunoassay detection is based on the labeling of an antibody
pair with a rare earth ion fluorophore donor and an acceptor fluorophore, thereby
producing a specific TR-FRET signal when the donor and acceptor labeled
antibodies bind to their antigen simultaneously. mHTT protein can be measured
and differentiated from wild type HTT using MW1, a polyQ-specific antibody
displaying higher avidity for expanded (mutant) polyQ HTT [27]. By performing
TR-FRET measurements on recombinant, purified amino-terminal fragments
comprising the first 548 residues of wild type HTT (N548 HTT) and mHTT at
different temperatures, we have discovered a temperature-dependent, reversible,
polyQ-dependent conformational change of wild type and mutant HTT proteins. We have confirmed the conformational nature of the effect by performing circular
dichroism spectroscopy on recombinant HTT proteins at different temperatures,
revealing striking temperature- and polyQ- dependent changes in the alpha-
helical structure of HTT’s N-terminal region. By investigating the temperature-
and polyQ-dependent variation in TR-FRET signal using antibody pairs targeted
at different N-terminal epitopes in HTT recombinant proteins and in lysates from
cells transfected with HTT cDNAs of different lengths (exon 1, N548 and full
length), we observed a consistent dependence of the effect on detection of the
polyQ region, irrespective of protein length. This novel assay can potentially be
employed to investigate HTT conformation in samples of different origins
(preclinical and clinical) as well as to identify modulators of HTT conformation. HTT conformation is reversibly affected by temperature as
measured by TR-FRET immunoassays We reasoned that the conformational flexibility of the N-terminal portion of HTT
may be detected by TR-FRET assays by interrogating relevant epitopes. In the
appropriate context, TR-FRET signals can inform on epitope accessibility as well
as relative distance between the antibodies themselves ([28], [29]), and therefore
can provide conformational information on the protein analyte. In the simplest
context, we elected to investigate conformational flexibility in recombinant,
purified HTT proteins by TR-FRET using an antibody pair comprising previously
characterized monoclonals (2B7 and MW1; Fig. 1A; [13, 27, 30–32]) interrogating
two regions of known relevance for HTT conformation and biological properties,
namely the N17 domain and the polyQ domain (e.g. see [17, 24, 24, 26]). As one
of the major factors influencing the stability of protein conformation is
temperature (e.g. see[33]), we pragmatically chose this variable and decided to
investigate HTT behaviour at a low temperature (4˚C) and at the temperature at
which TR-FRET assays are normally performed (room temperature, 20˚C, RT). In
order to avoid possible confounding effects due to aggregation, we chose to
employ recombinant, purified N548 HTT fragments of different polyQ length,
which should not aggregate or aggregate at a lower rate respective to the exon 1
under the conditions employed (Fig. 1B and C, [13]). Initially, we selected HTT We reasoned that the conformational flexibility of the N-terminal portion of HTT
may be detected by TR-FRET assays by interrogating relevant epitopes. In the
appropriate context, TR-FRET signals can inform on epitope accessibility as well
as relative distance between the antibodies themselves ([28], [29]), and therefore
can provide conformational information on the protein analyte. In the simplest
context, we elected to investigate conformational flexibility in recombinant, can provide conformational information on the protein analyte. In the simplest
context, we elected to investigate conformational flexibility in recombinant,
purified HTT proteins by TR-FRET using an antibody pair comprising previously
characterized monoclonals (2B7 and MW1; Fig. 1A; [13, 27, 30–32]) interrogating
two regions of known relevance for HTT conformation and biological properties,
namely the N17 domain and the polyQ domain (e.g. see [17, 24, 24, 26]). As one
of the major factors influencing the stability of protein conformation is
temperature (e.g. see[33]), we pragmatically chose this variable and decided to
investigate HTT behaviour at a low temperature (4˚C) and at the temperature at
which TR-FRET assays are normally performed (room temperature, 20˚C, RT). Introduction Huntington’s disease (HD) is a genetic neurodegenerative condition resulting
from the expansion of a polyglutamine (polyQ) encoding region within exon 1 of
the huntingtin gene (HTT; [1]). Mutant huntingtin protein (mHTT) can be
processed to generate amino-terminal (N-terminal) fragments which, owing to
the polyQ expansion, misfold and aggregate in the cytoplasm and nucleus of
distinct cell types in the CNS and the periphery [2–5]. HD is characterized by the
deposition of these insoluble macroaggregates, particularly in striatal and cortical
neurons and involving the formation of poorly characterized intermediate states,
including soluble oligomeric forms [6–8]. Although the relationship between
macroaggregates and neurotoxicity is still unclear [9–12], the correlation between
disease/phenotype status and aggregate load found in postmortem brains from
HD patients and animal models highlights the importance of investigating their
formation and properties [9, 13–15]. mHTT fragment aggregation is critically
influenced by sequences preceding (amino-terminal 17 residues; N17) and
following (proline-rich region; PRR) the polyQ stretch, as well as by post-
translational modifications within exon 1 [16–23]. In fact, structural studies have
revealed significant structural flexibility of the polyQ region [24], [25]. Recent
evidence indicates that expansion of the polyQ region in mHTT results in
decreased conformational flexibility of the polyQ region and decreased
interactions between and/or relative positioning of flanking sequences [26]. In this
study we probed the conformational flexibility of HTT taking advantage of robust,
sensitive, rapid time-resolved Fo¨rster resonance energy transfer (TR-FRET)
immunoassays we previously developed to quantify different HTT conformers 2 / 31 PLOS ONE | DOI:10.1371/journal.pone.0112262
December 2, 2014 HTT conformation is reversibly affected by temperature as
measured by TR-FRET immunoassays In
order to avoid possible confounding effects due to aggregation we chose to j
g
y
p
temperature (e.g. see[33]), we pragmatically chose this variable and decided to
investigate HTT behaviour at a low temperature (4˚C) and at the temperature at
which TR-FRET assays are normally performed (room temperature, 20˚C, RT). In
order to avoid possible confounding effects due to aggregation, we chose to
employ recombinant, purified N548 HTT fragments of different polyQ length,
which should not aggregate or aggregate at a lower rate respective to the exon 1
under the conditions employed (Fig. 1B and C, [13]). Initially, we selected HTT PLOS ONE | DOI:10.1371/journal.pone.0112262
December 2, 2014 3 / 31 Conformational Changes in Huntingtin Detected by Immunoassays and CD Figure 1. Epitopes of antibodies and N548 human recombinant HTT (hrHTT) proteins used in the study. A. Schematic positioning of epitopes for antibodies employed in these studies. 2B7 (recognizing the N17
domain), MW1 and 3B5H10 (specific for the polyQ region), 4C9 (recognizing an epitope within the PRR
region) and 2166 (specific for an epitope around residue K444). B. Coomassie gel staining of the N548 hrHTT
proteins, 5 mg of each protein bearing the indicated polyQ repeat length was loaded on the gel. C. Western
blot of N548 hrHTT proteins employed in the TR-FRET analyses, probed with 2B7, MW1 and an antibody
specific for a domain within the PRR region (4C9). 0.25 mg of protein bearing the indicated polyQ repeat
length. Figure 1. Epitopes of antibodies and N548 human recombinant HTT (hrHTT) proteins used in the study. Figure 1. Epitopes of antibodies and N548 human recombinant HTT (hrHTT) proteins used in the study. A. Schematic positioning of epitopes for antibodies employed in these studies. 2B7 (recognizing the N17
domain), MW1 and 3B5H10 (specific for the polyQ region), 4C9 (recognizing an epitope within the PRR
region) and 2166 (specific for an epitope around residue K444). B. Coomassie gel staining of the N548 hrHTT
proteins, 5 mg of each protein bearing the indicated polyQ repeat length was loaded on the gel. C. Western
blot of N548 hrHTT proteins employed in the TR-FRET analyses, probed with 2B7, MW1 and an antibody
specific for a domain within the PRR region (4C9). 0.25 mg of protein bearing the indicated polyQ repeat
length. doi:10.1371/journal.pone.0112262.g001 proteins bearing two polyQ lengths, representing a wild type (Q16) and an
expanded, or mutant (Q55), context. HTT conformation is reversibly affected by temperature as
measured by TR-FRET immunoassays We then performed TR-FRET using
antibodies 2B7 (labeled with a donor fluorophore, namely terbium) and MW1
(labeled with the acceptor fluorophore, D2), performing the incubation prior to
reading at RT and then shifting the sample to 4˚C for 2 hrs, first on a fixed
(3.3 ng) and then on a range of HTT protein analyte concentrations (Fig. 2 A–E). As expected, the specific (donor-acceptor energy transfer dependent) fluorescence
signal was significantly higher for mutant (Q55) HTT protein relative to wild-type
(Q16) HTT protein (Fig. 2A). On this substrate, at RT the fluorescence signal
obtained with the 2B7-Tb MW1-D2 antibody pair was close to that obtained from
acceptor labeled MW1 alone (Fig. 2A). This is consistent with the specificity of the
assay for mutant HTT protein ([13]). Interestingly, the TR-FRET signal obtained
with 2B7-MW1 on wild type (Q16) N548 HTT protein can be significantly
affected by temperature, where sample incubation at 4˚C results in significantly
higher TR-FRET signal than samples incubated at RT (Fig. 2B and C). However,
when the samples containing the mutant (Q55) N548 HTT protein are incubated
at 4˚C the increase in TR-FRET signal was significantly reduced relative to the
increase observed for the Q16 counterpart (Fig. 2B and C), suggesting that polyQ
expansion influences the effects of temperature on protein conformation and/or
antibody epitope recognition. The observed effect is not influenced by the proteins bearing two polyQ lengths, representing a wild type (Q16) and an
expanded, or mutant (Q55), context. We then performed TR-FRET using
antibodies 2B7 (labeled with a donor fluorophore, namely terbium) and MW1
(labeled with the acceptor fluorophore, D2), performing the incubation prior to
reading at RT and then shifting the sample to 4˚C for 2 hrs, first on a fixed
(3.3 ng) and then on a range of HTT protein analyte concentrations (Fig. 2 A–E). As expected, the specific (donor-acceptor energy transfer dependent) fluorescence
signal was significantly higher for mutant (Q55) HTT protein relative to wild-type
(Q16) HTT protein (Fig. 2A). On this substrate, at RT the fluorescence signal
obtained with the 2B7-Tb MW1-D2 antibody pair was close to that obtained from
acceptor labeled MW1 alone (Fig. 2A). This is consistent with the specificity of the
assay for mutant HTT protein ([13]). HTT conformation is reversibly affected by temperature as
measured by TR-FRET immunoassays Interestingly, the TR-FRET signal obtained
with 2B7-MW1 on wild type (Q16) N548 HTT protein can be significantly
affected by temperature, where sample incubation at 4˚C results in significantly
higher TR-FRET signal than samples incubated at RT (Fig. 2B and C). However,
when the samples containing the mutant (Q55) N548 HTT protein are incubated
at 4˚C the increase in TR-FRET signal was significantly reduced relative to the
increase observed for the Q16 counterpart (Fig. 2B and C), suggesting that polyQ
expansion influences the effects of temperature on protein conformation and/or
antibody epitope recognition. The observed effect is not influenced by the 4 / 31 PLOS ONE | DOI:10.1371/journal.pone.0112262
December 2, 2014 Conformational Changes in Huntingtin Detected by Immunoassays and CD Figure 3. The temperature dependence of the 2B7-MW1 TR-FRET signal is inversely proportional to polyQ length. A.-B. Fluorescence signals
obtained from dilution curves of N548 proteins bearing Q16, Q19, Q25, Q33, Q39 and Q55, tested by TR-FRET (1 ng/ml of 2B7-Tb and 10 ng/ml of MW1-d2)
at RT (A) and 4˚C (B). C. Ratio between the maximum value of the TR-FRETsignal obtained at 4˚C and the maximum value of the TR-FRET signal obtained
at RT, referred to the curves presented in A and B. D. Ratio obtained and expressed as in C after performing TR-FRET with 2B7-Tb (1 ng/ml) and MW1-d2
(10 ng/ml) antibody combination (as before) or MW1-Tb (1 ng/ml) and 2B7-Alexa647 (10 ng/ml) antibody combination on N548 proteins bearing Q16 and Q55
repeats. In C and D data are represented as mean ¡ S.D. of three independent experiments; significance was calculated using the one-way ANOVA test
and Bonferroni’s multiple comparison post-test (** p,0.01 and * p,0.05). doi:10.1371/journal.pone.0112262.g003 Figure 3. The temperature dependence of the 2B7-MW1 TR-FRET signal is inversely proportional to polyQ length. A.-B. Fluorescence signals
obtained from dilution curves of N548 proteins bearing Q16, Q19, Q25, Q33, Q39 and Q55, tested by TR-FRET (1 ng/ml of 2B7-Tb and 10 ng/ml of MW1-d2)
at RT (A) and 4˚C (B). C. Ratio between the maximum value of the TR-FRETsignal obtained at 4˚C and the maximum value of the TR-FRET signal obtained
at RT, referred to the curves presented in A and B. D. Ratio obtained and expressed as in C after performing TR-FRET with 2B7-Tb (1 ng/ml) and MW1-d2
(10 ng/ml) antibody combination (as before) or MW1-Tb (1 ng/ml) and 2B7-Alexa647 (10 ng/ml) antibody combination on N548 proteins bearing Q16 and Q55
repeats. In C and D data are represented as mean ¡ S.D. of three independent experiments; significance was calculated using the one-way ANOVA test
and Bonferroni’s multiple comparison post-test (** p,0.01 and * p,0.05). doi:10.1371/journal.pone.0112262.g003 acceptor label and MW1 with donor label. The results (Fig. 3D) indicate that the
influence of temperature on TR-FRET signal using 2B7 and MW1 antibodies is
independent of the nature of the acceptor antibody. The effect is rapidly (1 hr-
cycles) reversible (Fig. 4), indicating its dependence on fast operating events
compatible with conformational changes. Conformational Changes in Huntingtin Detected by Immunoassays and CD Figure 2. TR-FRET signals obtained using 2B7 and MW1 antibodies on hrHTT N548 wild type (Q16) and mutant (Q55) proteins are influenced by
temperature and polyQ length. A-B. Fluorescence signal, expressed as fluorescence ratio (665 nm/615 nm), obtained with the antibodies (2B7-Tb and
MW1-d2) tested individually or in combination with N548 hrHTT proteins (Q16 or Q55), at RT (A) and 4˚C (B). C. Ratio between the fluorescence signals
observed at the two temperatures (in A and B, 4˚C/RT). D. TR-FRET fluorescence signals from a dilution curve of N548 hrHTT proteins obtained using 1 ng
of 2B7-Tb and 10 ng of MW1-d2 antibody combination at RTor 4˚C. E. Same as D, TR-FRET performed using 1 ng of 2B7-Tb and 1 ng of MW1-d2 antibody
combination. Results from representative experiments are shown. Figure 2. TR-FRET signals obtained using 2B7 and MW1 antibodies on hrHTT N548 wild type (Q16) and mutant (Q55) proteins are influenced by
temperature and polyQ length. A-B. Fluorescence signal, expressed as fluorescence ratio (665 nm/615 nm), obtained with the antibodies (2B7-Tb and
MW1-d2) tested individually or in combination with N548 hrHTT proteins (Q16 or Q55), at RT (A) and 4˚C (B). C. Ratio between the fluorescence signals
observed at the two temperatures (in A and B, 4˚C/RT). D. TR-FRET fluorescence signals from a dilution curve of N548 hrHTT proteins obtained using 1 ng
of 2B7-Tb and 10 ng of MW1-d2 antibody combination at RTor 4˚C. E. Same as D, TR-FRET performed using 1 ng of 2B7-Tb and 1 ng of MW1-d2 antibody
combination. Results from representative experiments are shown. doi:10.1371/journal.pone.0112262.g002 stoichiometry of the donor and acceptor antibody pair, as performing the
experiments with a 1:1 instead of a 1:10 ratio of donor to acceptor antibody pair,
in which the acceptor-labeled MW1 is more limiting, still results in a temperature-
and polyQ dependent variation in the obtained TR-FRET signal (Fig. 2D and E). A clear polyQ dependence of the temperature effect in the 2B7-MW1 TR-FRET
signal was confirmed using HTT substrates of increasing polyQ length (Fig. 3A–
C). Additionally, to further avoid the possibility of different MW1 binding sites
between wild type and expanded HTT influencing the findings when using
acceptor-labeled MW1, experiments were performed where 2B7 was labeled with 5 / 31 PLOS ONE | DOI:10.1371/journal.pone.0112262
December 2, 2014 PLOS ONE | DOI:10.1371/journal.pone.0112262
December 2, 2014 Conformational Changes in Huntingtin Detected by Immunoassays and CD Figure 4. Reversibility of the temperature-dependent effect on TR-FRET signal. Following assembly of the TR-FRET cocktail (2B7-Tb and MW1-d2,
1:10 ratio), the same samples were subjected to 2 successive cycles of incubation (for 1 hr) and measurement, at the two different temperatures (RT and
4˚C). Note that the difference between fluorescence ratio values obtained at RT or 4˚C is inversely correlated with polyQ length. A-F. Reversibility for HTT
N548 HTT of increasing polyQ length (Q16, Q19, Q25, Q33, Q39, Q55). G. Correlation between fluorescence ratio values obtained in the two RT readings
performed during the cycles. H. Correlation between fluorescence ratio values obtained in the two 4˚C readings performed during the cycles. Figure 4. Reversibility of the temperature-dependent effect on TR-FRET signal. Following assembly of the TR-FRET cocktail (2B7-Tb and MW1-d2,
1:10 ratio), the same samples were subjected to 2 successive cycles of incubation (for 1 hr) and measurement, at the two different temperatures (RT and
4˚C). Note that the difference between fluorescence ratio values obtained at RT or 4˚C is inversely correlated with polyQ length. A-F. Reversibility for HTT
N548 HTT of increasing polyQ length (Q16, Q19, Q25, Q33, Q39, Q55). G. Correlation between fluorescence ratio values obtained in the two RT readings
performed during the cycles. H. Correlation between fluorescence ratio values obtained in the two 4˚C readings performed during the cycles. doi:10.1371/journal.pone.0112262.g004 We therefore concluded that, under
these experimental conditions, temperature can influence HTT epitope exposure/
recognition and/or protein analyte conformation, and investigated this further
employing a non immunoassay-based orthogonal readout. PLOS ONE | DOI:10.1371/journal.pone.0112262
December 2, 2014 6 / 31 PLOS ONE | DOI:10.1371/journal.pone.0112262
December 2, 2014 Conformational Changes in Huntingtin Detected by Immunoassays and CD HTT) to improve stability and solubility. We first sought to determine whether
such a heterologous N-terminal tag fused to the shorter HTT exon 1 fragment
might affect the temperature- and polyQ-dependent variation in 2B7-MW1 TR-
FRET values. The results, employing the 2B7-MW1 antibody pair and confirmed
with a second antibody pair (4C9-MW1), indicated that a temperature-
dependent, polyQ-dependent variation in TR-FRET signal is still observable with
THRX-HTT exon 1 Q25 protein, while it is virtually absent when THRX-HTT
exon 1 Q46 protein is used (Fig. 5A), coherent with what was observed using the
longer N548 HTT proteins as substrate. We therefore proceeded with exon 1 Q46 protein is used (Fig. 5A), coherent with what was observed using the
longer N548 HTT proteins as substrate. We therefore proceeded with
investigating the effects of temperature on the structure of THRX-HTT exon 1
proteins bearing 25 or 46 Q and chose to perform the experiments at 37˚C and
210˚C in order to explore a wide temperature range that encompassed the
conditions employed in the TR-FRET assays. The ensuing CD spectra for Q25 and
Q46 have significant changes with temperature that are reversible (Fig. 5B and C)
and independent of protein concentration in the range used (Fig. 5D), consistent
with the results of the immunoassay-based conformational assay. The difference
spectra for both proteins exhibit characteristic minima at 208 and 222 nm
indicating increased formation of alpha-helical structure at lower temperatures,
which is more pronounced for Q46 HTT protein (Fig. 6A), and all spectra (Fig. 5B
and C) show an isosbestic point near 203 nm, which is commonly observed for
conversions between random coil and alpha-helical structure. Another key feature
of alpha-helical CD spectra is a maximum near 190 nm, which was not possible to
view at high salt concentrations. We therefore recorded CD spectra for both
constructs in salt-free phosphate buffer. These spectra (Fig. 6B and C) also show
isosbestic points near 203 nm, and the difference spectra show the peaks near 190,
208, and 222 nm that are indicative of an alpha-helix (Fig. 6D). The temperature
dependent change in alpha-helicity is frequently plotted using the mean residue
ellipticity (MRE) observed at 222 nm. Using this measure, we find that cooling
increases helicity more strongly for the Q46 than the Q25 derivative (Fig. HTT conformation is affected by temperature as measured by
circular dichroism spectroscopy Circular dichroism spectroscopy (CD) measures differences in absorption of left-
handed polarized light versus right-handed polarized light due to structural
asymmetry in the analyte, and can inform on the presence of secondary/tertiary
structure in proteins (e.g., see [18]). CD data report on the overall secondary
structure content with contributions from all amino acids in the protein sequence. As the spectral contribution of residues around the polyQ region is progressively
diminished with increasing HTT fragment length, we initially decided to
investigate temperature effects on HTT proteins of different polyQ lengths using
the shorter exon 1 fragments. These fragments were produced recombinantly in
bacteria as previously described [18]. These proteins, available with normal (Q25)
or expanded (Q46) polyQ regions, bear a thioredoxin N-terminal fusion (THRX- PLOS ONE | DOI:10.1371/journal.pone.0112262
December 2, 2014 7 / 31 7A; red
asterisks indicate measurements performed at 4˚C and RT, for direct comparison
with TR-FRET data). The THRX tag is highly stable requiring more than 4 M urea
for denaturation [34]. In agreement with this notion, control experiments with
the fusion tag alone revealed no detectable change in the CD spectra between 210
and 37˚C (Fig. 7B), indicating that the exon-1 portion of the fusion protein
contains the region that undergoes structural changes with temperature. The
consistent isosbestic point across temperatures (Fig. 5B and C and 6B and C)
together with the typically alpha-helical difference spectra (Fig. 6A and D) suggest
that the conformational changes are dominated by a helix-coil transition. We
therefore applied a previously developed helix-coil model as one means to
calculate the number of residues that experienced a change in helicity [35, 36]. Using this model, we estimate that on the order of 23 and 31 residues become
helical upon cooling for the Q25 and Q46 constructs, respectively (Table 1A). To
further confirm the differential conformational changes in these constructs, we
analyzed their CD spectra using DICHROWEB software [37, 38] and thereby
again found the Q46 protein to experience a greater change in helicity, with investigating the effects of temperature on the structure of THRX-HTT exon 1
proteins bearing 25 or 46 Q and chose to perform the experiments at 37˚C and investigating the effects of temperature on the structure of THRX-HTT exon 1
proteins bearing 25 or 46 Q and chose to perform the experiments at 37˚C and 8 / 31 PLOS ONE | DOI:10.1371/journal.pone.0112262
December 2, 2014 Conformational Changes in Huntingtin Detected by Immunoassays and CD Figure 5. Temperature and polyQ length influence conformation of thioredoxin-exon 1 HTT proteins. A. Ratio between the maximum value of the TR-FRET curve obtained at 4˚C and the maximum value of the TR-
FRET curve obtained at RT, referred to the curves obtained with two different antibody combinations (2B7-
MW1 and 4C9-MW1) on recombinant, purified thioredoxin-exon 1 HTT proteins employed for circular
dichroism (CD) spectroscopy studies. CD spectra for THRX-HTT exon 1 Q25 (B) and Q46 (C) at all the
temperatures tested showing an isosbestic point around 203 nm (black arrows) and showing the spectra for
each construct at 210˚C after measurements had been taken at 37˚C to be nearly identical to those viewed
beforehand, indicating the reversibility of the temperature effect. D. MRE values at 222 nm for THRX-HTT
exon 1 Q25 and Q46 at indicated concentrations. Figure 5. Temperature and polyQ length influence conformation of thioredoxin-exon 1 HTT proteins. A. Ratio between the maximum value of the TR-FRET curve obtained at 4˚C and the maximum value of the TR-
FRET curve obtained at RT, referred to the curves obtained with two different antibody combinations (2B7-
MW1 and 4C9-MW1) on recombinant, purified thioredoxin-exon 1 HTT proteins employed for circular
dichroism (CD) spectroscopy studies. CD spectra for THRX-HTT exon 1 Q25 (B) and Q46 (C) at all the
temperatures tested showing an isosbestic point around 203 nm (black arrows) and showing the spectra for
each construct at 210˚C after measurements had been taken at 37˚C to be nearly identical to those viewed
beforehand, indicating the reversibility of the temperature effect. D. MRE values at 222 nm for THRX-HTT
exon 1 Q25 and Q46 at indicated concentrations. doi:10.1371/journal.pone.0112262.g005 estimates of 21 to 23 residues and 30 to 33 residues changing in Q25 and Q46,
respectively (Table 1A). From DICHROWEB analysis, both constructs are also
seen to undergo a loss in beta-sheet upon cooling (Table 2); however, this change
is relatively small and cannot readily be identified in the difference spectra. All
alpha-helicity estimates for the HTT Q46 protein are far beyond the number of
amino acids that are contained in the N17 domain, a region which has a
propensity to adopt an alpha-helical structure [24] ([39, 40]). This suggests that
the observed change in helicity cannot exclusively stem from structural changes in
the N17 region and likely involves the polyQ region (see below). The PRR can
adopt a polyproline type II (PPII) structure (e.g. [24] [19, 41]), a conformation
whose stability may also increase with decreasing temperature (e.g. see [18, 33]). However, the CD spectrum of a PPII structure is entirely different from that of an
alpha-helix. While we cannot exclude that more subtle changes might have also
occurred in the PRR, the structural changes observed by CD are dominated by
alpha-helical structure (Fig. 6A and D). Although the temperature-dependent
change in helicity is more pronounced for Q46, it is important to note that the
overall helicity still remains higher at 37˚C. Conformational Changes in Huntingtin Detected by Immunoassays and CD Figure 6. Temperature affects alpha-helical structure of thioredoxin-exon 1 HTT proteins. A. Difference spectra between 210˚C and 37˚C, the
temperature extremes shown in parts B and C of Figure 5, for Q25 and Q46. CD spectra plotted using MRE from 190 to 260 nm for THRX-HTT exon 1 Q25
(B) and Q46 (C) in salt-free phosphate buffer at all the temperatures tested showing an isosbestic point around 203 nm (black arrows) and reversibility of
structural changes upon cooling from 37˚C to 210˚C (post). D. Difference spectra between 210˚C and 37˚C for Q25 and Q46 tested in parts B and C. Each
of these spectra shows a maximum near 190 nm and minima at 208 and 222 nm. Figure 6. Temperature affects alpha-helical structure of thioredoxin-exon 1 HTT proteins. A. Difference spectra between 210˚C and 37˚C, the
temperature extremes shown in parts B and C of Figure 5, for Q25 and Q46. CD spectra plotted using MRE from 190 to 260 nm for THRX-HTT exon 1 Q25
(B) and Q46 (C) in salt-free phosphate buffer at all the temperatures tested showing an isosbestic point around 203 nm (black arrows) and reversibility of
structural changes upon cooling from 37˚C to 210˚C (post). D. Difference spectra between 210˚C and 37˚C for Q25 and Q46 tested in parts B and C. Each
of these spectra shows a maximum near 190 nm and minima at 208 and 222 nm. Figure 6. Temperature affects alpha-helical structure of thioredoxin-exon 1 HTT proteins. A. Difference spectra between 210˚C and 37˚C, the
temperature extremes shown in parts B and C of Figure 5, for Q25 and Q46. CD spectra plotted using MRE from 190 to 260 nm for THRX-HTT exon 1 Q25
(B) and Q46 (C) in salt-free phosphate buffer at all the temperatures tested showing an isosbestic point around 203 nm (black arrows) and reversibility of
structural changes upon cooling from 37˚C to 210˚C (post). D. Difference spectra between 210˚C and 37˚C for Q25 and Q46 tested in parts B and C. Each
of these spectra shows a maximum near 190 nm and minima at 208 and 222 nm. doi:10.1371/journal.pone.0112262.g006 apparent enhanced rigidity observed by the CRA on polyQ expanded HTT
proteins. Thus, Q46 has a higher helicity at all
temperatures and this enhanced structural ordering is likely related to the estimates of 21 to 23 residues and 30 to 33 residues changing in Q25 and Q46,
respectively (Table 1A). From DICHROWEB analysis, both constructs are also
seen to undergo a loss in beta-sheet upon cooling (Table 2); however, this change
is relatively small and cannot readily be identified in the difference spectra. All
alpha-helicity estimates for the HTT Q46 protein are far beyond the number of
amino acids that are contained in the N17 domain, a region which has a
propensity to adopt an alpha-helical structure [24] ([39, 40]). This suggests that
the observed change in helicity cannot exclusively stem from structural changes in
the N17 region and likely involves the polyQ region (see below). The PRR can
adopt a polyproline type II (PPII) structure (e.g. [24] [19, 41]), a conformation
whose stability may also increase with decreasing temperature (e.g. see [18, 33]). However, the CD spectrum of a PPII structure is entirely different from that of an
alpha-helix. While we cannot exclude that more subtle changes might have also
occurred in the PRR, the structural changes observed by CD are dominated by
alpha-helical structure (Fig. 6A and D). Although the temperature-dependent
change in helicity is more pronounced for Q46, it is important to note that the
overall helicity still remains higher at 37˚C. Thus, Q46 has a higher helicity at all
temperatures and this enhanced structural ordering is likely related to the estimates of 21 to 23 residues and 30 to 33 residues changing in Q25 and Q46,
respectively (Table 1A). From DICHROWEB analysis, both constructs are also
seen to undergo a loss in beta-sheet upon cooling (Table 2); however, this change
is relatively small and cannot readily be identified in the difference spectra. All
alpha-helicity estimates for the HTT Q46 protein are far beyond the number of
amino acids that are contained in the N17 domain, a region which has a
propensity to adopt an alpha-helical structure [24] ([39, 40]). This suggests that 9 / 31 PLOS ONE | DOI:10.1371/journal.pone.0112262
December 2, 2014 Red asterisks denote measurements taken at 4˚C and RT, the
temperatures used in the TR-FRET experiments. Black asterisks denote p-values as determined by the Student’s t-test. *p,0.05. **p,0.01. ns, not
significant. All data are represented as mean ¡ S.D. of three independent experiments. B. CD spectra for the THRX fusion partner alone at indicated
temperatures. C. MRE spectra for the Q25 N548 HTT protein at 210˚C and 37˚C, showing that the temperature effect can still be seen in this longer
fragment. D. MRE values at 222 nm at each indicated temperature relative to MRE at 37˚C for N548 HTT of increasing polyQ length (Q19, Q22, Q25, Q42,
Q52, Q55). doi:10.1371/journal.pone.0112262.g007 As TR-FRET observations employed N548 proteins, we next sought to
determine if such HTT protein fragments would display CD profiles compatible
with observations made using THRX-HTT exon 1 fragments. Although the
impact of the polyQ region on the CD spectra is significantly diluted out in the
larger N548 construct, we were, nonetheless, able to determine temperature-
dependent changes by CD on N548 Q25 HTT protein (Fig. 7C). Again, we found
that MRE values became more negative with decreasing temperature, indicating
increased alpha-helical structure at lower temperatures. Coherently with the
immunoassay-based observations, an influence of polyQ length on alpha-helical
content of N548 HTT proteins was also observed (Fig. 7D). Collectively, these
data pertaining to exon-1 and N548, two constructs of very different lengths, are
compatible with a role for both temperature and polyQ expansion on huntingtin
conformation, confirming the observation obtained using immunoassay-based
approaches. 10 / 31 PLOS ONE | DOI:10.1371/journal.pone.0112262
December 2, 2014 Conformational Changes in Huntingtin Detected by Immunoassays and CD Figure 7. Temperature-dependent alpha-helical variations in thioredoxin-tagged exon 1 HTT proteins are not due to variations in tag helicity, and
are also observed in untagged, larger N548 HTT purified recombinant proteins. A. MRE values at 222 nm (at which alpha-helical structure has a
signature peak) for Q25 and Q46 THRX-HTT exon 1 at each temperature tested. Red asterisks denote measurements taken at 4˚C and RT, the
temperatures used in the TR-FRET experiments. Black asterisks denote p-values as determined by the Student’s t-test. *p,0.05. **p,0.01. ns, not
significant. All data are represented as mean ¡ S.D. of three independent experiments. B. CD spectra for the THRX fusion partner alone at indicated
temperatures. C. MRE spectra for the Q25 N548 HTT protein at 210˚C and 37˚C, showing that the temperature effect can still be seen in this longer
fragment. D. MRE values at 222 nm at each indicated temperature relative to MRE at 37˚C for N548 HTT of increasing polyQ length (Q19, Q22, Q25, Q42,
Q52, Q55). Figure 7. Temperature-dependent alpha-helical variations in thioredoxin-tagged exon 1 HTT proteins are not due to variations in tag helicity, and
are also observed in untagged, larger N548 HTT purified recombinant proteins. A. MRE values at 222 nm (at which alpha-helical structure has a
signature peak) for Q25 and Q46 THRX-HTT exon 1 at each temperature tested. Red asterisks denote measurements taken at 4˚C and RT, the
temperatures used in the TR-FRET experiments. Black asterisks denote p-values as determined by the Student’s t-test. *p,0.05. **p,0.01. ns, not
significant. All data are represented as mean ¡ S.D. of three independent experiments. B. CD spectra for the THRX fusion partner alone at indicated
temperatures. C. MRE spectra for the Q25 N548 HTT protein at 210˚C and 37˚C, showing that the temperature effect can still be seen in this longer
fragment. D. MRE values at 222 nm at each indicated temperature relative to MRE at 37˚C for N548 HTT of increasing polyQ length (Q19, Q22, Q25, Q42,
Q52, Q55). Figure 7. Temperature-dependent alpha-helical variations in thioredoxin-tagged exon 1 HTT proteins are not due to variations in tag helicity, and
are also observed in untagged, larger N548 HTT purified recombinant proteins. A. MRE values at 222 nm (at which alpha-helical structure has a
signature peak) for Q25 and Q46 THRX-HTT exon 1 at each temperature tested. Role of the polyQ domain in temperature-dependent
conformational changes in HTT proteins detected by TR-FRET Based on the CD spectroscopy data, we reasoned that the most significant
conformational change conferred on HTT by temperature variations and detected
by TR-FRET would affect the N-terminal portion of the protein, and would be the
strongest when interrogating polyQ epitopes. Therefore, we investigated whether Table 1. Number of amino acids that gain alpha-helicity upon cooling from 37˚C to 210˚C, using different algorithms. Helix-Coil Model
SELCON3
CONTIN
Q25
23
21
23
Q46
31
33
30
Estimation of temperature-dependent structure of thioredoxin exon 1 HTT proteins. A. Number of amino acids in HTT exon 1 of each Q-length that
experienced a temperature-dependent change in alpha-helical structure, as indicated, calculated either by the helix-coil model or using DICHROWEB
software. Taking the varying results together, it is estimated that between 21 and 27 amino acids in the Q25 construct and between 30 and 37 amino acids in
the Q46 construct gain alpha-helical structure upon cooling from 37˚C to –10˚C. Table 1. Number of amino acids that gain alpha-helicity upon cooling from 37˚C to 210˚C, using different algorithms. Helix-Coil Model
SELCON3
CONTIN
Q25
23
21
23
Q46
31
33
30
Estimation of temperature-dependent structure of thioredoxin exon 1 HTT proteins. A. Number of amino acids in HTT exon 1 of each Q-length that
experienced a temperature-dependent change in alpha-helical structure, as indicated, calculated either by the helix-coil model or using DICHROWEB
software. Taking the varying results together, it is estimated that between 21 and 27 amino acids in the Q25 construct and between 30 and 37 amino acids in
the Q46 construct gain alpha-helical structure upon cooling from 37˚C to –10˚C. n alpha-helicity upon cooling from 37˚C to 210˚C, using different algorithms. Estimation of temperature-dependent structure of thioredoxin exon 1 HTT proteins. A. Number of amino acids in HTT exon 1 of each Q-length that
experienced a temperature-dependent change in alpha-helical structure, as indicated, calculated either by the helix-coil model or using DICHROWEB
software. Taking the varying results together, it is estimated that between 21 and 27 amino acids in the Q25 construct and between 30 and 37 amino acids in
the Q46 construct gain alpha-helical structure upon cooling from 37˚C to –10˚C. Estimation of temperature-dependent structure of thioredoxin exon 1 HTT proteins. A. Role of the polyQ domain in temperature-dependent
conformational changes in HTT proteins detected by TR-FRET Number of amino acids in HTT exon 1 of each Q-length that
experienced a temperature-dependent change in alpha-helical structure, as indicated, calculated either by the helix-coil model or using DICHROWEB
software. Taking the varying results together, it is estimated that between 21 and 27 amino acids in the Q25 construct and between 30 and 37 amino acids in
the Q46 construct gain alpha-helical structure upon cooling from 37˚C to –10˚C. Estimation of temperature-dependent structure of thioredoxin exon 1 HTT proteins. A. Number of amino acids in HTT exon 1 of each Q-length that
experienced a temperature-dependent change in alpha-helical structure, as indicated, calculated either by the helix-coil model or using DICHROWEB
software. Taking the varying results together, it is estimated that between 21 and 27 amino acids in the Q25 construct and between 30 and 37 amino acids in
the Q46 construct gain alpha-helical structure upon cooling from 37˚C to –10˚C. PLOS ONE | DOI:10.1371/journal.pone.0112262
December 2, 2014 PLOS ONE | DOI:10.1371/journal.pone.0112262
December 2, 2014 Conformational Changes in Huntingtin Detected by Immunoassays and CD Table 2. Percentage (and number) of amino acids in a given conformation at indicated temperature obtained from SELCON3 and CONTIN algorithms. Conformation
210˚C, SELCON3
37˚C, SELCON3
210˚C, CONTIN
37˚C, CONTIN
Q25
alpha-helix
32 (74)
23 (53)
33 (76)
23 (53)
beta-sheet
16 (37)
20 (46)
17 (39)
22 (51)
turns
15 (35)
15 (35)
13 (30)
14 (32)
unordered
37 (85)
39 (90)
37 (85)
41 (94)
Q46
alpha-helix
37 (93)
24 (60)
38 (95)
26 (65)
beta-sheet
12 (30)
20 (50)
15 (38)
21 (53)
turns
15 (38)
15 (38)
13 (33)
14 (35)
unordered
34 (85)
39 (98)
35 (88)
40 (100)
Estimated secondary structure percentages of amino acids in each construct at indicated temperatures according to DICHROWEB analyses. The
corresponding number of amino acids is given in parentheses. While the temperature-dependent changes are predominantly alpha-helical, small changes in
beta-sheet structure are also estimated. The number of residues that adopt a turn structure remains virtually unchanged. Note that the fusion proteins
contain a Trx tag, which based upon the Trx crystal structure (PDB: 1F6M) has 39 and 31 amino acids that adopt an alpha-helical and a beta-sheet structure,
respectively [70]. Thus, much of the alpha-helical and beta-sheet structure detected in the fusion proteins by DICHROWEB is contributed by the Trx moiety. ino acids in a given conformation at indicated temperature obtained from SELCON3 and CONTIN algorithms. ntage (and number) of amino acids in a given conformation at indicated temperature obtained from SELCON3 a Table 2. Percentage (and number) of amino acids in a given conformation at indicated temperature obtained Estimated secondary structure percentages of amino acids in each construct at indicated temperatures according to DICHROWEB analyses. The
corresponding number of amino acids is given in parentheses. While the temperature-dependent changes are predominantly alpha-helical, small changes in
beta-sheet structure are also estimated. The number of residues that adopt a turn structure remains virtually unchanged. Note that the fusion proteins
contain a Trx tag, which based upon the Trx crystal structure (PDB: 1F6M) has 39 and 31 amino acids that adopt an alpha-helical and a beta-sheet structure,
respectively [70]. Thus, much of the alpha-helical and beta-sheet structure detected in the fusion proteins by DICHROWEB is contributed by the Trx moiety. doi:10.1371/journal.pone.0112262.t002 an interrogation of two epitopes straddling (but not within) the polyQ region
would result in detection of a temperature-dependent conformational effect,
which as we described above is strongest for unexpanded polyQ (wild type) HTT. The 2B7 and 4C9 epitopes are specific for epitopes within the N17 and PRR
regions of HTT, respectively [13, 32]. Using this antibody pair, we investigated the
effects of temperature on TR-FRET signals obtained on N548 recombinant,
purified proteins with Q16 and Q55 lengths, as performed above for the 2B7 and
MW1 antibody pair. The 2B7-4C9 antibody pair does not detect a significant
temperature-dependent conformational effect on HTT protein (Fig. 8A and B),
lending support to the notion that the conformational change (alpha-helical
content change) observed by CD spectroscopy is principally affecting the polyQ
domain, with little detectable effect on other HTT regions. Consistent with this
notion, employing 2B7 in combination with 3B5H10, another antibody specific
for a polyQ epitope (e.g. [31, 42]) resulted in detection of the temperature-
dependent, polyQ-dependent variation in TR-FRET signals on the same protein
analytes (Fig. 8C and D). We further extended this analysis to determine whether
recognition of the N17 domain (with 2B7) is a critical factor in the
conformational change detected in TR-FRET with 2B7-MW1 using a variety of
antibody pair combinations (including the use of 4C9 as a donor). The results
clearly indicate that detection of the conformational variation is critically
dependent on interrogation of the polyQ region (Table 3). Conformational effects measured by the 2B7-MW1 TR-FRET assay
can be observed in HTT expressed in mammalian cells, and is
independent of HTT fragment length Conformational Changes in Huntingtin Detected by Immunoassays and CD Figure 8. Role of the polyQ domain in temperature-dependent conformational changes in hrHTT proteins detected by TR-FRET. A. Absolute ratios
between the maximum value of the TR-FRET signal obtained at 4˚C and the maximum value of the TR-FRET signal obtained at RT, referred to the curves
obtained with an antibody combination which does not interrogate directly the polyQ region (2B7 Tb, 4C9-A647, 1:10 ratio) on a dilution curve of hrN548 Q16
and Q55 proteins. B. Relative ratios, normalized with respect to ratios obtained for Q16 protein, for the same samples, illustrating the absence of a
temperature- and polyQ-dependent effect obtained with this antibody combination. C. Absolute ratios between the maximum value of the TR-FRET signal
obtained at 4˚C and the maximum value of the TR-FRET signal obtained at RT, referred to the curves obtained with 2B7-Tb, 3B5H10-A647 (1:10 ratio) on
hrN548 Q16 and Q55 proteins. D. Relative ratios, normalized with respect to ratios obtained for Q16 protein, for the same samples, illustrating the presence
of a temperature- and polyQ-dependent effect with this antibody combination. All data are represented as mean ¡ S.D. of three independent experiments,
significance was calculated using the one-way ANOVA test and Bonferroni’s multiple comparison post-test (*5p,0.05; **5p,0.01; ***5p,0.005;
****5p,0.001). doi:10 1371/journal pone 0112262 g008 Figure 8. Role of the polyQ domain in temperature-dependent conformational changes in hrHTT proteins detected by TR-FRET. A. Absolute ratios
between the maximum value of the TR-FRET signal obtained at 4˚C and the maximum value of the TR-FRET signal obtained at RT, referred to the curves
obtained with an antibody combination which does not interrogate directly the polyQ region (2B7 Tb, 4C9-A647, 1:10 ratio) on a dilution curve of hrN548 Q16
and Q55 proteins. B. Relative ratios, normalized with respect to ratios obtained for Q16 protein, for the same samples, illustrating the absence of a
temperature- and polyQ-dependent effect obtained with this antibody combination. C. Absolute ratios between the maximum value of the TR-FRET signal
obtained at 4˚C and the maximum value of the TR-FRET signal obtained at RT, referred to the curves obtained with 2B7-Tb, 3B5H10-A647 (1:10 ratio) on
hrN548 Q16 and Q55 proteins. D. Relative ratios, normalized with respect to ratios obtained for Q16 protein, for the same samples, illustrating the presence
of a temperature- and polyQ-dependent effect with this antibody combination. All data are represented as mean ¡ S.D. Conformational effects measured by the 2B7-MW1 TR-FRET assay
can be observed in HTT expressed in mammalian cells, and is
independent of HTT fragment length Having investigated the temperature- and polyQ-dependent conformational
change using recombinant, purified HTT proteins of different lengths (N548 and
exon 1), we investigated the effect in HTT proteins expressed in a biological PLOS ONE | DOI:10.1371/journal.pone.0112262
December 2, 2014 12 / 31 Conformational Changes in Huntingtin Detected by Immunoassays and CD Table 3. TR-FRET-based detection of conformational rigidity in HTT proteins can be detected when one of the antibodies in the TR-FRET pair detects the
polyQ region. Table 3. TR-FRET-based detection of conformational rigidity in HTT proteins can be detected when one of the
polyQ region. Donor antibody
Acceptor antibody
Epitopes
Temperature and polyQ-dependent TR-FRET variation
2B7
MW1
N17, polyQ
+
MW1
2B7
polyQ, N17
+
2B7
2166
N17, ,aa 445-459
2
2B7
4C9
N17, polyP
2
2B7
3B5H10
N17, polyQ
+
4C9
MW1
polyP, polyQ
+
4C9
2166
PolyP, ,aa 445-459
2
Donor (Tb) and acceptor (d2 or Alexa647) labeled antibodies are indicated, as well as the epitopes recognized. Further details of antibody characterization
and references thereof can be found in the materials and methods section. Donor (Tb) and acceptor (d2 or Alexa647) labeled antibodies are indicated, as well as the epitopes recognized. Further details of antibody characterization
and references thereof can be found in the materials and methods section. Donor (Tb) and acceptor (d2 or Alexa647) labeled antibodies are indicated, as well as the epitopes recognized. Further details of antibody characterization
and references thereof can be found in the materials and methods section. Donor (Tb) and acceptor (d2 or Alexa647) labeled antibodies are indicated, as well as the epitopes recognized
and references thereof can be found in the materials and methods section. antibody pair that did not detect the temperature- and polyQ-dependent
conformational change (2B7-4C9). Consistent with our results using recombi-
nant, purified N548 HTT proteins, the conformational change was observed when
lysates from HEK293T cells transfected with an HTT construct encoding the N548
fragment were analyzed using 2B7-MW1 (Fig. 10A-D), but not when the same antibody pair that did not detect the temperature- and polyQ-dependent
conformational change (2B7-4C9). Consistent with our results using recombi-
nant, purified N548 HTT proteins, the conformational change was observed when
lysates from HEK293T cells transfected with an HTT construct encoding the N548
fragment were analyzed using 2B7-MW1 (Fig. 10A-D), but not when the same Figure 9. HTT proteins of defined fragment length and polyQ content expressed in transfected cells. Western blot of lysates from transfected
HEK293Tcell samples probed with antibody 4C9, demonstrating expression of constructs encoding full length HTT with Q18 or Q83 (arrow), N548 HTTand
exon-1 HTT with or without C-terminal EGFP with Q16, Q39 and Q72. of three independent experiments,
significance was calculated using the one-way ANOVA test and Bonferroni’s multiple comparison post-test (*5p,0.05; **5p,0.01; ***5p,0.005;
****5p,0.001). doi:10.1371/journal.pone.0112262.g008 context, using lysates from HEK293T cells transiently expressing wild type or
mutant HTT cDNAs of different lengths (full length, N548 or exon 1; Fig. 9A, B
and C, respectively). In order to demonstrate temperature- and polyQ-
dependence of the effect in HTT proteins expressed in a cellular context (rather
than on purified recombinant proteins), we elected to use a transient transfection
system which, although clearly artificial, allows rapid and robust expression of
HTT proteins of defined length and polyQ content, as produced by selected and
characterized plasmid constructs. We analyzed these lysates using the same 2B7-
MW1 antibody pair used on recombinant proteins (see above), as well as a control PLOS ONE | DOI:10.1371/journal.pone.0112262
December 2, 2014 13 / 31 doi:10.1371/journal.pone.0112262.t003 doi:10.1371/journal.pone.0112262.g009 Conformational Changes in Huntingtin Detected by Immunoassays and CD Figure 10. Temperature- and polyQ- dependent conformational changes detected on HTT N548 protein (Q16, Q39 and Q72) using the 2B7-MW1
TR-FRET assay in a biological sample (lysates from transfected cells). A–B. TR-FRET values obtained at RT (A) or 4˚C (B) using 2B7-Tb (1 ng/ml) -
MW1-d2 (10 ng/ml) on different protein concentrations of lysates, confirming the temperature- and polyQ-dependent conformational effect. C. Absolute ratios
between the maximum value of the TR-FRETsignal obtained at 4˚C and the maximum value of the TR-FRETsignal obtained at RT, referred to the curves in
A and B. D. Absolute ratios between the maximum value of the TR-FRET signal obtained at 4˚C and the maximum value of the TR-FRET signal obtained at
RT, referred to the curves achieved using a 1:1 ratio of 2B7-Tb and MW1-d2 (1 ng/ml for both the antibodies). E-F. TR-FRET values obtained at RT (E) or 4˚C
(F) using 2B7-Tb and 4C9-Alexa647 on the same lysates, confirming the requirement for polyQ interrogation to observe conformational changes. G. Absolute ratios between the maximum value of the TR-FRET signal obtained at 4˚C and the maximum value of the TR-FRET signal obtained at RT, referred
to the curves in G and F. H. Maximum value of the TR-FRET signals obtained at RT, normalized with respect to the maximum value of the TR-FRET signals
obtained for N548 Q16, referred to the curves in A (upper graph) and E (lower graph). In C, D and G, data are represented as mean ¡ S.D. of three
independent experiments. In H the three different experiments are represented as single point together with their mean. Significance was calculated using
the one-way ANOVA test and Bonferroni’s multiple comparison post-test (*5p,0.05; **5p,0.01; ***5p,0.005). d i 10 1371/j
l
0112262 010 Figure 10. Temperature- and polyQ- dependent conformational changes detected on HTT N548 protein (Q16, Q39 and Q72) using the 2B7-MW1
TR-FRET assay in a biological sample (lysates from transfected cells). A–B. TR-FRET values obtained at RT (A) or 4˚C (B) using 2B7-Tb (1 ng/ml) -
MW1-d2 (10 ng/ml) on different protein concentrations of lysates, confirming the temperature- and polyQ-dependent conformational effect. C. Absolute ratios
between the maximum value of the TR-FRETsignal obtained at 4˚C and the maximum value of the TR-FRETsignal obtained at RT, referred to the curves in
A and B. D. Blots were also probed with an anti-GAPDH antibody as loading control. Figure 9. HTT proteins of defined fragment length and polyQ content expressed in transfected cells. Western blot of lysates from transfected
HEK293Tcell samples probed with antibody 4C9, demonstrating expression of constructs encoding full length HTT with Q18 or Q83 (arrow), N548 HTTand
exon-1 HTT with or without C-terminal EGFP with Q16, Q39 and Q72. Blots were also probed with an anti-GAPDH antibody as loading control. doi:10.1371/journal.pone.0112262.g009 PLOS ONE | DOI:10.1371/journal.pone.0112262
December 2, 2014 PLOS ONE | DOI:10.1371/journal.pone.0112262
December 2, 2014 14 / 31 doi:10.1371/journal.pone.0112262.g010 Conformational Changes in Huntingtin Detected by Immunoassays and CD construct encoding exon 1 HTT fragments (Fig. 11). As the employed exon 1 HTT
constructs included a carboxyl-terminal (C-terminal) EGFP fusion, we investi-
gated the effects of the presence of a heterologous protein domain (EGFP) on
detection of the temperature- and polyQ-dependent conformational change by
comparing EGFP tagged and untagged exon 1 HTT expression constructs,
transiently transfected in HEK293T cells. The results (Fig. 12) clearly suggested
that the detected conformational change was again independent of regions outside
the N-terminal part of HTT, even when such regions are heterologous. Finally, we
investigated the conformational effect in cell lysates expressing full length (Q18,
Q83) HTT. Again, the temperature- and polyQ-dependent conformational change
was observed when lysates from HEK293T cells transfected with an HTT construct
encoding the full length protein were analyzed using 2B7-MW1, but not when the
same lysates were analyzed with the 2B7-4C9 antibody pair (Fig. 13). When Figure 11. Temperature- and polyQ- dependent conformational changes detected on HTT exon 1 protein (Q16, Q39 and Q72) using the 2B7-MW1
TR-FRET assay in a biological sample (lysates from transfected cells). A–B. TR-FRET values obtained at RT (A) or 4˚C (B) using 2B7-Tb (1 ng/ml) -
MW1-d2 (10 ng/ml) on lysates, confirming the temperature- and polyQ-dependent conformational effect. C. Absolute ratios between the maximum value of
the TR-FRET signal obtained at 4˚C and the maximum value of the TR-FRET signal obtained at RT, referred to the curves in A and B. D. Absolute ratios
between the maximum value of the TR-FRET signal obtained at 4˚C and the maximum value of the TR-FRET signal obtained at RT, referred to the curves
achieved using a 1:1 ratio of 2B7-Tb and MW1-d2 (1 n/ml for both the antibodies). E-F. TR-FRET values obtained at RT (E) or 4˚C (F) using 2B7-Tb and 4C9-
Alexa647 on the same lysates, confirming the requirement for polyQ detection to observe conformational changes. In C, D and G data are represented as
mean ¡ S.D. of three independent experiments; significance was calculated using the one-way ANOVA test and Bonferroni’s multiple comparison post-test
(**5p,0.01; ***5p,0.005). Figure 11. Temperature- and polyQ- dependent conformational changes detected on HTT exon 1 protein (Q16, Q39 and Q72) using the 2B7-MW1
TR-FRET assay in a biological sample (lysates from transfected cells). A–B. Absolute ratios between the maximum value of the TR-FRET signal obtained at 4˚C and the maximum value of the TR-FRET signal obtained at
RT, referred to the curves achieved using a 1:1 ratio of 2B7-Tb and MW1-d2 (1 ng/ml for both the antibodies). E-F. TR-FRET values obtained at RT (E) or 4˚C
(F) using 2B7-Tb and 4C9-Alexa647 on the same lysates, confirming the requirement for polyQ interrogation to observe conformational changes. G. Absolute ratios between the maximum value of the TR-FRET signal obtained at 4˚C and the maximum value of the TR-FRET signal obtained at RT, referred
to the curves in G and F. H. Maximum value of the TR-FRET signals obtained at RT, normalized with respect to the maximum value of the TR-FRET signals
obtained for N548 Q16, referred to the curves in A (upper graph) and E (lower graph). In C, D and G, data are represented as mean ¡ S.D. of three
independent experiments. In H the three different experiments are represented as single point together with their mean. Significance was calculated using
the one-way ANOVA test and Bonferroni’s multiple comparison post-test (*5p,0.05; **5p,0.01; ***5p,0.005). doi:10.1371/journal.pone.0112262.g010 lysates were analyzed with the 2B7-4C9 antibody pair (Fig. 10E–G). As expected,
the difference in behaviour between the MW1 and 4C9 antibodies on wild type
and mutant HTT substrates was clearly observed, with the acceptor-labeled MW1
producing an increased TR-FRET signal with increasing polyQ length and the
acceptor-labeled 4C9 displaying a decreased TR-FRET signal with increasing
polyQ length (Fig. 10H), consistent with published observations [26]. Similar
results were obtained with lysates from HEK293T cells transfected with a PLOS ONE | DOI:10.1371/journal.pone.0112262
December 2, 2014 15 / 31 Conformational Changes in Huntingtin Detected by Immunoassays and CD Figure 12. Presence of a C-terminal heterologous tag does not influence the temperature- and polyQ dependent conformational effect. A. Schem
of proteins expressed by constructs encoding exon 1 HTT cDNAs with or without C-terminally fused EGFP. B. TR-FRET values (665 nm/615 nm) obtaine
at RT or 4˚C using 2B7-Tb (1 ng/ml)- MW1-d2 (10 ng/ml) on lysates of HEK293T cells transfected with exon 1 HTT expression constructs (Q16, Q39 or Q7
with or without a C-terminal EGFP moiety, confirming the temperature- and polyQ-dependent conformational effect irrespective of the presence of a C-
terminal EGFP fusion. C. Absolute ratios between the maximum value of the TR-FRETsignal obtained at 4˚C and the maximum value of the TR-FRETsign
obtained at RT, referred to the curves in C. Figure 12. Presence of a C-terminal heterologous tag does not influence the temperature- and polyQ dependent conformational effect. A. Scheme
of proteins expressed by constructs encoding exon 1 HTT cDNAs with or without C-terminally fused EGFP. B. TR-FRET values (665 nm/615 nm) obtained
at RT or 4˚C using 2B7-Tb (1 ng/ml)- MW1-d2 (10 ng/ml) on lysates of HEK293T cells transfected with exon 1 HTT expression constructs (Q16, Q39 or Q72)
with or without a C-terminal EGFP moiety, confirming the temperature- and polyQ-dependent conformational effect irrespective of the presence of a C-
terminal EGFP fusion. C. Absolute ratios between the maximum value of the TR-FRETsignal obtained at 4˚C and the maximum value of the TR-FRETsignal
obtained at RT, referred to the curves in C. doi:10.1371/journal.pone.0112262.g012 considered with the data demonstrating that N-terminal fusion with thioredoxin
does not prevent detection of the conformational change, the data collectively
suggest a pronounced conformational flexibility of the N-terminal portion of
HTT that is not affected by natural or heterologous flanking sequences. TR-FRET values obtained at RT (A) or 4˚C (B) using 2B7-Tb (1 ng/ml) -
MW1-d2 (10 ng/ml) on lysates, confirming the temperature- and polyQ-dependent conformational effect. C. Absolute ratios between the maximum value of
the TR-FRET signal obtained at 4˚C and the maximum value of the TR-FRET signal obtained at RT, referred to the curves in A and B. D. Absolute ratios
between the maximum value of the TR-FRET signal obtained at 4˚C and the maximum value of the TR-FRET signal obtained at RT, referred to the curves
achieved using a 1:1 ratio of 2B7-Tb and MW1-d2 (1 n/ml for both the antibodies). E-F. TR-FRET values obtained at RT (E) or 4˚C (F) using 2B7-Tb and 4C9-
Alexa647 on the same lysates, confirming the requirement for polyQ detection to observe conformational changes. In C, D and G data are represented as
mean ¡ S.D. of three independent experiments; significance was calculated using the one-way ANOVA test and Bonferroni’s multiple comparison post-test
(**5p,0.01; ***5p,0.005). doi:10.1371/journal.pone.0112262.g011 16 / 31 PLOS ONE | DOI:10.1371/journal.pone.0112262
December 2, 2014 N17 mutations do not significantly influence temperature-
dependent conformational changes in HTT We next asked if variations within the N-terminal portion of HTT, and specifically
within the N17 domain, could influence the temperature- and polyQ-dependent
conformational event detected by the 2B7 and MW1 TR-FRET antibody
combination. Post-translational modifications represent an obvious candidate
modifier of mutant HTT properties, in particular those addressing the first 17
amino acids of the protein, as this region has been shown to have profound 17 / 31 PLOS ONE | DOI:10.1371/journal.pone.0112262
December 2, 2014 17 / 31 doi:10.1371/journal.pone.0112262.g013 influences on subcellular localization, aggregation and toxicity (e.g. reviewed in
[43, 44]). Mutations within the N17 region have been utilized to mimic the
presence of PTMs; for instance, D mutations of T3, S13 and S16 residues mimic
phosphorylation at these positions and have been used to provide support
towards the hypothesis that N17 phosphorylation can influence HTT properties
pertinent to toxicity of the mutant protein [17, 20, 23, 26, 45–47]. In order to
determine if mutational phosphomimicry can affect HTT conformation as
detected by the temperature-dependent TR-FRET assay, we employed lysates from
HEK293T cells transfected with HTT exon 1 cDNA constructs bearing T3, S13
and S16 mutations in a Q16, Q39 and Q72 context (Fig. 14A). Instead of
employing the 2B7-Tb/MW1-D2 antibody pair, we employed a 4C9-Tb-MW1-D2
antibody pair in order to avoid potential confounding issues arising from the use
of 2B7, whose epitope is within the N17 region (where the residues affected by the
mutations are present). When analyzed with the 4C9-MW1 TR-FRET assay, none
PLOS ONE | DOI:10.1371/journal.pone.0112262
December 2, 2014
18 / 31 influences on subcellular localization, aggregation and toxicity (e.g. reviewed in
[43, 44]). Mutations within the N17 region have been utilized to mimic the
presence of PTMs; for instance, D mutations of T3, S13 and S16 residues mimic
phosphorylation at these positions and have been used to provide support
towards the hypothesis that N17 phosphorylation can influence HTT properties
pertinent to toxicity of the mutant protein [17, 20, 23, 26, 45–47]. In order to
determine if mutational phosphomimicry can affect HTT conformation as
detected by the temperature-dependent TR-FRET assay, we employed lysates from
HEK293T cells transfected with HTT exon 1 cDNA constructs bearing T3, S13
and S16 mutations in a Q16, Q39 and Q72 context (Fig. 14A). Instead of
employing the 2B7-Tb/MW1-D2 antibody pair, we employed a 4C9-Tb-MW1-D2
antibody pair in order to avoid potential confounding issues arising from the use
of 2B7, whose epitope is within the N17 region (where the residues affected by the
mutations are present). When analyzed with the 4C9-MW1 TR-FRET assay, none influences on subcellular localization, aggregation and toxicity (e.g. reviewed in
[43, 44]). Conformational Changes in Huntingtin Detected by Immunoassays and CD Figure 13. Temperature- and polyQ- dependent conformational changes detected on HTT full length protein (Q18 and Q83) using the 2B7-MW1
TR-FRET assay in a biological sample (lysates from transfected cells). A–B. TR-FRET values obtained at RT (A) or 4˚C (B) using 2B7-Tb (1 n/ml) -
MW1-d2 (10 ng/ml) on lysates, confirming the temperature- and polyQ-dependent conformational effect. C. Absolute ratios between the maximum value of
the TR-FRET signal obtained at 4˚C and the maximum value of the TR-FRET signal obtained at RT, referred to the curves in A and B. D. Absolute ratios
between the maximum value of the TR-FRET signal obtained at 4˚C and the maximum value of the TR-FRET signal obtained at RT, referred to the curves
achieved using a 1:1 ratio of 2B7-Tb and MW1-d2 (1 n/ml for both antibodies). E-F. TR-FRET values obtained at RT (E) or 4˚C (F) using 2B7-Tb and 4C9-
Alexa647 on the same lysates, confirming the requirement for polyQ detection to observe conformational changes. In C, D and G data are represented as
mean ¡ S.D. of three independent experiments; significance was calculated using the one-way ANOVA test and Bonferroni’s multiple comparison post-test
(**5p,0.01; ***5p,0.005). Figure 13. Temperature- and polyQ- dependent conformational changes detected on HTT full length protein (Q18 and Q83) using the 2B7-MW1
TR-FRET assay in a biological sample (lysates from transfected cells). A–B. TR-FRET values obtained at RT (A) or 4˚C (B) using 2B7-Tb (1 n/ml) -
MW1-d2 (10 ng/ml) on lysates, confirming the temperature- and polyQ-dependent conformational effect. C. Absolute ratios between the maximum value of
the TR-FRET signal obtained at 4˚C and the maximum value of the TR-FRET signal obtained at RT, referred to the curves in A and B. D. Absolute ratios
between the maximum value of the TR-FRET signal obtained at 4˚C and the maximum value of the TR-FRET signal obtained at RT, referred to the curves
achieved using a 1:1 ratio of 2B7-Tb and MW1-d2 (1 n/ml for both antibodies). E-F. TR-FRET values obtained at RT (E) or 4˚C (F) using 2B7-Tb and 4C9-
Alexa647 on the same lysates, confirming the requirement for polyQ detection to observe conformational changes. In C, D and G data are represented as
mean ¡ S.D. of three independent experiments; significance was calculated using the one-way ANOVA test and Bonferroni’s multiple comparison post-test
(**5p,0.01; ***5p,0.005). Conformational Changes in Huntingtin Detected by Immunoassays and CD Figure 14. N17 mutations do not affect temperature- and polyQ- dependent conformational changes as detected by a 4C9-MW1 antibody
combination in TR-FRET. A. Western blot of lysates from transfected HEK293T cell samples probed with antibody 4C9, demonstrating expression of
constructs encoding wild type or N-terminal mutant HTT exon 1 overexpressed in HEK293T cells with Q16, Q39 and Q72. The blot was also probed with an
anti-GAPDH antibody as loading control. B. Absolute ratios between the maximum value of the TR-FRET signal obtained at 4˚C and the maximum value of
the TR-FRET signal obtained at RT, referred to the curves obtained using 2B7-Tb (1 ng/ml) and MW1-d2 (10 ng/ml) on the same lysates. Data are
represented as mean ¡ S.D. of three independent experiments; significance was calculated using the one-way ANOVA test and Bonferroni’s multiple
comparison post-test (**5p,0.01; ***5p,0.005). d i 10 1371/j
l
0112262 014 Figure 14. N17 mutations do not affect temperature- and polyQ- dependent conformational changes as detected by a 4C9-MW1 antibody
combination in TR-FRET. A. Western blot of lysates from transfected HEK293T cell samples probed with antibody 4C9, demonstrating expression of
constructs encoding wild type or N-terminal mutant HTT exon 1 overexpressed in HEK293T cells with Q16, Q39 and Q72. The blot was also probed with an
anti-GAPDH antibody as loading control. B. Absolute ratios between the maximum value of the TR-FRET signal obtained at 4˚C and the maximum value of
the TR-FRET signal obtained at RT, referred to the curves obtained using 2B7-Tb (1 ng/ml) and MW1-d2 (10 ng/ml) on the same lysates. Data are
represented as mean ¡ S.D. of three independent experiments; significance was calculated using the one-way ANOVA test and Bonferroni’s multiple
comparison post-test (**5p,0.01; ***5p,0.005). doi:10.1371/journal.pone.0112262.g014 of the mutants influenced the temperature-dependent behaviour of either wild
type (Q16) or mutant (Q39 and Q72) HTT exon 1 (Fig. 14B). Together with the
observation that the detected conformational event is independent of HTT
fragment length (see Figs. 10, 11, 12, and 13), this finding suggests that it may also
be independent of changes within the N17 region. Mutations within the N17 region have been utilized to mimic the
presence of PTMs; for instance, D mutations of T3, S13 and S16 residues mimic
phosphorylation at these positions and have been used to provide support
towards the hypothesis that N17 phosphorylation can influence HTT properties
pertinent to toxicity of the mutant protein [17, 20, 23, 26, 45–47]. In order to
determine if mutational phosphomimicry can affect HTT conformation as
detected by the temperature-dependent TR-FRET assay, we employed lysates from
HEK293T cells transfected with HTT exon 1 cDNA constructs bearing T3, S13
and S16 mutations in a Q16, Q39 and Q72 context (Fig. 14A). Instead of
employing the 2B7-Tb/MW1-D2 antibody pair, we employed a 4C9-Tb-MW1-D2
antibody pair in order to avoid potential confounding issues arising from the use
of 2B7, whose epitope is within the N17 region (where the residues affected by the
mutations are present). When analyzed with the 4C9-MW1 TR-FRET assay, none 18 / 31 PLOS ONE | DOI:10.1371/journal.pone.0112262
December 2, 2014 Conformational Changes in Huntingtin Detected by Immunoassays and CD handedness), with its N-terminal residues amenable to adopting the right-handed
alpha-helical structure of the N17 module while its C-terminal residues can adopt
the structure of the proline repeat module[24] [25, 48]. The N17 and PRR
domains can have a substantial (and opposing) effect on the intramolecular and
intermolecular properties of N-terminal HTT, with consequences on protein
aggregation and cell toxicity [26, 50–55]. This is likely to result at least in part
from structural interplay amongst the three modules, allowing a range of possible
conformations and intramolecular/intermolecular interactions for N-terminal
HTT fragments [55, 56]. Indeed, recent evidence proposes that the N-terminal
portion of monomeric, wild type HTT is conformationally flexible, with the
relatively unstructured polyQ domain acting as a flexible hinge between the N17
and downstream HTT domains (e.g. the PRR region), and becomes rigid when
expanded in mHTT [26, 57]. This conformational dynamism is likely essential to
modulate a range of intra- and intermolecular interactions which, when
abnormal, might result in altered protein associations and abnormal HTT
function [26]. The possibility of interrogating HTT conformation with simple
robust assays would allow investigations on the role of mHTT conformational
changes in HD, its determinants (e.g. post-translational modifications, protein-
protein interactions) and its modulation (genetic or pharmacological) to
influence mutant protein function, aggregation, and/or toxicity. With appropriate
antibodies, TR-FRET assays provide the sensitivity, robustness and scalability
required for screening applications. We have previously developed TR-FRET
assays to quantify different HTT conformers [13] and reasoned that such assays
might be able to detect conformational changes in response to variations in polyQ
length and/or conditions known to modulate protein conformation, such as
temperature. We reasoned that interrogating HTT epitopes including the polyQ
repeat region would provide the most significant information, as this latter
represents the pathologically relevant module. Using such immunoassays, we
identified a reversible, polyQ-dependent effect of temperature on the TR-FRET
signal of recombinant, N548 HTT proteins bearing wild-type or mutant polyQ
expansions. Lowering the incubation temperature increased TR-FRET signal when
wild type HTT was employed, and this increase was inversely proportional to
polyQ length, reaching a minimum in proximity of the CAG length associated
with HD (e.g. see [58, 59]). The rapid reversibility of the effect is compatible with
a conformational event, and suggests decreased flexibility of mutant HTT relative
to the wild type protein, consistent with recent observations [26]. Discussion The N-terminal portion (exon 1 encoded) of the HTT protein is a key
determinant of HD as it comprises the polyQ repeat expansion. Structural,
biophysical, and functional studies of HTT exon 1 suggest that in the HTT
monomer this region can be viewed as a modular unit comprised of at least three
major structural domains — the N-terminal N17, the polyQ, and the PRR [24]
[17–19, 25, 41, 48]. The N17 region displays a propensity to adopt a right handed
amphipathic alpha-helical structure [24] [24, 39, 40]. This region is followed by
the polyQ repeat module, which can take up a beta-sheet structure in fibrils, but
which has been thought to be predominantly unfolded in solution (reviewed in
[25, 49]). The polyQ module is immediately followed by a pure proline repeat
encoding region which is likely to adopt a left-handed polyproline II helix
structure [19, 41]. In both crystallographic and computational structural studies,
the less ordered polyQ repeat is conformationally influenced by the flanking
regions (which can form, interestingly, two protein helices of opposite 19 / 31 PLOS ONE | DOI:10.1371/journal.pone.0112262
December 2, 2014 For example, the affinity or avidity of MW1 for its epitope may be
higher at lower temperature (for instance, through more stable hydrogen bonds)
and such difference might be more readily detectable by TR-FRET on polyQ
substrates where fewer epitopes are present and the signal is lower (e.g. wild type
HTT). On substrates with longer polyQ regions, where multiple MW1 binding
events are possible, this antibody-specific effect of temperature might be
overridden by multiple IgGs binding to the (mutant) HTT antigen. A detailed
investigation of the effects of temperature on the affinity and avidity of MW1 and
3B5H10 would ideally need to be performed in solution to be comparable to the
TR-FRET assay, and under conditions where HTT conformation would need to
be irreversibly fixed in order to eliminate antigen conformation as a confounding
variable. The complexities of such analysis led us to apply an orthogonal, non-
immunoassay biophysical readout to determine if temperature could influence
antigen (HTT) conformation in a polyQ-dependent fashion. Our CD studies
indicate that HTT exon 1 can take up an extensive helical structure that likely
includes the N17 as well as substantial portions of the polyQ region. This notion is
in agreement with prior crystallographic studies, which showed that alpha-helical
structure could extend from the N17 into the polyQ region in some, but not all
structures [24] [25 [24]]. Our data indicate that the observed alpha-helical and
unfolded structures are in equilibrium with one another. Perhaps not surprisingly,
lower temperatures favor the helical state. This effect is more pronounced at
higher Q length and, additionally, higher Q lengths result in overall higher helicity
at any given temperature. One possibility is that this residual helical structure
directly causes apparent rigidity in proteins with higher Q lengths, a notion
consistent with previous fluorescence studies which showed that intramolecular
FRET is strongly polyQ length dependent [26 [26]]. However, we cannot exclude
other more indirect mechanisms. For example, it is possible that epitope
recognition by MW1 (and 3B5H10) is somewhat influenced by the extent of
alpha-helical structure present in the polyQ region (Fig. 15). For instance, polyQ
alpha-helical content could influence the exposure, number and/or relative
distance/orientation of epitopes from the donor (Tb labeled) antibody. We are
therefore further investigating the observed phenomenon using higher resolution, results are due to temperature-dependent changes in the affinity, flexibility,
binding modality, position and number of labels and/or conformation of the
IgGs, although such effects would have to be exquisitely specific for MW1 and
3B5H10 as the temperature- and polyQ-dependent TR-FRET effect is not
observed when IgGs other than MW1 and 3B5H10 are used, even if labeled and
employed in the same exact way (e.g. 2B7, 4C9 and 2166). A similar consideration
applies to potential effects due to influences due to IgG conjugation aspects. However, the temperature-dependent kinetics of antibody-antigen interactions
are indeed variable from antibody to antibody, and these kinetics are likely to be
complex with antibodies such as MW1 which can recognize multiple epitopes in
polyQ regions. For example, the affinity or avidity of MW1 for its epitope may be
higher at lower temperature (for instance, through more stable hydrogen bonds)
and such difference might be more readily detectable by TR-FRET on polyQ
substrates where fewer epitopes are present and the signal is lower (e.g. wild type
HTT). On substrates with longer polyQ regions, where multiple MW1 binding
events are possible, this antibody-specific effect of temperature might be
overridden by multiple IgGs binding to the (mutant) HTT antigen. A detailed
investigation of the effects of temperature on the affinity and avidity of MW1 and
3B5H10 would ideally need to be performed in solution to be comparable to the
TR-FRET assay, and under conditions where HTT conformation would need to
be irreversibly fixed in order to eliminate antigen conformation as a confounding
variable. The complexities of such analysis led us to apply an orthogonal, non-
immunoassay biophysical readout to determine if temperature could influence
antigen (HTT) conformation in a polyQ-dependent fashion. Our CD studies
indicate that HTT exon 1 can take up an extensive helical structure that likely
includes the N17 as well as substantial portions of the polyQ region. This notion is
in agreement with prior crystallographic studies, which showed that alpha-helical
structure could extend from the N17 into the polyQ region in some, but not all
structures [24] [25 [24]]. Our data indicate that the observed alpha-helical and
unfolded structures are in equilibrium with one another. Perhaps not surprisingly,
lower temperatures favor the helical state. Conformational Changes in Huntingtin Detected by Immunoassays and CD results are due to temperature-dependent changes in the affinity, flexibility,
binding modality, position and number of labels and/or conformation of the
IgGs, although such effects would have to be exquisitely specific for MW1 and
3B5H10 as the temperature- and polyQ-dependent TR-FRET effect is not
observed when IgGs other than MW1 and 3B5H10 are used, even if labeled and
employed in the same exact way (e.g. 2B7, 4C9 and 2166). A similar consideration
applies to potential effects due to influences due to IgG conjugation aspects. However, the temperature-dependent kinetics of antibody-antigen interactions
are indeed variable from antibody to antibody, and these kinetics are likely to be
complex with antibodies such as MW1 which can recognize multiple epitopes in
polyQ regions. For example, the affinity or avidity of MW1 for its epitope may be
higher at lower temperature (for instance, through more stable hydrogen bonds)
d
h diff
i h b
dil d
bl b TR FRET
l Q results are due to temperature-dependent changes in the affinity, flexibility,
binding modality, position and number of labels and/or conformation of the
IgGs, although such effects would have to be exquisitely specific for MW1 and
3B5H10 as the temperature- and polyQ-dependent TR-FRET effect is not
observed when IgGs other than MW1 and 3B5H10 are used, even if labeled and
employed in the same exact way (e.g. 2B7, 4C9 and 2166). A similar consideration
applies to potential effects due to influences due to IgG conjugation aspects. results are due to temperature-dependent changes in the affinity, flexibility,
binding modality, position and number of labels and/or conformation of the
IgGs, although such effects would have to be exquisitely specific for MW1 and
3B5H10 as the temperature- and polyQ-dependent TR-FRET effect is not
observed when IgGs other than MW1 and 3B5H10 are used, even if labeled and
employed in the same exact way (e.g. 2B7, 4C9 and 2166). A similar consideration
applies to potential effects due to influences due to IgG conjugation aspects. However, the temperature-dependent kinetics of antibody-antigen interactions
are indeed variable from antibody to antibody, and these kinetics are likely to be
complex with antibodies such as MW1 which can recognize multiple epitopes in
polyQ regions. This effect is more pronounced at
higher Q length and, additionally, higher Q lengths result in overall higher helicity
at any given temperature. One possibility is that this residual helical structure
directly causes apparent rigidity in proteins with higher Q lengths, a notion
consistent with previous fluorescence studies which showed that intramolecular
FRET is strongly polyQ length dependent [26 [26]]. However, we cannot exclude
other more indirect mechanisms. For example, it is possible that epitope
recognition by MW1 (and 3B5H10) is somewhat influenced by the extent of
alpha-helical structure present in the polyQ region (Fig. 15). For instance, polyQ
alpha-helical content could influence the exposure, number and/or relative
distance/orientation of epitopes from the donor (Tb labeled) antibody. We are
therefore further investigating the observed phenomenon using higher resolution,
non-immunoassay based methods such as electron paramagnetic resonance (EPR;
e.g. see [18]). Based on these observations, it is likely that repeat length- and
temperature-dependent behaviour of polyglutamine repeats may be observable pp
p
g
j g
p
However, the temperature-dependent kinetics of antibody-antigen interactions
are indeed variable from antibody to antibody, and these kinetics are likely to be
complex with antibodies such as MW1 which can recognize multiple epitopes in
polyQ regions. For example, the affinity or avidity of MW1 for its epitope may be
higher at lower temperature (for instance, through more stable hydrogen bonds) p
p
p
g
( g
yp
HTT). On substrates with longer polyQ regions, where multiple MW1 binding
events are possible, this antibody-specific effect of temperature might be
overridden by multiple IgGs binding to the (mutant) HTT antigen. A detailed
investigation of the effects of temperature on the affinity and avidity of MW1 and
3B5H10 would ideally need to be performed in solution to be comparable to the
TR-FRET assay, and under conditions where HTT conformation would need to
be irreversibly fixed in order to eliminate antigen conformation as a confounding
variable. The complexities of such analysis led us to apply an orthogonal, non-
immunoassay biophysical readout to determine if temperature could influence
antigen (HTT) conformation in a polyQ-dependent fashion. Our CD studies
indicate that HTT exon 1 can take up an extensive helical structure that likely
includes the N17 as well as substantial portions of the polyQ region. Interestingly,
the effect appears to be detectable only when the polyQ domain is interrogated,
and is not influenced by HTT fragment length or by mutations affecting
neighbouring domains (such as the N17 domain). Importantly, levels of soluble
antigen are not significantly different at 4˚C vs room temperature, as detected by
TR-FRET interrogating epitopes other than the polyQ region. There are several
possible explanations for the observed temperature- and polyQ-dependent 20 / 31 PLOS ONE | DOI:10.1371/journal.pone.0112262
December 2, 2014 Q55) polyQ
expansion, the polyQ region adopts a higher alpha-helical content relatively to wild type HTT, a relatively
larger number of epitopes for the acceptor-labeled MW1 antibody are present and, consequently, TR-FRET
signals are higher. The significant alpha-helical structure of the mutant polyQ region is less influenced by
temperature, the number of MW1 epitopes remains relatively constant and therefore FRET between donor
and acceptor labels is relatively unchanged. Figure 15. Model of structural changes observed in HTT proteins upon temperature change. The N17
(orange) and polyproline (blue) regions are depicted as alpha- and PPII helices, respectively, while the
polyglutamine region is illustrated as a domain which can adopt temperature- and polyQ-dependent
conformations (red). The epitopes of the 2B7 and MW1 antibodies are graphically illustrated. A. The N-
terminal region of a HTT protein bearing a wild type (e.g. Q16) polyQ expansion can adopt variable degrees of
structure in the polyQ region, with alpha-helical content being higher at lower temperatures. At higher
temperatures, alpha-helical content is lower and results in a 1: #1 ratio of 2B7Tb-MW1D2 bound to HTT. At
lower temperatures, this ratio becomes 1: >1 due to increased alpha-helical content in the polyglutamine
region, an increased number of epitopes for the acceptor-labeled MW1 antibody and, consequently, an
increase in TR-FRET signal. B. In the N-terminal region of a HTT protein bearing a mutant (e.g. Q55) polyQ
expansion, the polyQ region adopts a higher alpha-helical content relatively to wild type HTT, a relatively
larger number of epitopes for the acceptor-labeled MW1 antibody are present and, consequently, TR-FRET
signals are higher. The significant alpha-helical structure of the mutant polyQ region is less influenced by
temperature, the number of MW1 epitopes remains relatively constant and therefore FRET between donor
and acceptor labels is relatively unchanged. doi:10.1371/journal.pone.0112262.g015 more generally with other polyQ proteins, such as those associated with
spinocerebellar ataxias, spinal bulbar muscular atrophy (SBMA) and dentator-
ubral-pallidoluysian atrophy (DRPLA) [60–62]. Additionally, our studies high-
light the importance of carefully controlling temperature in measuring levels of
proteins comprising polyglutamine repeats, particularly where such assays involve
the use of antibodies interrogating the polyQ repeat [13]. Although the precise
mechanisms regulating N-terminal HTT conformation are only starting to be
understood,the N17 region is subject to post-translational modifications (PTMs),
including phosphorylation, acetylation, ubiquitination and SUMOylation which
significantly alter HTT N-terminal fragment biological properties in vitro and in
cells [17, 22, 23, 45, 63]. This notion is
in agreement with prior crystallographic studies, which showed that alpha-helical
structure could extend from the N17 into the polyQ region in some, but not all
structures [24] [25 [24]]. Our data indicate that the observed alpha-helical and
unfolded structures are in equilibrium with one another. Perhaps not surprisingly,
lower temperatures favor the helical state. This effect is more pronounced at
higher Q length and, additionally, higher Q lengths result in overall higher helicity
at any given temperature. One possibility is that this residual helical structure
directly causes apparent rigidity in proteins with higher Q lengths, a notion
consistent with previous fluorescence studies which showed that intramolecular
FRET is strongly polyQ length dependent [26 [26]]. However, we cannot exclude
other more indirect mechanisms. For example, it is possible that epitope
recognition by MW1 (and 3B5H10) is somewhat influenced by the extent of
alpha-helical structure present in the polyQ region (Fig. 15). For instance, polyQ
alpha-helical content could influence the exposure, number and/or relative
distance/orientation of epitopes from the donor (Tb labeled) antibody. We are
therefore further investigating the observed phenomenon using higher resolution,
non-immunoassay based methods such as electron paramagnetic resonance (EPR;
e.g. see [18]). Based on these observations, it is likely that repeat length- and
temperature-dependent behaviour of polyglutamine repeats may be observable 21 / 31 PLOS ONE | DOI:10.1371/journal.pone.0112262
December 2, 2014 Conformational Changes in Huntingtin Detected by Immunoassays and CD Figure 15. Model of structural changes observed in HTT proteins upon temperature change. The N17
(orange) and polyproline (blue) regions are depicted as alpha- and PPII helices, respectively, while the
polyglutamine region is illustrated as a domain which can adopt temperature- and polyQ-dependent
conformations (red). The epitopes of the 2B7 and MW1 antibodies are graphically illustrated. A. The N-
terminal region of a HTT protein bearing a wild type (e.g. Q16) polyQ expansion can adopt variable degrees of
structure in the polyQ region, with alpha-helical content being higher at lower temperatures. At higher
temperatures, alpha-helical content is lower and results in a 1: #1 ratio of 2B7Tb-MW1D2 bound to HTT. At
lower temperatures, this ratio becomes 1: >1 due to increased alpha-helical content in the polyglutamine
region, an increased number of epitopes for the acceptor-labeled MW1 antibody and, consequently, an
increase in TR-FRET signal. B. In the N-terminal region of a HTT protein bearing a mutant (e.g. Conformational Changes in Huntingtin Detected by Immunoassays and CD phosphorylation on specific N17 residues has a significant impact on HD-related
pathology in vivo and can alter N-terminal HTT flexibility [20, 26]. Although the
temperature- and polyQ-dependent conformational event reported here does not
appear to be influenced by such mutations, the availability of rapid, robust,
scalable assays sampling HTT conformational aspects in purified proteins of a
defined nature (fragment length, polyQ length, presence of PTMs; e.g. see [16]) as
well as relevant biological samples allows the interrogation of the role of
conformational aspects of HTT in HD pathology, for instance to determine
whether HTT conformational differences can explain variations in disease
progression in patients with identical polyQ expansions, as well as enable drug
discovery efforts aimed at modulating mutant HTT conformation through genetic
or pharmacological means. In conclusion, we have demonstrated using different
methodologies that the conformation of HTT proteins (studied either as
recombinant proteins or as proteins expressed in cell lysates) can, when supported
by orthogonal methods, be interrogated using appropriately controlled immu-
noassays, and that HTT protein structure may be sensitive to genetic (e.g., polyQ
length) as well as non-genetic (e.g., temperature) factors. Although further
structural studies are necessary, these data confirm previous studies suggesting
that the N-terminal region of HTT protein is flexible and becomes less so upon
polyQ expansion [26], and converge on the hypothesis that HTT protein
conformation may be amenable to modulation. Finally, heterogeneity in disease
manifestation amongst patients bearing the same polyglutamine expansion
supports the possible existence of disease modifiers which may include events that
affect HTT protein conformation, a hypothesis that can be tested with the assays
presented here. Note added during revision Following submission of this manuscript, a manuscript appeared illustrating
largely comparable findings to those presented in this present report [64]. For example, mimicking the presence or absence of 22 / 31 PLOS ONE | DOI:10.1371/journal.pone.0112262
December 2, 2014 Plasmid constructs cDNAs encoding N-terminal fragments (exon 1, N548) or full length human HTT
bearing different polyQ lengths were obtained as follows. cDNAs encoding human
HTT exon 1 fragments (Q16, Q39 or Q72) were synthesized (Genscript,
Piscataway, NJ) and subcloned into pCDNA3.1 either with or without a C-
terminal EGFP translational fusion. Mammalian expression constructs for cDNAs
encoding human HTT N548 fragments (Q16, Q39 or Q72) or full length HTT
(Q18, Q83) in pCDNA3.1 were reported previously [13]. 23 / 31 PLOS ONE | DOI:10.1371/journal.pone.0112262
December 2, 2014 Conformational Changes in Huntingtin Detected by Immunoassays and CD Cell lines HEK293T cells were cultured in DMEM, 10% FBS, 1% Penicillin and
Streptomycin (all from Gibco by Life Technologies) as per supplier’s instructions. Cells were routinely transfected with plasmid constructs using Lipofectamine2000
(Life Technologies) as per manufacturer’s instructions. Forty-eight hours later,
transfection cells were harvested and lysed in lysis buffer (PBS, 0.4% Triton X-
100) supplemented with 1X protease inhibitor cocktail (Roche). Protein expression and purification Recombinant human proteins containing the N-terminal sequence of HTT with
548 amino acids (N548) and a polyQ repeat of different length were generated as
previously described [13]. THRX-HTT exon 1-Q46 and Q25 were expressed by
the previously used pET32a-HD46Q [18] and pET32a-HD25Q [65] parent
constructs. Expression and subsequent lysis of cell pellets were performed as
previously described [18]. Then, similarly to the previously described protocol,
proteins were purified by centrifuging the lysates at 19,0006g for 30 min;
incubating with nickel-nitrilotriacetic acid-agarose beads (Qiagen) on a rocker at
4˚C for 1 hr; washing with several column volumes of 20 mM Tris-HCl, pH 8.0,
300 mM NaCl, 50 mM imidazole; and eluting with 20 mM Tris-HCl, pH 7.4,
300 mM NaCl, 250 mM imidazole. Following concentration of the proteins via
Amicon Ultra-15 10,000 MWCO centrifugal filters (Millipore), proteins were re-
diluted in 20 mM Tris, pH 7.4, then purified on a HiTrap Q XL column (GE
Healthcare) with an AKTA FPLC system (Amersham Pharmacia Biotech), taking
1-ml fractions and using a phosphate-buffered saline gradient from 20 mM
sodium phosphate, pH 7.4, 50 mM NaCl, to 20 mM sodium phosphate, pH 7.4,
1 M NaCl. For isolation of the THRX fusion partner, the fusion protein was
cleaved with EKmax (Life Technologies, Grand Island, NY) at room temperature
for 30 min. Then, 1 M urea was added to the resulting fragments, which were
subsequently applied to nickel-nitrilotriacetic acid-agarose beads and briefly
centrifuged in a tabletop Eppendorf 5415D centrifuge at 13,200 rpm. The
supernatant was diluted 1:10 in 20 mM, Tris pH 7.4, and further purified by FPLC
as described for THRX-HTT exon 1 above. Antibodies The MW1 antibody recognizing an epitope within the polyQ stretch of HTT was
developed by Paul Patterson [27] and obtained from the Developmental Studies
Hybridoma Bank developed under the auspices of the NICHD and maintained by
The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242. 2B7 antibody binds to the N17 region of HTT; its generation and characterization
were described previously [32]. 4C9 antibody was raised against the human PRR
region in exon1 of the HTT protein [66]. 3B5H10 antibody is specific for the
polyQ stretch of HTT and was generated and characterized by Steve Finkbeiner
[67, 68]. 2B7, 4C9 and MW1 antibodies were obtained from the CHDI 24 / 31 PLOS ONE | DOI:10.1371/journal.pone.0112262
December 2, 2014 Conformational Changes in Huntingtin Detected by Immunoassays and CD Foundation (New York, NY). Antibodies MAB2166 and 3B5H10 were obtained
from a commercial source (Millipore cat. n. MAB2166 and Sigma cat. n. P1874,
respectively). Custom terbium cryptate and D2-fluorophore antibody labeling was
performed by CisBio (Bagnols, France). Depending on the batch used, antibodies
were cross-linked to 5 to 7 mol of terbium cryptate or D2-fluorophore per mole
of antibody. Alexa-647 labeled 2B7 antibody was generated using the Alexa Fluor
647 Monoclonal Antibody Labeling Kit from Life Technologies (cat.n. A20186) as
per manufacturer’s instructions. Antibody against GAPDH was from Sigma
(cat.n. G9545). Western blot assay and Coomassie staining Total protein levels of lysates from HEK293T cells were quantified using the BCA
Protein Assay kit (Novagen) as per manufacturer’s protocol. 20 mg of total lysates
were denatured at 95˚C in 4X loading buffer (125 mM TrisHCl pH 6.8, 6% SDS,
4 M urea, 4 mM EDTA, 30% glycerol, 4% b-mercaptoethanol and Bromophenol
blue) and loaded on NuPAGE 4-12% Bis-Tris Gel (Life Technologies). Proteins
were transferred on PVDF membranes using the transfer apparatus from Life
Technologies or Trans-Blot Turbo Transfer System from Biorad following
manufacturer’s protocol. Membranes were stained for 30 minutes in TBS, 0.1%
Tween, 0.4% PFA, before blocking (1 hr) in TBS, 0.1% Tween, 5% non fat milk. Incubations with primary antibodies were performed overnight at 4˚C. Incubations with secondary anti-mouse or -rabbit horseradish peroxidase
conjugated antibodies were carried out for 1 hour at room temperature. Protein
bands were detected using chemiluminescence substrate (ECL from Life
Technologies). Similarly 0.25 mg or 2–5 mg of human recombinant HTT N548
proteins were loaded on NuPAGE 4–12% Bis-Tris Gel (Life Technologies) for
Western blotting analysis and for fast coomassie-G250 staining respectively. Total protein levels of lysates from HEK293T cells were quantified using the BCA
Protein Assay kit (Novagen) as per manufacturer’s protocol. 20 mg of total lysates
were denatured at 95˚C in 4X loading buffer (125 mM TrisHCl pH 6.8, 6% SDS,
4 M urea, 4 mM EDTA, 30% glycerol, 4% b-mercaptoethanol and Bromophenol
blue) and loaded on NuPAGE 4-12% Bis-Tris Gel (Life Technologies). Proteins
were transferred on PVDF membranes using the transfer apparatus from Life
Technologies or Trans-Blot Turbo Transfer System from Biorad following
manufacturer’s protocol. Membranes were stained for 30 minutes in TBS, 0.1%
Tween, 0.4% PFA, before blocking (1 hr) in TBS, 0.1% Tween, 5% non fat milk. Incubations with primary antibodies were performed overnight at 4˚C. Conformational Changes in Huntingtin Detected by Immunoassays and CD replicates and the bars represent the standard deviations among these replicates. The dilution points of each sample were fitted in a 4 parameters function that
describes the curves. In each experiment, in order to obtain a unique value
describing the temperature- and polyQ-dependent variation in TR-FRET signal,
the ratio between the maximum fluorescence value of the fitted curve at 4˚C and
the maximum fluorescence value of the fitted curve at room temperature was
calculated. The columns in the presented graphs correspond to the averages of
such ratios obtained in different TR-FRET experiments. The bars represent the
standard deviations among values obtained in different experiments. The ratios
were also expressed as relative fold differences with respect to ratios obtained for
wild type HTT protein (Q16). Significance was calculated using the one-way
ANOVA test and Bonferroni’s multiple comparison post-test (*5p,0.05;
**5p,0.01; ***5p,0.005; ****5p,0.001). TR-FRET assays TR-FRET assays were performed as described previously [32]. In brief, 5 ml of
each sample was transferred to a low volume 384 well plate (Greiner) in serial
dilutions starting from a defined concentration (1–4 mg/ml for total lysates and
2 ng/ml for recombinant proteins). 1 ml of antibody cocktail was then added. Soluble HTT was measured with 2B7-Tb/MW1-D2, 2B7-Tb/3B5H10-D2, 2B7-Tb/
4C9-D2 using 1 ng/ml of 2B7-Tb and 10 ng/ml of D2 labeled antibody. TR-FRET
measurements were routinely performed following incubation for 1 hour at room
temperature and following incubation for 1 hour or overnight at 4˚C using an
EnVision Reader (Perkin Elmer) following excitation at 320 nm (time delay
100 msec, window 400 msec, 100 flashes/well). Values were collected as the
background subtracted ratio between fluorescence emission at 665 nm and
615 nm where the background signal corresponds to the ratio (665/615) measured
for the antibodies in lysis buffer. The points in the graphs correspond to the
averages of the background subtracted fluorescence ratio relative to the sample 25 / 31 PLOS ONE | DOI:10.1371/journal.pone.0112262
December 2, 2014 Conformational Changes in Huntingtin Detected by Immunoassays and CD hC~2220{53T
ð2aÞ hC~2220{53T ð2aÞ and hH~ {44,000z250T
ð
Þ 13=Nr
ð
Þ
ð2bÞ ð2bÞ given that T is the temperature in ˚C and Nr is the number of residues. For
computation using DICHROWEB, which requires input spectral data with the
lowest wavelength point at 190 nm or lower, spectral data sets collected for the
Q25 and Q46 constructs in salt-free buffer at 210˚C and 37˚C were entered and
analyzed with the SELCON3 and CONTIN analysis programmes, using the
reference set SP175 [37, 38]. Programs were chosen to provide best fit of the data. Values resulting from CONTIN analysis are those of the closest matching solution
with all proteins. Author Contributions Conceived and designed the experiments: AC AW RL VF. Performed the
experiments: VF MV CC LA LP MD RB NCK JMI. Analyzed the data: VF AC RB
CC AP RK JMI NCK RL AW. Contributed reagents/materials/analysis tools: RP
HP RCS DM. Wrote the paper: AC VF RL NCK. Conceived and designed the experiments: AC AW RL VF. Performed the
experiments: VF MV CC LA LP MD RB NCK JMI. Analyzed the data: VF AC RB
CC AP RK JMI NCK RL AW. Contributed reagents/materials/analysis tools: RP
HP RCS DM. Wrote the paper: AC VF RL NCK. Acknowledgments We gratefully acknowlegde Prof. Paul Patterson for the MW1 antibody. CD study CD was performed using a Jasco 815 spectropolarimeter (Jasco Inc., Easton, MD). Temperature was regulated by a Jasco PFD-425S Peltier type FDCD attachment
connected to a PolyScience recirculator (PolyScience, Niles, IL). For spectra at
each temperature, measurements were taken every 1 nm from 200 to 260 nm,
scanning at 50 nm/min and an averaging time of 1 sec. For spectra spanning from
190 to 260 nm, protein was eluted into 20 mM sodium phosphate, pH 7.4, no salt,
via a PD-10 column (GE Healthcare) and CD measurements taken with the
parameters denoted above. Ten scans, or twelve scans for 190-to-260-nm
readings, were averaged for each sample spectrum; background spectra were
obtained by averaging twenty scans and the appropriate ones were subtracted
from the respective sample spectra. Spectra were smoothed by the Savitsky-Golay
algorithm. Single-wavelength readings at 222 nm were obtained at 210˚C, 0˚C,
4˚C, 10˚C, 20˚C, 30˚C, and 37˚C. In each case, ellipticity was measured every 1 sec
for 300 sec; the 301 readings for each sample at each temperature were averaged. The number of amino acids in each htt exon 1 sample to experience a change in
helicity from at 210˚C to 37˚C was estimated using a previously developed helix-
coil transition model, which gives the change in fraction of helicity (DfH) by the
following equation [36, 69]: DfH~fH,{100C{fH,370C where fH at each temperature is given as follows:
fH~ h222 hC
ð
Þ= hH hC
ð
Þ where fH at each temperature is given as follows: fH~ h222 hC
ð
Þ= hH hC
ð
Þ where h222 describes the helix coil transition in exon-1, obtained from the
difference between the observed MRE at 222 nm of the fusion protein at the given
temperature and the product of the MRE at 222 nm of Trx alone at the same
temperature and the fraction of residues comprised by Trx in that particular
construct, and 26 / 31 PLOS ONE | DOI:10.1371/journal.pone.0112262
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using site-directed spin labeling. J Biol Chem 279: 48420–48425. 10.1074/jbc.M406853200 [doi];
M406853200 [pii]. 70. Lennon BW, Williams CH Jr, Ludwig ML (2000) Twists in catalysis: alternating conformations of
Escherichia coli thioredoxin reductase. Science 289: 1190–11948731 [pii]. 31 / 31
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Vol. 34 No. 2 September 2022, pp. 345-355
Le Recours Aux Structures De Microfinance Dans Les Stratégies
De Résilience Aux Effets De La Pandémie Du Covid-19 Chez
Les Commerçantes De Produits Vivriers Au Marché Cocovico
ANDOH Amognima Armelle Tania and TEBAGLO Guéablehon Débora
Departmant of sociology, UNIVERSITE FELIX HOUPHOUET BOIGNY (COTE D'IVOIRE)
Résumé – Cette étude analyse les logiques sociales rattachées au non recours des services de la microfinance « Sirius Finance » par les
commerçantes du vivrier comme stratégie de résilience aux effets négatifs du covid-19. En effet, les commerçantes du marché Cocovico
n’adoptent pas les services d’une microfinance dénommée malgré les effets économiques de la pandémie sur leur commerce. Celles-ci
développent des stratégies pour contourner les services de cette structure en dépit des avantages financiers qu’elle offre. Sirius Finance
par les commerçantes. Fondée essentiellement sur une approche qualitative, l’étude montre que la non adoption se justifie par un
ensemble de systèmes de représentations associées aux propriétés et au fonctionnement de la microfinance. Comme stratégie de
résilience, les commerçantes ont recours aux associations locales et aides traditionnelles. De plus, la reconversion professionnelle, la
diversification des produits en vente sont mobilisées comme stratégies. Pour finir, l’étude a révélé que la non adoption des services de la
microfinance est aussi associée à des enjeux d’acquisition de l’assurance en soi et de la dignité.
Mots Clés – Stratégie De Résilience, Covid-19, Logiques Sociales, Commerçante Du Vivrier, Abidjan
I. INTRODUCTION
Le coronavirus (COVID19) est une maladie infectieuse due au virus SARS-CoV-2. La plupart des personnes infectées par le
virus présentent une maladie respiratoire d’intensité légère modérée et se rétablissent sans avoir besoin d’un traitement particulier.
N’importe qui, à n’importe quel âge, peut contracter la COVID-19 et tomber gravement malade ou en mourir. (OMS, 2020) selon
l’OMS.
Cette pandémie qui voit la moitié de la population mondiale confinée, a impacté l'économie mondiale en générale et celle de la
Côte d'Ivoire en particulier.
La situation sanitaire mondiale a fortement affecté les ménages et les entreprises ivoiriennes. Par conséquent, la croissance
économique devrait ralentir pour s’établir à près de 1,8 % en 2020 contre les 7 % attendus (Banque mondiale, 2020). Se basant
sur les résultats d’une enquête auprès de 800 ménages réalisée en avril 2020, le rapport souligne que 71 % des ménages ont
déclaré avoir subi une baisse de revenus et ne savent pas comment faire face à leurs dépenses courantes de base. Seulement
trois ménages sur dix ont suffisamment d’économies pour payer les charges régulières comme le loyer, les factures d’eau et
d’électricité et la nourriture. Les données de l’enquête montrent clairement que l’impact a été plus important et étendu qu’anticipé
et a aggravé les conditions de vie des ménages déjà précaires. À court terme, cet écart de revenu sera difficile à compenser pour
de nombreux ménages. Le programme de soutien du gouvernement est donc essentiel pour atténuer le choc. L’épidémie de
COVID-19 a eu plusieurs répercussions directes sur les entreprises : réduction du temps de travail, baisse des ventes et des
revenus, voire cessation totale ou partielle d’activités. Dans l’ensemble, 37,7 % des entreprises ont dû cesser leurs activités (dont
2,4 % définitivement et 35,3 % temporairement.
Corresponding Author: ANDOH Amognima Armelle Tania
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Ces conséquences socioéconomiques et les chiffres qui illustrent l’ampleur de la pandémie en Côte d’Ivoire, justifient le fait que
ce pays fasse partie des pays faisant objet d’étude pour la recherche sur les impacts des politiques publiques liées à la pandémie
sur les populations de nos pays. En raison de la progression rapide de ce fléau, à l’instar de la plupart des pays affectés, le
gouvernement ivoirien a pris des mesures de prévention et des mesures sanitaires pour ralentir, voire éviter la propagation du
coronavirus. Il s’agit, entre autres du lavage fréquent des mains à l’eau et au savon, la distanciation sociale, le confinement, la
fermeture des frontières, la fermeture des espaces publics (restaurants, bars, églises…), l’instauration du couvre-feu et les
enseignements scolaires à distance. Ces différentes décisions affectent l’économie du pays en ce sens que des activités
économiques étaient suspendues et des commerces étaient fermés privant ainsi des populations de revenus.
Ces mesures de prévention ont impacté la majorité des secteurs, en général mais celui du commerce du vivriers en particulier en
ce sens que ces secteurs d'activités de l'économie informelle ont été affectés par un manque d’approvisionnement en raison de
l’isolement de la ville d’Abidjan. En effet, les commerçantes de viviers ont été confrontées à des difficultés d’approvisionnement
à cause de l’isolement de d’Abidjan. Il s’agit entre autres du manque de marchandises, de l’augmentation des coûts de production.
En 2020, ces difficultés ont occasionné l’arrêt momentané du commerce de vivriers chez certaines commerçantes. Ces différents
impacts sont aussi observables dans les activités économiques des commerçantes du marché de Cocovico. Cependant, il existe
dans ce marché, des commerçantes qui utilisent des stratégies adaptatives à la situation afin de pallier aux difficultés qu’elles
rencontrent. Ces stratégies sont soit des tontines, soit des prêts entre commerçantes. Ce sont des mesures alternatives aux services
de la microfinance Sirius Finance, qui propose ses prestations à ces commerçantes en matière d’aide. En effet, cette microfinance
s’est installée dans la période d’apparition du coronavirus en Côte-d’Ivoire mars 2020 en face du marché Cocovico et propose aux
commerçants et commerçantes des services qui leur permettront de relancer leur commerce ou d’agrandir leur commerce afin de
maximiser leur revenu.
Des études sur les conséquences économiques de la Covid-19 ont été réalisées. Zahonogo et Ahouré (2021) relèvent que
l’évaluation des conséquences économiques et financières du Covid-19 sur les économies dans le monde est cependant, pour
l’instant rendue difficile par l’incertitude sur : (i) la durée de la crise, (ii) la vitesse de propagation du Covid-19 et (iii) l’efficacité
de la réponse que les pays y apporteront, aussi bien de manière individuelle que concertée. Toutefois, les premières études
démontrent que cette urgence sanitaire de portée mondiale dégradera les perspectives macroéconomiques de tous les pays en 2020
et au-delà.
Par ailleurs, le bien-être des populations se détériore surtout celui des populations vulnérables, notamment les femmes, les jeunes
et les acteurs du secteur informel. Les impacts de la covid-19 sont globalement négatifs d’après les résultats du rapport de
l’Institut National des Statistiques sur la covid-19 (2020) en Côte d’Ivoire. (CAPEC, 2021)
Aussi, le secteur informel a été affecté par la modification des horaires de travail en application du couvre-feu (20%) et
l’obligation de fermeture imposée par les décisions gouvernementales (18%). Un autre effet de la covid-19 sur les entreprises
informelles est la baisse du chiffre d’affaires de 79% et du bénéfice de 84% comparativement au mois de février 2020. Par
ailleurs, les rémunérations versées en mars 2020 sont de 62% inférieures à celles de février 2020. Les ménages, quant à eux,
observent aussi des changements concernant leurs conditions de vie du fait de la pandémie. En effet, ils ont augmenté durant la
période de la crise sanitaire leur consommation alimentaire (47%) et leur communication (26%). En outre, ils ont subi une baisse
de revenu de 72%. En plus de ces effets, le rapport de l’INS montre qu’à la date du 30 Avril, 27,5% des chefs de ménages étaient
au chômage technique (CAPEC, 2021).
Aussi, les différents secteurs d’activités de l’économie nationale en générale sont très touchés et les activités des commerçantes en
particulier et il est constaté une fragilisation des activités du commerce de vivriers due aux mesures de lutte contre la COVID-19.
Cette présente étude constitue une contribution sur les impacts des mesures prisent en raison du coronavirus sur les activés
économiques des commerçantes. Aussi, permettra-t-elle de comprendre les logiques sociales rattachées à la non utilisation des
services de la microfinance SIRIUS FINANCE par certaines femmes malgré les difficultés qu’elles rencontrent dans leurs
activités et de montrer les stratégies de résilience aux effets négatifs du Covid-19. De façon spécifique, il s’agit d’identifier les
représentations sociales servant à justifier la non adoption des services de la microfinance SIRUIS FINANCE par les
commerçantes de vivriers du marché de Cocovico, de décrire les stratégies de résiliences alternatives à la microfinance des
commerçantes aux conséquences de la Covid-19 et de présenter les enjeux de la non adoption des services de la microfinance.
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Les travaux sur le recours à des structures de microfinance par des commerçants lors d’une crise sanitaire n’existent pas en tant
que tels. Par contre, des études sur des représentations sur les microfinances, sur les stratégies de résilience des commerçantes ont
été menées.
Les auteurs tels que Wamba et Takoudjou (2014) dans leur analyse sur les MPE et les instituts de la microfinance stipulent que, le
caractère très sélectif des conditions d’accès aux services financiers des IMF est à l’origine du faible niveau d’utilisation des
services de ces institutions par les micro-entrepreneurs. D’où les normes qui encadrent le processus d’acquisition de crédit
financier par les microfinances, justifient les différents comportements des individus en vers cette structure financière.
De même, pour Tche (2009) ; Godquin ( 2004), c’est une suite de malentendus issus des chocs ou évènements inattendus
affectant l’emprunteur ou l’inefficacité du fonctionnement de l’IMF qui pose un véritable problème d’asymétrie d’information.
Cette asymétrie informationnelle ne permet pas un mariage parfait entre les MPE demandeurs de crédit et les IMF offreurs de
crédit en ce sens que la demande n’est pas toujours égale à l’offre. Les raisons de ces malentendus sont divergentes selon le point
de vue des offreurs (IMF) et des demandeurs (MPE). Le problème d’asymétrie information fait référence aux informations
imparfaites et d’une inégalité qui peut être la cause d’une posture de méfiance et d’adhésion des emprunteurs à la microfinance.
Ensuite, selon Camileri (2005) ; Wampfler (2000) ; Aryeetey (1988), les MPE, dans le souci de leur développement, ont besoin en
permanence des services financiers des IMF soit pour constituer le fonds de roulement (achats des matières premières, des
marchandises…), soit pour acheter les équipements ou des pièces de rechange. Malgré leur besoin accru, très peu de microentrepreneurs demandent les services financiers des IMF au profit de micro financement purement informel (tontines, usuriers et
des prêts familiaux). Ceci étant dû au caractère sélectif des conditions d’accès aux services financiers des IMF et de l’offre
souvent mal adaptée. La raison est que certaines MPE estiment que la plupart des IMF octroie des prêts sur le court terme, d’un
montant faible et assortis de taux d’intérêt relativement élevés qui servent plus à financer des fonds de roulement que du matériel
d’équipement. Cependant, les MPE ne trouvent pas la nécessité de se tourner vers les microfinances qui dont prestations
l’obtention de prêts ne sont pas favorables aux IMF. Cette situation traduit les raisons qui légitiment le recours des individus aux
moyens de financement informel que ceux de la microfinance.
Enfin, Fiebig (2001) stipule que les risques auxquels doivent faire face les IMF quant au financement de la MPE peuvent être
classés en quatre catégories à savoir : les risques liés au crédit (problème de remboursement), les risques de liquidité et de taux
d’intérêt (volume de crédit sollicités en déséquilibre avec le volume d’épargne mobilisé), les risques de gestion et les risques de
gouvernance. L’ensemble de ces risques explique le fait que les IMF soient prudentes à l’égard du financement de certaines MPE.
Les analyses de ces auteurs relèvent une des caractéristiques qui met en lumière, les raisons de méfiance des individus vers la
microfinance.
Aussi, d’autres travaux concernent les stratégies de résilience des commerçantes afin de surmonter et s’adapter aux différents
bouleversements dans leur activité.
Les chocs de santé et leurs coûts associés ont un impact à la fois à court et à long terme sur le bien-être des ménages. A court
terme, les ménages confrontés à des chocs de santé sont obligés de se substituer les dépenses de consommation et de production
pour les soins de santé. A long terme, les flux nets d'investissement dans les activités ont tendance à diminuer. Les chocs de santé
conduisent les ménages à la pauvreté ou les rendre encore plus pauvres. (Somi et al., 2009)
Aussi, Bonfrer et Gustafsson-Wright(2017) analysent les stratégies d’adaptation aux chocs de santé par les ménages, la réduction
de la consommation (alimentaire), la vente d’actifs ou de bétail, la souscription de prêts formels et informels, la diversification du
travail d’activités et de substitution du travail au sein des ménages comme stratégies d’adaptation des ménages. En effet, les
ménages en raison des différents chocs modifiaient leurs différentes pratiques. Au niveau de l’alimentation, il y a eu une réduction
de la consommation comme par exemple réduire le nombre de fois de consommation par jour afin de pouvoir s’adapter à la
situation.
Au niveau des ventes d’actifs et de bétails et la diversification du travail d’activités et de substitution du travail, l’auteur stipule
que les ménages se reconvertissent dans ces différentes pratiques afin de diversifier leur activité et avoir des rentabilités pour faire
face aux chocs. Ils entreprennent une réorganisation des taches au sein de leur ménage.
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Au niveau des prêt formels et informels, Zahonogo et Ahouré (2021) montrent que les ménages adoptent comme stratégie le
recours à l’emprunt vers les services de financements afin de se ressourcer au plan financier et vers les associations familiales afin
d’avoir un appui financier.
Dans la même ligne, Fomba et Ahouré (2021), évoquent l’aspect du recours au stockage des denrées alimentaires et des
médicaments, la réduction des dépenses courantes comme stratégie de résilience des populations et l’amélioration de la
comptabilité, l’amélioration de la qualité et de la visibilité des produits et l’utilisation des réseaux sociaux pour la prospection des
produits comme stratégies de résilience chez les micros moyens et petites entreprises. En effet, face aux effets négatifs de la
pandémie, certains ménages ont recouru par anticipation au stockage des denrées alimentaires afin de réduire les sorties et faire
face à une éventuelle pénurie.
Ensuite, concernant l’aspect de la réduction des dépenses courantes, les mêmes auteurs montrent que cette pratique adoptée par
d’autres ménages consiste à diminuer le nombre de repas par jour ou à vendre des biens afin de faire face aux effets négatifs de la
pandémie.
Enfin, la nouvelle façon de contacter les potentiels fournisseurs, de partager les expériences avec les collègues, mais surtout de
prospecter la clientèle est l’utilisation du numérique par les macros, moyennes et petites entreprises afin de moins ressentir les
effets du coronavirus.
Pour l’analyse des logiques sociales rattachées au non recours aux services de la microfinance SIRIUS FINANCE par les
commerçantes de vivrier comme stratégie de résilience aux effets négatifs du Covid-19, l’on a recours à la théorie de la résilience
de Bernard Michallet (2009). En effet, selon Michallet (2009), la résilience est un concept émanant de la réalité vécue et
permettant de mieux comprendre les divers facteurs, qui en interactions, aident l’individu, la famille ou la communauté, à vivre
malgré un sombre pronostic et à s’en sortir, parfois grandie. Aussi, la résilience est à la fois selon (Tisseron, 2007), la capacité de
se délier des effets d’un traumatisme et celle de se reconstruire après un choc.
En somme, l’adoption des stratégies alternatives à la microfinance par les femmes sont analysées comme résilience en tant que
capacité, processus et résultat. Il s’agit donc d’analyser les logiques sociales rattachées au non recours aux services de
microfinance par les commerçantes de vivriers comme stratégies de résilience aux effets négatifs du Covid-19, à partir de
l’approche de la résilience comme stratégie d’adaptation qui est le reflet des expériences individuelles et des pratiques sociales
des individus. Elle permet de comprendre et d’agir sur la structure en vue de sa transformation.
De façon spécifique, il s’agit d’ identifier les représentations sociales servant à justifier la non adoption des services de la
microfinance SIRUIS FINANCE par les commerçantes de vivriers du marché de Cocovico, de décrire les stratégies de résiliences
alternatives à la microfinance des commerçantes aux conséquences de la Covid-19 et d’ identifier les enjeux de la non adoption
des services de la microfinance.
II. MÉTHODOLOGIE
La présente étude se déroule à Abidjan plus précisément dans la commune Cocody Angré 8me tranche. Le marché de Cocovico
appartient administrativement à la commune de Cocody et est situé dans le sous-quartier d’Angré Djibi, ce marché est né d’une
coopérative des femmes exerçant dans le commerce des produits vivriers et a été créé en 1994. La commune de Cocody tirerait sa
dénomination de certaines déformations linguistiques telles que, "coli" qui signifierait petit bourg ou encore "cocoli" qui serait le
nom du génie protecteur des Tchaman, et premier occupants de cette cité (Union des villes et communes de la Côte d’Ivoire,
2017).
Le choix de la localité s’explique par le fait que le marché de Cocovico est l’un des marchés les plus touchés par le Covid-19 en
général et les commerçantes du vivriers de ce marché particulièrement victimes des impacts de la situation sanitaire.
Ainsi, la population choisie dans notre étude est constituée de commerçantes ayant été impactées par les effets des mesures
barrières dues au coronavirus, de la présidente du marché de Cocovico en raison des informations sur la gestion du marché et le
représentant du directeur de la microfinance SIRIUS FINANCE par rapport aux services offerts par leur structure.
L’échantillonnage par choix raisonné a été car le choix des enquêtés est basé sur le jugement du chercheur par rapport à leurs
caractères atypiques ou typique. Ainsi, l’échantillon de l’étude constitué de 27 individus, a été structuré en différentes catégories
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d’acteurs dont les commerçantes de vivriers du marché de Cocovico, la présidente du marché et le représentant du directeur de la
microfinance SIRIUS FINANCE.
Les données liées à l’étude ont été collectés à partir des techniques ressortant de la perspective qualitative. Ce sont : la recherche
documentaire, l’observation directe et l’entretien. Comme outils, les fiches de lecture, la grille d’observation, le guide d’entretien
et l’enregistrement des données.
1.
La recherche documentaire
La recherche documentaire a permis de collecter des informations sur la pandémie de la Covid-19 et particulièrement sur les
impacts socioéconomiques de cette pandémie sur les populations. Des articles en ligne, des ouvrages, des rapports d’étude ont été
explorés.
2. L’observation directe
Au cours de cette étude, les comportements des commerçantes, leurs réactions face à la structure de microfinance et la façon
dont elles vivent la situation de la crise sanitaire ont été observés à partir d’une grille d’observation au sein du marché du marché
Cocovico.
3. L’entretien semi-directif et le guide d’entretien
L’entretien semi-directif a été mobilisé. Cette approche a permis de mieux identifier les différents effets négatifs du Covid19 sur
les activités économiques des commerçantes de vivriers et de comprendre les raisons du non recours aux services de la
microfinance comme stratégie de résilience, ceci à l’aide d’un guide d’entretien.
Deux (2) guides d’entretien ont été construits dans notre recherche en rapport avec les différentes catégories d’acteurs. Ainsi, un
guide a été adressé aux commerçantes de vivriers et la présidente du marché et un autre guide adressé à la microfinance. Cette
technique a permis d’avoir des informations pertinentes liées à l’étude.
4. L’enregistrement et la retranscription des données
Les entretiens ont été enregistrés avec la permission des enquêtés. Puis, une retranscription des entretiens à consister à saisir
manuellement et le plus fidèlement les propos des enquêtés. Ces informations obtenues nous ont permis d’aborder la phase
d’analyse des données.
5. L’analyse de contenu, technique de traitement de données
L’analyse de contenu a permis d’analyser le discours des enquêtés à partir des thématiques du guide d’entretien constitué en trois
(3) points que sont :
L’identification des représentations sociales servant à justifier la non adoption des services de la microfinance SIRUIS
FINANCE par les commerçantes de vivriers du marché de cocovico.
La description des stratégies de résiliences alternatives des commerçantes aux conséquences de la Covid-19.
L’identification des enjeux de la non adoption des services de la microfinance
III. RÉSULTATS
1.
Les représentations sociales liées à la non adoption des services de la microfinance
Plusieurs représentations sont mises en évidence par les acteurs justifiant la non adoption des services de la microfinance. Ce sont
entre autres : la pression liée au dispositif de recouvrement de microfinance ; les conditions d’accès aux crédits dans les
microfinances ; les difficultés liées des termes techniques ; la rupture du secret autour de son état financier.
1.1 La pression liée au dispositif de recouvrement de microfinance comme forme de justification de la non adoption
La pression liée au dispositif de recouvrement de microfinance est l’une des représentations que les commerçantes ont de cette
structure bancaire. En effet, la microfinance est considérée comme étant un outil de pression pour les différents acteurs qui ne
l’adoptent pas comme stratégie de résilience face aux effets négatifs de la Covid-19. La microfinance est un instrument financier
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qui à partir de ses prestations, prête des services en termes de crédit aux différents individus qui sont encadrés par des normes de
remboursement.
Ce moyen d’aide financier est vu comme un endettement pour les commerçantes qui se voient obligées de fournir plus d’efforts,
s’abstenir de la liberté de faire leur commerce selon leur volonté en raison du crédit que ces acteurs doivent rembourser. Elles
doivent toujours se mettre au travail dans le but d’avoir des ressources afin de respecter leur engagement d’où une forme
d’esclavage.
Aussi, les agents de la microfinance, étant toujours présents sur les lieux de commerce, constitue un stress et représente un
manque de liberté pour les acteurs.
Les mécanismes de remboursement des prêts attribués par la microfinance, conduisent l’individu à penser qu’il ne vend plus pour
lui-même, pour s’épanouir et même pour répondre à ses besoins de liberté mais qu’il exerce son activité seulement pour pouvoir
être en règle avec la microfinance. C’est à dire rembourser le crédit emprunté.
« Eh ma fille, quand tu vas prendre l’argent avec ceux-là, ça devient esclavage parce que matin, midi, soir ils sont là. Tu ne dors
plus, tu es stressé, tu ne peux pas vendre en paix parce que tu dois rembourser crédit donc ça te met la pression même. Moi je
veux être libre. » (Extrait d’entretien avec une commerçante du marché de cocovico)
« Les gens de la microfinance là, même quand tu n’es pas là, ils viennent et puis quand tu les manques, ça devient
beaucoup parce que c’est par jour que tu rembourses. Moi je veux être libre, parce que je vais à 04h à Adjamé pour prendre
bagage et si je suis fatiguée, si je ne dois pas, je peux me reposer mais si je dois, je suis obligée de venir vendre pour pouvoir
rembourser. Je peux plus librement maintenant.» (Extrait d’entretien avec une commerçante au marché de cocovico)
1.2. Les conditions d’accès aux crédits dans les microfinances : un obstacle idéologique
Les commerçantes n’adoptant pas les services de la microfinance, comme stratégie de résilience en raison des conditions d’accès
aux crédits. En effet, selon les acteurs, il n’est pas facile d’avoir les prêts en ce sens que le temps de traitement des demandes de
crédits met beaucoup de temps dans l’attribution des prêts aux différents acteurs. Pour les commerçantes, c’est un processus lent,
qui leur faire perdre du temps pourtant, elles ont besoin de financement d’urgence afin de surmonter les effets néfastes du
coronavirus. Selon elles, le fonctionnement de la microfinance dans la distribution des prêts n’est pas favorable dans le contexte
d’accès d’urgence de crédit financier. D’où la raison de la non adoption des services de cette structure financière. Cette perception
s’illustre par les propos suivants :
« Ceux-là, pour donner crédit là c’est difficile dèh. Leur truc la est bien et moi-même j’étais dans une microfinance là mais à
cause de la difficulté pour avoir crédit là, ça m’a découragé et puis je suis quittée là-bas. Toujours quand tu t’en vas on dit on
traite ton dossier mais ça fini jamais » (Extrait d’entretien avec une commerçante du marché de cocovico).
« Leur affaire de microfinance là, quand tu demandes prêt,, ils vont te faire tourner seulement, ça dur trop avant de te donner
crédit, .il te font tourner pourtant, je dois régler mon problème, donc ça m’énerve. Donc pour éviter tout ça, pendant corona je
suis restée dans mon association, j’ai pu vite avoir l’argent pour acheter mes marchandises, hum je n’ai pas de problème. »
(Extrait d’entretien avec une commerçante du marché de cocovico).
1.3. Difficultés liées à l’interprétation des termes techniques, élément justificatif de non adoption
De l’analyse des données, il ressort que les difficultés liées à l’interprétation aux instruments techniques, pour les commerçantes,
justifie la non adoption des services de cette structure financière. En effet, les enquêtées affirment qu’elles n’arrivent pas à saisir
le sens réel des propos des agents de la microfinance, elles n’arrivent pas à comprendre le langage avec lequel les agents de la
microfinance leur présentent les différentes prestations. Cette situation fait prendre des décisions rapides aux commerçantes en
disant oui, c’est-à-dire faire croire qu’elles ont cerné les différents propos mais en réalité c’est un moyen de se débarrasser des
agents de la microfinance. Les commerçantes évoquent qu’elles n’ont pas assez d’information sur le fonctionnement de la
microfinance, sur le processus d’acquisition de prêts. Les agents de la microfinance ne les informent pas réellement sur le
fonctionnement de la microfinance. Ce qui limite les personnes dans la connaissance du mécanisme de fonctionnement de la
microfinance. Cette situation les empêche d’adopter les services de cette structure. Les verbatim suivants l’ illustrent :
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« Quand les agents de la microfinance là viennent, en tout cas je n’arrive pas à comprendre leur français qu’ils parlent là.
Ils viennent tu dis oui hein mais tu ne sais pas ce que ça veut dire même, donc pour te débarrasser d’eux tu dis oui. Vraiment leur
affaire-là est trop compliqué, moi ça me fatigue parce que je comprends pas ce qu’ils disent donc je peux pas aller là-bas »
(Extrait d’entretien avec une commerçante du marché de cocovico).
« Oui on dit qu’ils prêtent l’argent aux commerçants pour pouvoir s’en sortir surtout à cause de coronavirus là mais je ne
comprends pas bien le français qu’ils parlent là, c’est ce qu’ils ont dit quand ils sont venus. Quand ils viennent parler, je les
regarde et je dis oui pourtant j’ai rien compris. Et c’est ça même qui fait que je ne suis pas trop convaincu de ce qu’ils disent
pour prendre crédit là-bas » (Extrait d’entretien avec une commerçante du marché de cocovico)
1.4. la rupture du secret autour de son état financier comme une forme de justification de la non adoption
La rupture du secret autour de son état financier est la dernière représentation que les commerçantes ont de la microfinance. En
effet, les enquêtées affirment que le fait de se rendre dans une agence de microfinance, traduit un manque de moyen ou une
demande d’aide financière. Selon les commerçantes, elles peuvent se faire remarquer par d’autres commerçantes, lorsqu’elles se
rendent à la microfinance située en face du marché. Elles craignent de subir les moqueries. Ainsi, préfèrent-elles ne pas s’y
rendre :
« Je ne veux pas que on sache que j’ai des problèmes, je veux que mes problèmes restent dans la maison, faut pas quelqu’un va
savoir que j’ai problème c’est pour ça que je ne vais pas à la microfinance là. Là-bas là tu peux croiser ta voisine commerçante
comme toi ow, et puis elle va savoir que tu as problème. Donc je veux vraiment pas qu’on sache que j’ai problème » (Extrait
d’entretien avec une commerçante du marché de cocovico)
« Je sais que je ne vais jamais aller à la justice pour régler problème de crédits, et si tu vas prendre crédits et que tu croises ta
camarade là-bas, elle va dire que c’est avec l’argent de crédits que tu vends, ça la ça fait trop honte et puis ça te diminue même.
Pourtant tu sais chez nous les Gouro1 là c’est la grandeur hein, quand on sait que tu es femme capable là on te respecte bien.
Voilà donc si je vais pas j’ai la liberté et puis le respect » (Extrait d’entretien avec une commerçante du marché de cocovico)
En fin de compte, la non adoption des services de la microfinance par les commerçantes du marché de cocovico se justifie par les
propriétés et les conditions d’accès aux crédits.
2. Les stratégies de résilience des commerçantes
Les commerçantes concernées par l’étude, adoptent plusieurs stratégies afin de faire face aux conséquences de la crise sanitaire
dans leurs activités économiques. Celles-ci n’adoptant pas les services de la microfinance comme moyens de résilience aux effets
du coronavirus, ont recours à d’autres formes de stratégies. Ces stratégies de résiliences alternatives aux services de la
microfinance sont les suivantes : le recours aux associations locales et aide traditionnelle ; une reconversion d’activité
professionnelle ; une diversification du produit en vente.
2.1. Le recours aux associations locales et aides traditionnelles
Selon les enquêtes réalisées, le recours aux associations locales et aux types d’aides traditionnelles est l’une des stratégies de
résilience alternative qui justifient la non adoption des services de la microfinance par les commerçantes. Concernant les
associations locales (tontines), les commerçantes dans le marché, ont des associations entre femmes commerçantes dans
lesquelles elles se cotisent des montants levés par la gérante de cette association. En effet, ces types d’association constituent des
formes de tontine entre femmes du marché. Les commerçantes se cotisent et tour à tour, chacune prend son argent. Parlant du
recours aux types d’aides traditionnelles, selon les commerçantes, elles tournent vers des personnes au sein du marché et qui leur
prêtent de l’argent afin qu’elles puissent régler leur problème. Pour elles, c’est un processus dans lequel, elles se sentent à l’aise
car tout se passe entre commerçantes du marché.
Ainsi, ces différentes formes d’associations, leurs ont permis d’avoir facilement les moyens pour faire face aux effets négatifs de
la crise sanitaire sur leurs activités économiques :
1
Gouro : c’est un groupe ethnique de la Côte d’Ivoire
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« Moi je suis dans les associations au marché ici, genre parie quoi. On se donne tour à tour, genre si c’est à ton tour, vous
cotisez pour moi et après pour l’autre. C’est comme ça on a fait pour avoir un peu de moyen pour continuer nos commerces là
pendant temps de corona là » (Extrait d’entretien avec une commerçante du marché de cocovico)
« Les trucs de microfinance là, on dit c’est bon hein mais moi je suis habituée là mon gars-là. Si ça ne va pas il vient te
donner crédit. Pendant corona j’ai pris plus de 200000 avec lui. Dès que je l’ai appelé, c’est venu et puis j’ai pris pour me
débrouiller un peu un peu » (Extrait d’entretien avec une commerçante du marché de cocovico)
2.2 La reconversion professionnelle comme stratégie de résilience
L’analyse des données montre que la reconversion dans d’autres activités commerciales a été une stratégie de résilience. En effet,
selon les commerçantes, la situation du corona était difficile pour elles, pour pouvoir s’en sortir avec la vente des produits
vivriers. Ainsi, elles ont observé ce qui marchait le plus selon elles et elles ont commencé à s’intéresser à ces autres types de
commerce comme la vente des cache-nez et la vente de riz en fermant temporairement la vente de produits vivriers. Pour elles,
acteurs, la vente des cache-nez était le commerce idéal pour avoir des ressources financières qui leur permettra de relancer leurs
activités économiques (vente des produits vivriers). Et pour d’autres, c’est la vente de riz. La reconversion professionnelle pour
les commerçantes, a été une stratégie favorable afin d’amortir les effets négatifs de la crise sanitaire:
« Moi ma stratégie quand corona était venu et puis tout était chaud là, je suis rentrée un peu à la maison pour vendre cache-nez
parce que ça marchait bien et je m’en suis sortie. Si j’ai eu un peu je reviens, je vends mes marchandises. C’est ce que j’ai fait
pour m’en sortir, je n’ai pas pris crédit à la microfinance » (Propos d’une commerçante)
« Moi j’ai pu m’en sortir en vendant le riz et puis j’ai laissé les produits vivriers là un peu. C’est après je suis revenu mais ça fait
que j’ai eu l’argent pour acheter mes marchandises » (Extrait d’entretien avec une commerçante du marché de cocovico)
3. Diversification du produit en vente comme stratégie de résilience
Il ressort que la diversification du produit en vente a été l’une des stratégies de résilience des commerçantes pendant le Covid-19.
En effet, elles ont acheté d’autres produits différents des produits vivriers afin de constituer un plus sur ce qu’elles vendent déjà.
Ce système leur a permis d’attirer plus de clientèle à partir des nouveaux produits qui pour elles, étaient incontournables c’est-àdire nécessaire pour les clients.
Aussi, en agissant ainsi, ces commerçantes font remarquer leurs produits vivriers aux clients pour qu’ils puissent les acheter.
C’est ainsi qu’elles ont réussi à maintenir leur commerce :
« C’est ça je suis en train de te dire là. Quand je dis l’Eternel est bon là, parce que malgré c’était dur j’ai eu une technique pour
pouvoir maintenir mon commerce par la grâce de Dieu. Au fait là, j’ai agrandi mon commerce. Tu vois, avant c’était tomate,
piment, pomme de terre, oignon avec les petits petits trucs là non, mais produits pour avoir toujours même et puis les clients c’est
dur donc quand je vends par exemple un peu je prends l’argent et j’achète d’autres produits que les gens vont acheter. Donc, je
vends plusieurs choses en même temps, tu vois maintenant il y a huile, il y a riz donc c’est ça qui a fait que je me suis en sortie un
peu pendant corona » (commerçante du marché de cocovico)
Somme toute, il ressort de cette analyse, qu’il existe des stratégies de résilience alternatives à la microfinance que les
commerçantes du marché de Cocovico ont mobilisées pour faire face à la crise sanitaire. Ces stratégies se déclinent en différentes
formes de pratique que sont entre autres : le recours aux associations locales et aide traditionnelle, une reconversion d’activité
professionnelle et la diversification du produit en vente.
4. Les enjeux liés à la non adoption des services de la microfinance
La non adoption des services de la microfinance est reliée à deux (2) enjeux : l’acquisition de l’assurance en soi et l’acquisition de
la dignité.
4.1. L’acquisition de l’assurance en soi
L’un des enjeux liés à la non adoption des services de la microfinance est l’acquisition de l’assurance en soi. En effet, selon les
commerçantes, lorsqu’elles arrivent à vendre avec leurs propres moyens, et qu’elles arrivent à s’en sortir pendant les moments
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difficiles comme la crise sanitaire (Covid-19), cela représente une grande victoire pour elles. Elles savent qu’elles ont la capacité,
sans l’aide d’une microfinance, à surmonter les épreuves ce qui leur donne de l’assurance :
« J’ai plus d’assurance en moi, parce que je sais que je peux faire face aux problèmes avec mes moyens. Donc vraiment, ce que
gagne quand je ne prends pas crédit à la microfinance, me donne une confiance en moi-même et puis je marche sans regarder
derrière, je suis libre » (commerçante)
4.2. L’acquisition de la dignité comme enjeu
Le respect, la dignité et la paix constituent un enjeu lié à la non adoption des services de la microfinance comme stratégie de
résilience. En effet pour les commerçantes, le fait de ne pas se rendre dans une agence de microfinance, les préserve de la
rencontre d’autres commerçantes se retrouvant dans la même structure. Alors, personne ne saura qu’elles ont un quelconque
problème financier. Le fait d’être aperçu dans une microfinance signifie le manque de moyens, la dépendance, le rabaissement en
demandant du crédit. Par contre, ne pas solliciter une microfinance induit une liberté et impose un respect. Cette façon de faire
permet d’avoir le respect de tous et avoir de la dignité. Aussi, stipulent-elles, que le fait de ne pas avoir recours à une
microfinance, permet de vendre librement, sans stress et sans pression :
« je sais que je vais jamais aller à la justice pour régler problème de crédits, et si tu vas prendre crédits et que tu croises ta
camarade là-bas, elle va dit que c’est avec l’argent de crédits que tu vends, ça la ça fait trop honte et puis ça te diminue même.
Pourtant tu sais chez nous les Gouro là c’est la grandeur hein, quand on sait que tu es femme capable là on te respecte bien.
Voilà donc si je vais pas j’ai la liberté et puis le respect (toutes éclatent de rire) » (Extrait d’entretien avec une commerçante).
« Je gagne l’autonomie, la dignité, la paix parce que on ne va pas venir m’embrouiller et puis contrôler ce que je fais »
(commerçante)
En somme, la non adoption des services de la microfinance, est sous-tendue par deux enjeux, à savoir : l’acquisition de
l’assurance en soi ; le respect, dignité et la paix.
IV. DISCUSSION
La discussion s’articulera en deux (2) axes : les capacités d’adaptation des commerçantes comme fondement de la non adoption
des services de la microfinance et les représentations sociales servant à justifier la non adoption des services de la microfinance.
1.
Les capacités d’adaptation comme fondement de la non adoption des services de la microfinance
Dans cette étude, les capacités d’adaptation des commerçantes constituent un fondement de non adoption des services de
microfinance. En effet, ces capacités sont constituées d’un ensemble de stratégies qui sont entre autres : le recours aux
associations locales et aide traditionnelle, une reconversion d’activité professionnelle et la diversification du produit en vente. Il
faut noter que ces stratégies construites par les commerçantes émanent de leur capacité ou mobilisation à faire face aux différents
effets négatifs de la crise sanitaire sur leurs activités économiques. L’ensemble de ces pratiques sociales, organisationnelles et
économiques, favorise-la non adoption des services de la microfinance.
Les résultats obtenus et interprétés s’opposent aux résultats de Sosso (2013) sur les capacités d’adaptation des exploitants
agriculteurs commerçants afin de survenir à leurs difficultés économiques. En effet, il révèle que face aux besoins des
exploitations, les individus font recours aux institutions de microfinance comme stratégie d’amélioration et de développement de
leurs activités. Cependant, les crédits accordés concernent en grande partie de faibles montants et ne sont pas totalement
satisfaisants. Les investissements productifs qui constituent les investissements à moyen et long termes pouvant induire des
améliorations notables de la production agricole sont quasi insatisfaits. Ainsi, cela engendre le facteur qui traduit à un certain
niveau, la capacité des exploitants agricoles à fournir les garanties financières exigées par les institutions de microfinance. Aussi,
la capacité du producteur à investir, sa constance au niveau de la structure et la conformité aux règles des institutions de
microfinance permettent d’accéder à des crédits élevés pour la satisfaction de leurs besoins d’accès.
2- Les représentations sociales servant à justifier la non adoption des services de la microfinance
Il ressort que les logiques sociales rattachées à la non adoption des services de la microfinance Sirius finance par les
commerçantes du vivier est une stratégie de résilience aux effets négatifs du covid-19 ». Dans un premier temps, la pression liée
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au dispositif de recouvrement de microfinance justifie la non adoption des services de la microfinance. En effet, cette
représentation relève du caractère des normes qui encadre le processus de remboursement des crédits et même du type de
structure que la microfinance représente. A cela s’ajoute, les conditions d’accès aux crédits dans les microfinances. Selon les
commerçantes, l’acquisition des prêts renvoie à un processus d’une longue attente.
Aussi, les difficultés liées à l’interprétation aux termes techniques ne permettent pas aux acteurs, la mobilisation de s’engager à la
microfinance. D’où le fait que les différentes techniques et procédures d’approches aux commerçantes, ne sont pas adaptées au
champ social dans lequel les agents de la microfinance se trouvent. Cette situation crée une hésitation et un manque d’insertion
des commerçantes dans cette structure financière.
La dernière logique, est que les commerçantes considèrent la microfinance comme un espace social de rupture de son état
financier. Se retrouver dans une structure de microfinance, renvoie à présenter son état financier dans une posture de manque de
moyen et de faiblesse.
Les résultats de l’étude vont dans le même sens que ceux obtenus par Gandré (2012). Ses travaux montrent que la microfinance
est apparue à ses débuts comme la solution idéale face à l’exclusion bancaire et financière qui interdit aux plus pauvres toute
possibilité d’enrichissement durable, pourtant ses effets sont controversés en pratique. En effet, de façon générale, son étude
montre que les effets et pratiques de la microfinance sur le terrain commercial, ne sont pas favorables pour les emprunteurs. Cette
situation justifie leur refus d’insertion dans les différentes structures de microfinance.
V. CONCLUSION
L’objectif de cette étude était d’analyser les logiques sociales rattachées au non recours des services de la microfinance Sirius
Finance par les commerçantes du vivrier comme stratégie de résilience aux effets négatifs du Covid-19. Les commerçantes du
marché Cocovico ont développé différentes stratégies pour faire face aux effets de la Covid-19 sans avoir recours aux services
d’une microfinance. Pour comprendre cette réalité sociale, la théorie de la résilience de Bernard Michallét (2009) explique le non
recours aux structures de microfinance. Aussi se justifie par un ensemble de systèmes de représentations associées aux propriétés
et au fonctionnement de la microfinance. En effet, la non adoption des services de la microfinance se justifie par la pression du
dispositif de recouvrement de microfinance. Les conditions d’accès aux crédits dans la microfinance sont idéologiquement
construites comme des obstacles. Aussi, les difficultés liées à l’interprétation des instruments techniques sont considérées comme
des éléments justificatifs de la non adoption. La rupture du secret autour de l’état financier des commerçantes justifie également la
non adoption des services de la microfinance. Aussi, les commerçantes ont- elles développé des stratégies de résilience qui sont le
recours aux associations locales et aides traditionnelle, la reconversion professionnelle et la diversification des produits en vente.
En agissant de la sorte, elles acquièrent une assurance en soi et une dignité.
REFERENCES
[1] Boukaira S. et Daamouch M. (2021). COVID-19 : Etude d’impact sur l’activité des TPE et plan de relance, Revue Française
d’Economie et de Gestion, 2 (3), 267-291.
[2] CAPEC, (2021). Impacts des politiques publiques liées à la pandémie de la Covid-19 sur le secteur informel, les femmes et
les jeunes : cas du Burkina Faso, du Cameroun, de la cote d’ivoire et du Sénégal. Cellule d’Analyse des politiques
Economiques du CIRES.
[3] Laissus P. et Lallau B., (2013). Résilience spontanée, résilience suscitée. Les complexités de l’action humanitaire en zone
LRA (Est de la République Centrafricaine). Ethique et économie/Ethics and Economics, 10 (1), 95-118.
[4] Lallau B. (2008). Les agriculteurs africains entre la vulnérabilité et la résilience. Pour une approche par les capacités de la
gestion des risques. Revue française de socio-économie, 1(1) 177-198.
[5] Djoutsa W. L. & Takoujou N. A. (2014). Les MPE et Institutions de Microfinance en Zone Urbaine au Cameroun : un
mariage de confiance ou de méfiance ? Gestion 2000, 1 (31), 33-51.
[6] Michellet, B., (2009). Perspective historique, défis théoriques et enjeux cliniques. Frontière, 22 (1-2), 10-18.
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Commerçantes De Produits Vivriers Au Marché Cocovico
[7] Morgan F. de C., (2013). Les Associations Villageoises d’Epargne crédit : une approche adaptée aux ménages les plus
pauvres ? Solidarité Internationale, www.solidarités.org.
[8] Pam Z. et Ahouré A. A. E (2021), Impacts des politiques publiques liées à la pandémie de la Covid-19 sur le secteur
informel, les femmes et les jeunes : cas du Burkina Faso, du Cameroun, de la Côte d’Ivoire et du Sénégal. DESKTOP
REVIEW DU BURKINA-FASO, 56p.
[9] Sene I, (2021) « Impact des mesures de prévention de la pandémie de Covid-19 sur les travailleurs du secteur informel au
Sénégal » Revue Internationale du chercheur « Volume 2 : Numero1 » pp : 507-520
[10] Ulrichs M. (2016). Accroitre la résilience des populations grâce à la protection sociale, édition BRACED, 3, 16p.
[11] Yevesse D, Yaovi T. (2021). Effet de la COVID-19 sur la variation du revenu et la sécurité alimentaire des ménages au
Togo. Revue du développement en Afrique, 33 (1) 194-206.
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© 2014, International Textile and Apparel Association, Inc. ALL RIGHTS RESERVED
ITAA Proceedings, #71 – www.itaaonline.org 2014 Proceedings Charlotte, North Carolina The sleeves of the jacket are
made from
quilted leather
that I quilted
myself to
represent the
crisscrossing
of lines found
on the
bridges. An unexpected challenge was that rust particles
left in the dyed fabrics made needle penetration difficult,
especially on the thin lining fabric. The jacket is paired with high waisted black denim jeans
using flat patterning to create the pattern. The seaming in
the jeans reflects the structure of rusted bridges. 2014 Proceedings Charlotte, North Carolina Charlotte, North Carolina Charlotte, North Carolina Rusted
Sarah Corlett, Mount Mary University, USA
Keywords: Sustainability, Architecture, Design, Transformation
The goal of this ensemble was to transform an element that represents deterioration into an
element that is part of something new. Old rusted
bridges provided the inspiration for this ensemble. I
was drawn to the contrast between the structure and
stability with the slow corrosion of rusting. White
silk dupion was dyed by wrapping the fabric in and
around rusted objects such as chains, shovels, bolts
and washers to reflect the beautiful patterns and
colors created by oxidation of metal over the years. The resulting fabric takes a rust-colored print
reminiscent of tie dye. The pattern for the short
blazer was draped. The curved style lines of the
jacket reflect the large arches from my inspiration. The strong shoulders and silhouette are used to
give the look of strength and structure to contrast
the look of rusted deterioration. The silk jacket
lining was also dyed. The sleeves of the jacket are
made from
quilted leather
that I quilted
myself to
represent the
crisscrossing
of lines found
on the
bridges. An unexpected challenge was that rust particles
left in the dyed fabrics made needle penetration difficult,
especially on the thin lining fabric. The jacket is paired with high waisted black denim jeans
using flat patterning to create the pattern. The seaming in
h j
fl
h
f
d b id Rusted
Sarah Corlett, Mount Mary University, USA
Keywords: Sustainability, Architecture, Design, Transformation
The goal of this ensemble was to transform an element that represents deterioration into an
element that is part of something new. Old rusted
bridges provided the inspiration for this ensemble. I
was drawn to the contrast between the structure and
stability with the slow corrosion of rusting. White
silk dupion was dyed by wrapping the fabric in and
around rusted objects such as chains, shovels, bolts
and washers to reflect the beautiful patterns and
colors created by oxidation of metal over the years. The resulting fabric takes a rust-colored print
reminiscent of tie dye. The pattern for the short
blazer was draped. The curved style lines of the
jacket reflect the large arches from my inspiration. The strong shoulders and silhouette are used to
give the look of strength and structure to contrast
the look of rusted deterioration. The silk jacket
lining was also dyed. Rusted Rusted
Sarah Corlett, Mount Mary University, USA
Keywords: Sustainability, Architecture, Design, Transformation
The goal of this ensemble was to transform an element that represents deterioration into an
element that is part of something new. Old rusted
bridges provided the inspiration for this ensemble. I
was drawn to the contrast between the structure and
stability with the slow corrosion of rusting. White
silk dupion was dyed by wrapping the fabric in and
around rusted objects such as chains, shovels, bolts
and washers to reflect the beautiful patterns and Page 1 of 1
The goal of this ensemble was to transform an element that represents deterioration into an
element that is part of something new. Old rusted
bridges provided the inspiration for this ensemble. I
was drawn to the contrast between the structure and
stability with the slow corrosion of rusting. White
silk dupion was dyed by wrapping the fabric in and
around rusted objects such as chains, shovels, bolts
and washers to reflect the beautiful patterns and
colors created by oxidation of metal over the years. The resulting fabric takes a rust-colored print
reminiscent of tie dye. The pattern for the short
blazer was draped. The curved style lines of the
jacket reflect the large arches from my inspiration. The strong shoulders and silhouette are used to
give the look of strength and structure to contrast
the look of rusted deterioration. The silk jacket
lining was also dyed. The sleeves of the jacket are
made from
quilted leather
that I quilted
myself to
represent the
crisscrossing
of lines found
on the
bridges. An unexpected challenge was that rust particles
left in the dyed fabrics made needle penetration difficult,
especially on the thin lining fabric. The jacket is paired with high waisted black denim jeans
using flat patterning to create the pattern. The seaming in
the jeans reflects the structure of rusted bridges. A corseted leotard was also created using flat patterning to
be worn with the blazer. The black cotton lycra was treated
with a bleaching technique to further reflect the rusted
effect. Boning and padding are used to give the top shape. Page 1 of 1
014, International Textile and Apparel Association, Inc. Rusted ALL RIGHTS RESERVED
ITAA Proceedings, #71 – www.itaaonline.org
made from
quilted leather
that I quilted
myself to
represent the
crisscrossing
of lines found
on the
n unexpected challenge was that rust particles
dyed fabrics made needle penetration difficult,
on the thin lining fabric. is paired with high waisted black denim jeans
patterning to create the pattern. The seaming in
eflects the structure of rusted bridges. leotard was also created using flat patterning to
ith the blazer. The black cotton lycra was treated
ching technique to further reflect the rusted
ning and padding are used to give the top shape. The jacket is paired with high waisted black denim jeans
using flat patterning to create the pattern. The seaming in
the jeans reflects the structure of rusted bridges. The jacket is paired with high waisted black denim jeans
using flat patterning to create the pattern. The seaming in
the jeans reflects the structure of rusted bridges. A corseted leotard was also created using flat patterning to
be worn with the blazer. The black cotton lycra was treated
with a bleaching technique to further reflect the rusted
effect. Boning and padding are used to give the top shape. A corseted leotard was also created using flat patterning to
be worn with the blazer. The black cotton lycra was treated
with a bleaching technique to further reflect the rusted
effect. Boning and padding are used to give the top shape. Page 1 of 1 29
|
https://openalex.org/W3001243572
|
https://repozitorij.uni-lj.si/Dokument.php?id=151146&dn=
|
English
| null |
Microstructure and Texture Evolution with Relation to Mechanical Properties of Compared Symmetrically and Asymmetrically Cold Rolled Aluminum Alloy
|
Metals
| 2,020
|
cc-by
| 10,384
|
Received: 17 December 2019; Accepted: 17 January 2020; Published: 21 January 2020 Abstract: The impact of asymmetric cold rolling was quantitatively assessed for an industrial
aluminum alloy AA 5454. The asymmetric rolling resulted in lower rolling forces and higher strains
compared to conventional symmetric rolling. In order to demonstrate the positive effect on the
mechanical properties with asymmetric rolling, tensile tests, plastic-strain-ratio tests and hardness
measurements were conducted. The improvements to the microstructure and the texture were
observed with a light and scanning electron microscope; the latter making use of electron-backscatter
diffraction. The result of the asymmetric rolling was a much lower planar anisotropy and a more
homogeneous metal sheet with finer grains after annealing to the soft condition. The increased
isotropy of the deformed and annealed aluminum sheet is a product of the texture heterogeneity and
reduced volume fractions of separate texture components. Keywords: asymmetric rolling; aluminum alloy; planar anisotropy; mechanical properties; microstructures Metals 2020, 10, 156; doi:10.3390/met10020156 Microstructure and Texture Evolution with Relation
to Mechanical Properties of Compared Symmetrically
and Asymmetrically Cold Rolled Aluminum Alloy
Jakob Kraner 1,2,* Peter Fajfar 2 Heinz Palkowski 3
Goran Kugler 2 Matjaž Godec 1 and Jakob Kraner 1,2,*, Peter Fajfar 2, Heinz Palkowski 3
, Goran Kugler 2, Matjaž Godec 1 and
Irena Paulin 1 1
Institute of Metals and Technology, Lepi pot 11, SI-1000 Ljubljana, Slovenia; matjaz.godec@imt.si (M.G. irena.paulin@imt.si (I.P.) 1
Institute of Metals and Technology, Lepi pot 11, SI-1000 Ljubljana, Slovenia; matjaz.godec@imt.si (M.G
i
li @i
i (I P) 2
Department of Materials and Metallurgy, Faculty of Natural Sciences and Engineering,
University of Ljubljana, Aškerˇceva cesta 12, SI-1000 Ljubljana, Slovenia; peter.fajfar@ntf.uni-lj.si (P.F.);
goran.kugler@ntf.uni-lj.si (G.K.) g
g
j
3
Department of Metal Forming and Processing, Institute of Metallurgy, Faculty of Natural and Materials Science,
Clausthal University of Technology, Robert-Koch-Straße 42, DE-38678 Clausthal-Zellerfeld, Germany;
heinz.palkowski@tu-clausthal.de p
*
Correspondence: jakob.kraner@imt.si; Tel.: +386-1-4701-990 metals metals 1. Introduction Rolling is one of the most common metal-forming processes, frequently used for steels and
aluminum alloys [1–5]. With their lower density and higher corrosion resistance, aluminum alloys
will be used for increasing numbers of components in the automotive industry [6–8]. Of particular
interest is the AA 5xxx series, because its mechanical properties have the potential to be improved by
asymmetric rolling, endowing it with mechanical properties that are closer to those of steels [9–11]. Asymmetry in the rolling process can be introduced in different ways. Kinetics, geometry and
friction are the major parameters that can be influenced to create the asymmetry. This can involve rolling
with different roller diameters, rolling with the uneven use of lubricants, rolling with single-drive
roller and rolling with different rotation speeds of the rollers. The latter two are more appropriate
for rolling mills in the industry [12–17]. Rolling with different rotation speeds, where the so-called
“grabbing problems” are less significant than with a single-drive roller, have an impact on changes
to the workpiece’s thickness as a consequence of the uneven distribution of the longitudinal velocity. Higher contact and shear stresses as well as friction differences on the contact surfaces were introduced
while rolling with a speed difference between the upper and lower rollers of 5 m·s−1. A larger difference www.mdpi.com/journal/metals Metals 2020, 10, 156 2 of 14 between the velocity of the upper and lower work roller contributes to a greater reduction of the
rolling force [18–22]. At the same time, the speed difference results in the creation of a larger shear
deformation area, which appears as a consequence of the changed position of the neutral points in
the deformation zone [23,24]. On the other hand, a negative consequence of asymmetric rolling is the
bending of the workpiece, more often known as the “ski effect” [25–27]. The asymmetric rolling of aluminum was in most cases investigated for thin sheets with small
dimensions [28,29]. This means it would be interesting to study the impact of asymmetry in the rolling
process on thicker sheets. In addition to achieving higher strains and creating a more homogeneous
microstructure with smaller crystal grains, investigations of asymmetric rolling need to focus on
the texture components. It would be particularly useful to know which of the rolling, shear and
recrystallization texture components are the reason for the more effective heat treatments and the lower
anisotropy [30–35]. 2.1. Material and Geometry The experimental materials were industrially produced via vertical direct chill casting with
a subsequent homogenization of the slab, which was further hot rolled and coiled. A sheet with
a thickness of 6.7 mm was cut into plates with an entry size of approximately 510 mm × 230 mm. The chemical composition of the AA 5454 aluminum alloy plates (Table 1) was controlled in the
laboratory with X-ray fluorescence (XRF) and with inductively coupled plasma, optical emission
spectrometry (ICP-OES). Table 1. Chemical composition of AA 5454 aluminum alloy (wt.%). Mg
Mn
Fe
Si
Cr
Al
2.43
0.61
0.25
0.18
0.09
Bal. Table 1. Chemical composition of AA 5454 aluminum alloy (wt.%). Table 1. Chemical composition of AA 5454 aluminum alloy (wt.%). Mg
Mn
Fe
Si
Cr
Al
2.43
0.61
0.25
0.18
0.09
Bal. 1. Introduction For thinner sheets, the Erichsen test is normally used to assess the formability
properties. In the case of thicker sheets, the plastic-strain-ratio test can be performed as an alternative. The calculated values of the so-called Lankford factor were in some cases, in previous investigations,
much smaller for asymmetrically rolled samples than for symmetrically rolled samples, which means
the formability is improved [36–39]. In our investigation the influences of asymmetric rolling in comparison to symmetric rolling were
studied with respect to the technological, mechanical and metallographic perspectives. The interactions
between the measured rolling forces, the achieved strains, the tensile and yield strength, the hardness,
the indicators of planar anisotropy, the microstructures with an average size of grains and
crystallographic textures with volume fractions of different rolling, shear and recrystallization texture
components were investigated, with the aim to determine appropriate asymmetric rolling conditions
for industrial applications. 2.3. Mechanical Tests The mechanical tests and the metallographic analyses were carried out on deformed and
heat-treated samples. The heat treatment involved 400 ◦C for 1 h, which is common in the industry for
this alloy. The mechanical properties from the tensile test, in accordance with the ASTM E8 M standard,
and the plastic-strain-ratio test, in accordance with the ASTM E517 standard, were evaluated along
the rolling direction (0◦), the transverse direction (90◦) and the diagonal direction (45◦). The Brinell
hardness was measured on the surfaces on the top and the bottom of the plates and in the centre
position of the cross-section. 2.2. Rolling Parameters The plates were rolled using two different roll gaps, i.e., 4.0 mm and 3.1 mm. With the roll gap of
4.0 mm the imposed strains were around 33%, and with the roll gap of 3.1 mm, around 44%. The rolling
asymmetry results from the different rotation speeds of the two identical rollers of diameter 295 mm. In the laboratory experiments the rotation speed of the upper roller was kept constant at 10 rpm,
while the rotation speed of the lower roller was set at 10 rpm, 15 rpm and 20 rpm. The corresponding
asymmetry factors (ωlower/ωupper), as a consequence of the speed difference between the upper and
lower rollers, were equal to 1.0, 1.5 and 2.0. The asymmetry factor 1.0 represents symmetric rolling, while
the 1.5 and 2.0 factors of asymmetry represent two different types of asymmetric rolling. To recreate
industrially relevant conditions a special lubricant, SomentorTM 32, was used. Minimally and for each
rolling type same quantity of lubricant was added on both rollers, with intention to avoid the adhesion, 3 of 14 Metals 2020, 10, 156 which is frequent for rolled aluminum. With lubrication at symmetric and asymmetric rolling the
friction was reduced and consequently the factors of asymmetry were maintained. When lubrication
applied, the factors of asymmetry are expected to be lower than the design value. On the other hand,
the cold dry rolling is very rarely used and without lubrication the right response of material will not be
reached and comparable between symmetric and asymmetric rolling with industry-similar conditions. 3.1. Rolling Force and Ski Effect
3.1. Rolling Force and Ski Effect An undesirable ski effect only appeared with the asymmetric rolling. Stated is clearly shown
with the comparison of numerical simulations for symmetric (Figure 1a) and asymmetric (Figure 1b)
rolling. The same phenomena were observed also at laboratory rolling. Comparing simulations and
experiments the same functions, effects and phenomena were observed. Asymmetric rolling provided
thinner rolled plates at the same roll gap set and that made the rolling process faster than symmetric
rolling. With a higher factor of asymmetry exit thicknesses of plates were closer to the set roll gap,
what was noted at simulations and laboratory rolling as well. An undesirable ski effect only appeared with the asymmetric rolling. Stated is clearly shown
with the comparison of numerical simulations for symmetric (Figure 1a) and asymmetric (Figure 1b)
rolling. The same phenomena were observed also at laboratory rolling. Comparing simulations and
experiments the same functions, effects and phenomena were observed. Asymmetric rolling
provided thinner rolled plates at the same roll gap set and that made the rolling process faster than
symmetric rolling. With a higher factor of asymmetry exit thicknesses of plates were closer to the set
roll gap, what was noted at simulations and laboratory rolling as well. Fi
1 N
i
l i
l
i
f
i
lli
( )
d
i
lli
(b)
Figure 1. Numerical simulation of symmetric rolling (a) and asymmetric rolling (b). Figure 1. Numerical simulation of symmetric rolling (a) and asymmetric rolling (b). Figure 1. Numerical simulation of symmetric rolling (a) and asymmetric rolling (b). Figure 2 shows the influence of the rolling-speed asymmetry factor on the strain of the plates
and the rolling force for two set roll gaps of 4.0 mm and 3.1 mm. It is clear that with an increasing
asymmetry factor the rolling forces were decreasing with the increasing strain. Explanation for that
occurrence is in a much lower mean contact pressure at asymmetric rolling than in the case of
symmetric rolling. That phenomena functioned by the denature shape of the deformation zone,
which creates favorable circumstances for evolving longitudinal tensile stresses, whose action above
the workpiece during the rolling match the previous and subsequent applied stress. Stated
extensively reduces the mean pressure exerted on the contact surface of the rolls and as significance
also rolling forces [22]. 2.4. Microscopy Prior to the light microscopy (LM) and the electron-backscatter diffraction (EBSD), the samples
were mechanically grinded and polished. The last preparation step was polishing for 10 min with OPS
and ion etching 50 min. For the metallographic analyses and the determination of the average size of
the grains, in the top, bottom and centre positions of the specimen’s cross-section, a ZEISS AXIO Imager
M2m microscope (Carl Zeiss AG, Oberkochen, Germany) was used. A JEOL JSM-6500F field-emission
scanning electron microscope (JEOL, Tokio, Japan) equipped with an HKL Nordlys II EBSD camera
using Channel5 version 5.0.9.0 software (Oxford Instruments HKL, Hobro, Denmark) was used to
detect the texture components in the symmetrically and asymmetrically rolled samples. Twelve major
texture components in the face-centered cubic (fcc) material were searched for. Four rolling texture
components (C, S, B and D), three shear texture components (H, E and F) and five recrystallized
texture components (G, R, P, Q and Cube) were investigated and their volume fractions determined. The crystallographic planes and directions, as well as the angles in Euler space, are listed in Table 2. The instrument was operated at 15 kV and a 1.3 nA current for the EBSD analysis, with a tilting angle
of 70◦. Individual diffraction patterns were obtained together with mapping of the areas of interest. The detection was set to 5–7 bands, with 4 × 4 binning. For each sample, a map 1235 µm × 1005 µm, in
the cross-section centre and bottom surfaces, which was in contact with the faster roller, was measured
with a maximum step size of 5 µm. The volume fraction of the texture components was determined
with a 10◦deviation. Table 2. Major texture components in face-centered cubic (fcc) material [30]. Designation
Miller Indices {hkl} <uvw>
Euler Angles (φ1, Φ, φ2)
B
{011} <211>
(35◦, 45◦, 0◦)
S
{123} <634>
(59◦, 34◦, 65◦)
C
{112} <111>
(90◦, 35◦, 45◦)
D
{4411} <11118>
(90◦, 27◦, 45◦)
H
{001} <110>
(0◦, 0◦, 45◦)
E
{111} <110>
(60◦, 54.7◦, 45◦)
F
{111} <112>
(90◦, 54.7◦, 45◦)
Cube
{001} <100>
(0◦, 0◦, 0◦)
G
{110} <001>
(0◦, 45◦, 0◦)
R
{124} <211>
(53◦, 36◦, 60◦)
P
{011} <112>
(65◦,45◦, 0◦)
Q
{013} <231>
(58◦, 18◦, 0◦) Table 2. Major texture components in face-centered cubic (fcc) material [30]. Metals 2020, 10, 156
Metals 2020 10 156 4 of 14
4 of 14 2.5. Numerical Simulation
2.5. Numerical Simulation The
commercial
finite
element
package
ABAQUS
CAE
2018
(Dassault
Systémes,
Vélizy-Vallacoublay, France) was used with a model modified from an explicit three-dimensional
rolling model. For numerical simulations all initial dimensions of workpieces and rolling mill were
the same as at experimental laboratory rolling. The density (2690 kg·m−3), modulus of elasticity
(70.5 × 109 Pa), Poisson’s ratio (0.33) and stress-strain values of initial (hot rolled) material, with the
yield stress 151.0 MPa, were set as mechanical properties of specific aluminum alloy. The set time
period was 2.5 s and the used friction coefficient were 0.1, 0.15 or 0.2, what is in accordance with the
velocity of rollers. Rotation speed of rollers (0.1667, 0.2500 and 0.3333 radians·time−1) and roll gap set
(4.0 mm and 3.1 mm) were the same as at laboratory rolling, that way the factors of asymmetry and
strain were approximately equal. The commercial finite element package ABAQUS CAE 2018 (Dassault Systémes, Vélizy-
Vallacoublay, France) was used with a model modified from an explicit three-dimensional rolling
model. For numerical simulations all initial dimensions of workpieces and rolling mill were the same
as at experimental laboratory rolling. The density (2690 kg·m−3), modulus of elasticity (70.5 × 109 Pa),
Poisson’s ratio (0.33) and stress-strain values of initial (hot rolled) material, with the yield stress 151.0
MPa, were set as mechanical properties of specific aluminum alloy. The set time period was 2.5 s and
the used friction coefficient were 0.1, 0.15 or 0.2, what is in accordance with the velocity of rollers. Rotation speed of rollers (0.1667, 0.2500 and 0.3333 radians·time−1) and roll gap set (4.0 mm and 3.1
mm) were the same as at laboratory rolling, that way the factors of asymmetry and strain were
approximately equal. 3. Results and Discussion
3. Results and Discussion The results are presented as a comparison between symmetric and asymmetric rolling. The technological, mechanical and metallographic findings were examined with respect to the
strain and the samples’ condition, i.e., deformed or annealed. The results are presented as a comparison between symmetric and asymmetric rolling. The
technological, mechanical and metallographic findings were examined with respect to the strain and
the samples’ condition, i.e., deformed or annealed. 3.1. Rolling Force and Ski Effect
3.1. Rolling Force and Ski Effect Measured rolling forces and calculated strains according to the two different roll gaps
Figure 2. Measured rolling forces and calculated strains according to the two different roll gaps. g
g
g
g p
The ski effect only appeared on the asymmetrically rolled plates, and it was described using the
length and the angle. The length of the ski effect was measured from the bended edge to the place
where the plate became straight. The angle of the ski effect was defined as the highest angle of the
plates’ curvature. Ski effect on rolled plates is presented in Figure 3a. In Figure 3b the schematic
presentation of the ski effect length and angle for separate rolling type is shown. Plates with exit
dimensions from 684 mm to 785 mm had average values of the bended length from 57.0 to 63.6 mm
(±3 mm). The differences between the measured angles of the bending area were small. At the same
time, it should be noted that the factor of asymmetry had a higher impact on the formation of greater
ski effect than the strain. Larger angles of 20.3 ± 0.5° and 21 ± 0°, as well as longer lengths of 60.3 ± 0.6
mm and 63.6 ± 1 mm, were measured on the plates rolled with a factor of asymmetry equal to 2.0
Shorter lengths of the ski effect equal to 57.0 ± 3 mm and 57.6 ± 0.6 mm were measured at a factor of
asymmetry equal to 1.5. For this factor of asymmetry, the observed angles were between 18.3 ± 0.6°
and 19 ± 0°. Asymmetric rolling with a faster lower roller results in a down-orientated ski effect. This
can result in damage to the rolling table at the exit of the laboratory rolling mill or the transport rollers
on industrial rolling mills, if in such cases asymmetric rolling is even possible. The ski effect
represents a bent, useless area of rolled workpiece and must be cut off or made flat with mechanical
intervention. The length of the ski effect, depending on selected asymmetric rolling type, will be
significantly influenced by the vertical space between the deformation zone and the rolling table. The
smaller the height between the workpiece on the exit and rolling table the faster the bending process
The ski effect only appeared on the asymmetrically rolled plates, and it was described using the
length and the angle. 3.1. Rolling Force and Ski Effect
3.1. Rolling Force and Ski Effect In the case of the symmetric rolling for a set roll gap of 4.0 mm, the rolling
Figure 2 shows the influence of the rolling-speed asymmetry factor on the strain of the plates
and the rolling force for two set roll gaps of 4.0 mm and 3.1 mm. It is clear that with an increasing
asymmetry factor the rolling forces were decreasing with the increasing strain. Explanation for that
occurrence is in a much lower mean contact pressure at asymmetric rolling than in the case of symmetric
rolling. That phenomena functioned by the denature shape of the deformation zone, which creates
favorable circumstances for evolving longitudinal tensile stresses, whose action above the workpiece
during the rolling match the previous and subsequent applied stress. Stated extensively reduces the
mean pressure exerted on the contact surface of the rolls and as significance also rolling forces [22]. In the case of the symmetric rolling for a set roll gap of 4.0 mm, the rolling force was 1128 ± 3 kN
at a 30.6 ± 0.6% strain. Compared to asymmetric rolling for the same set roll gap, the strain was 5 of 14
5 of 14 Metals 2020, 10, 156
Metals 2020, 10, 15 higher and the rolling forces were lower. The obtained rolling force for a 1.5 factor of asymmetry
was 1099 ± 2 kN, and for a factor of asymmetry 2.0 it was 1083 ± 7 kN. The differences between the
strains were not significant. The achieved strain for the factor of asymmetry 1.5 was 31.4 ± 0.5% and
for the factor of asymmetry 2.0 it was 31.5 ± 0.2%. Somewhat smaller differences in the strains for
the asymmetric rolling were obtained for the set roll gap of 3.1 mm. In both cases the strain was up
to 44.9 ± 0.5%. In spite of the similar strain, a rolling force of 1341 ± 2 kN for the lower factor of
asymmetry and 1309 ± 3 kN for the higher factor of asymmetry were measured. Compared to the
symmetric rolling, a rolling force of 1377 ± 2 kN and a strain of 42.7 ± 0.1% were obtained. The increase
in the strain and the decrease of the rolling force with asymmetric rolling was the case for both set roll
gaps. For the higher set roll gap, the difference in the achieved strain between the symmetric and the
asymmetric rolling process was around 2.5%. 3.1. Rolling Force and Ski Effect
3.1. Rolling Force and Ski Effect At a lower set roll gap the difference was 5%. According
to the presented strains, the decrease of the rolling force in the case of a factor of asymmetry equal
to 1.5 was 2.5% and for a factor of asymmetry equal to 2.0 it was 5%. In general, the increases of the
achieved strains and the decreases of the rolling forces were not significant, but in the case of more
passes, with asymmetric rolling, the desired thickness would be achieved with a smaller number of
passes, which has an important economic impact for the rolling mill’s function and wear. between the strains were not significant. The achieved strain for the factor of asymmetry 1.5 was 31.4
± 0.5% and for the factor of asymmetry 2.0 it was 31.5 ± 0.2%. Somewhat smaller differences in th
strains for the asymmetric rolling were obtained for the set roll gap of 3.1 mm. In both cases the strain
was up to 44.9 ± 0.5%. In spite of the similar strain, a rolling force of 1341 ± 2 kN for the lower facto
of asymmetry and 1309 ± 3 kN for the higher factor of asymmetry were measured. Compared to th
symmetric rolling, a rolling force of 1377 ± 2 kN and a strain of 42.7 ± 0.1% were obtained. The increase
in the strain and the decrease of the rolling force with asymmetric rolling was the case for both se
roll gaps. For the higher set roll gap, the difference in the achieved strain between the symmetric and
the asymmetric rolling process was around 2.5%. At a lower set roll gap the difference was 5%
According to the presented strains, the decrease of the rolling force in the case of a factor o
asymmetry equal to 1.5 was 2.5% and for a factor of asymmetry equal to 2.0 it was 5%. In general, th
increases of the achieved strains and the decreases of the rolling forces were not significant, but in
the case of more passes, with asymmetric rolling, the desired thickness would be achieved with a
smaller number of passes, which has an important economic impact for the rolling mill’s function
and wear. Figure 2. Measured rolling forces and calculated strains according to the two different roll gaps
Figure 2. Measured rolling forces and calculated strains according to the two different roll gaps. Figure 2. 3.2. Tensile and Yield Strength
3.2. Tensile and Yield Strength With both set roll gaps the highest tensile-strength values were in the transverse direction. At
the same time the tensile-strength values in the rolling direction with both set roll gaps were the
lowest. For the 4.0 mm set roll gap the tensile-strength values were 279 ± 5 MPa for an asymmetry
factor of 1.0 and 280 ± 5 MPa for asymmetry factors of 1.5 and 2.0. At a 3.1 mm set roll gap the average
tensile strength values were higher. A factor of asymmetry of 1.0 brought us a tensile strength of 290
± 6 MPa, with a factor of asymmetry of 1.5 the average value was 293 ± 6 MPa and for a factor of
asymmetry 2.0 the tensile strength was 291 ± 7 MPa. Regarding the heat-treated samples for both set
roll gaps, the highest values of the tensile strength were in the rolling direction. At the same time the
lowest tensile-strength values after the heat treatment were observed in the diagonal direction. The
average tensile-strength values after the heat treatment were similar for both set roll gaps and all
three directions. Measured tensile strength was around 220 ± 3 MPa. In both cases of the roll-gap
settings the highest values of the yield strength were in the rolling direction. The values for the factor
of asymmetry equal to 2.0 were for both set roll gaps the highest and reached 235 ± 8 MPa with a set
roll gap of 4.0 mm and 253 ± 1 MPa with a set roll gap of 3.1 mm. All the other yield-strength values
for the deformed samples were for a higher set roll gap between 216 ± 6 MPa and 234 ± 5MPa and for
a lower set roll gap between 244 ± 7 MPa and 251 ± 8 MPa. The highest yield-strength values were for
a set roll gap of 3.1 mm with the heat-treated samples in the diagonal direction. All the values of the
yield strength for heat-treated samples and all rolling types were lower and relatively even for both
set roll gaps, with small deviations between 86 ± 0 MPa and 91 ± 1 MPa. The symmetrically and
asymmetrically achieved strengths before and after heat treatment were very similar, with just
smaller increases in both properties with asymmetric rolling. 3.2. Tensile and Yield Strength
3.2. Tensile and Yield Strength On the other hand, the tensile and yield
strengths with asymmetric rolling stayed in the range of created strengths with symmetric rolling
With both set roll gaps the highest tensile-strength values were in the transverse direction. At the
same time the tensile-strength values in the rolling direction with both set roll gaps were the lowest. For the 4.0 mm set roll gap the tensile-strength values were 279 ± 5 MPa for an asymmetry factor
of 1.0 and 280 ± 5 MPa for asymmetry factors of 1.5 and 2.0. At a 3.1 mm set roll gap the average
tensile strength values were higher. A factor of asymmetry of 1.0 brought us a tensile strength of
290 ± 6 MPa, with a factor of asymmetry of 1.5 the average value was 293 ± 6 MPa and for a factor
of asymmetry 2.0 the tensile strength was 291 ± 7 MPa. Regarding the heat-treated samples for both
set roll gaps, the highest values of the tensile strength were in the rolling direction. At the same time
the lowest tensile-strength values after the heat treatment were observed in the diagonal direction. The average tensile-strength values after the heat treatment were similar for both set roll gaps and
all three directions. Measured tensile strength was around 220 ± 3 MPa. In both cases of the roll-gap
settings the highest values of the yield strength were in the rolling direction. The values for the factor
of asymmetry equal to 2.0 were for both set roll gaps the highest and reached 235 ± 8 MPa with a set
roll gap of 4.0 mm and 253 ± 1 MPa with a set roll gap of 3.1 mm. All the other yield-strength values
for the deformed samples were for a higher set roll gap between 216 ± 6 MPa and 234 ± 5MPa and for
a lower set roll gap between 244 ± 7 MPa and 251 ± 8 MPa. The highest yield-strength values were
for a set roll gap of 3.1 mm with the heat-treated samples in the diagonal direction. All the values of
the yield strength for heat-treated samples and all rolling types were lower and relatively even for
both set roll gaps, with small deviations between 86 ± 0 MPa and 91 ± 1 MPa. 3.1. Rolling Force and Ski Effect
3.1. Rolling Force and Ski Effect On the other hand, it is necessary to emphasize that besides a larger
percentage of a bent workpiece, the plates were longer because higher strains were reached with
asymmetric rolling and the material will have better properties
will finish and the workpiece with a limited ski effect will slide forward on the rolling table. In the
cases of asymmetric rolling from 7 ± 1% to 9 ± 1% of the ski effect according to the whole rolled plate
were calculated. Comparing the symmetric and asymmetric plates, where, with the asymmetric
rolling, between 2% and 5% more useless material was produced. On the other hand, it is necessary
to emphasize that besides a larger percentage of a bent workpiece, the plates were longer because
higher strains were reached with asymmetric rolling and the material will have better properties Figure 3. Ski effect on rolled plates (a) and schematic presentation of ski effect with measured lengths
and angles (b). Figure 3. Ski effect on rolled plates (a) and schematic presentation of ski effect with measured lengths
and angles (b). Figure 3. Ski effect on rolled plates (a) and schematic presentation of ski effect with measured lengths
and angles (b). Figure 3. Ski effect on rolled plates (a) and schematic presentation of ski effect with measured lengths
and angles (b). 3.1. Rolling Force and Ski Effect
3.1. Rolling Force and Ski Effect The length of the ski effect was measured from the bended edge to the place
where the plate became straight. The angle of the ski effect was defined as the highest angle of the
plates’ curvature. Ski effect on rolled plates is presented in Figure 3a. In Figure 3b the schematic
presentation of the ski effect length and angle for separate rolling type is shown. Plates with exit
dimensions from 684 mm to 785 mm had average values of the bended length from 57.0 to 63.6 mm
(±3 mm). The differences between the measured angles of the bending area were small. At the same
time, it should be noted that the factor of asymmetry had a higher impact on the formation of greater ski
effect than the strain. Larger angles of 20.3 ± 0.5◦and 21 ± 0◦, as well as longer lengths of 60.3 ± 0.6 mm
and 63.6 ± 1 mm, were measured on the plates rolled with a factor of asymmetry equal to 2.0. Shorter
lengths of the ski effect equal to 57.0 ± 3 mm and 57.6 ± 0.6 mm were measured at a factor of asymmetry
equal to 1.5. For this factor of asymmetry, the observed angles were between 18.3 ± 0.6◦and 19 ± 0◦. Asymmetric rolling with a faster lower roller results in a down-orientated ski effect. This can result in
damage to the rolling table at the exit of the laboratory rolling mill or the transport rollers on industrial
rolling mills, if in such cases asymmetric rolling is even possible. The ski effect represents a bent, useless
area of rolled workpiece and must be cut offor made flat with mechanical intervention. The length of
the ski effect, depending on selected asymmetric rolling type, will be significantly influenced by the
vertical space between the deformation zone and the rolling table. The smaller the height between the
workpiece on the exit and rolling table, the faster the bending process will finish and the workpiece 6 of 14
6
f 14 Metals 2020, 10, 156 with a limited ski effect will slide forward on the rolling table. In the cases of asymmetric rolling from
7 ± 1% to 9 ± 1% of the ski effect according to the whole rolled plate were calculated. Comparing
the symmetric and asymmetric plates, where, with the asymmetric rolling, between 2% and 5% more
useless material was produced. 3.3. Anisotropy For thicker sheets, the Erichsen test for deep-drawing properties could not be performed. For that
reason the alternative plastic-strain-ratio test was conducted. The planar anisotropy indicator ∆r was
calculated from the Lankford factor r. The values of ∆r could be positive or negative, depending on
the relationship between the elongation and width increase of the tested sample. More important is
that the ∆r values were close to 0 [38]. For all the rolling types and for both set heights of the roll gap,
the ∆r values were negative (Table 3). The deformed sample’s ∆r values for both set roll gaps were, in
the case of asymmetric rolling, higher in the negative section than with symmetric rolling. Higher
negative values of ∆r with the asymmetric rolling also mean a higher planar anisotropy, because
the total isotropy of the material appears when ∆r is 0. Comparing the ∆r values of the deformed
and heat-treated samples there was a strong contrast. The ∆r values for the deformed samples were
decreasing with an increasing factor of asymmetry, and the values of ∆r for the heat-treated samples
were increasing with an increasing factor of asymmetry. After the heat treatment ∆r was −0.237 ± 0
for a factor of asymmetry equal to 1.0 at a 4.0 mm set roll gap. This was a more negative ∆r value
than for the same sample in the just-deformed condition. The same phenomenon was observed with
a factor of asymmetry equal to 1.5 and a larger set roll gap, where the ∆r after heat treatment was
−0.206 ± 0.01. For a factor of asymmetry equal to 2.0 ∆r was −0.169 ± 0.02, which was less negative
than the ∆r of the same, just-deformed sample. A more significant negative decrease in the values
was visible with a smaller set roll gap. Of particular note is that for factors of asymmetry equal to 1.5
and 2.0 the ∆r values were −0.052 ± 0 and −0.050 ± 0. This is a very good indicator of the very low
anisotropy for the asymmetric deformed samples after heat treatment, knowing that the ∆r after heat
treatment with the same set roll gap and factor of asymmetry equal to 1.0 was −0.112 ± 0.02. 3.2. Tensile and Yield Strength
3.2. Tensile and Yield Strength The symmetrically and
asymmetrically achieved strengths before and after heat treatment were very similar, with just smaller
increases in both properties with asymmetric rolling. On the other hand, the tensile and yield strengths
with asymmetric rolling stayed in the range of created strengths with symmetric rolling. Metals 2020, 10, 156 7 of 14 7 of 14 3.3. Anisotropy Regarding
the higher ∆r values of the asymmetrically rolled samples in the deformed condition, after the heat
treatment the asymmetrically rolled samples in all cases had a lower ∆r than the symmetrically rolled
samples. This phenomenon could be attributed to a more appropriate texture for the successful heat
treatment [29]. Table 3. Planar anisotropy indicator ∆r for deformed and heat-treated samples. Roll Gap Set
ωlower/ωupper
∆r
Deformed Condition
∆r
Heat-Treated Condition
4.0 mm
1.0
−0.145 ± 0.05
−0.237 ± 0
1.5
−0.197 ± 0.05
−0.206 ± 0.01
2.0
−0.276 ± 0.02
−0.169 ± 0.02
3.1 mm
1.0
−0.134 ± 0.01
−0.112 ± 0.02
1.5
−0.157 ± 0.02
−0.052 ± 0
2.0
−0.155 ± 0.01
−0.050 ± 0
Hardness Table 3. Planar anisotropy indicator ∆r for deformed and heat-treated samples. 3.5. Microstructure Comparing the microstructures of the deformed samples (Figure 4a–c) with the microstructures
of the heat-treated samples (Figure 4d–f), some differences were observed. Since higher strains were
achieved with the asymmetric rolling, the average size of the grains, as a consequence, was smaller. The centre band of the longitudinally deformed grains was for the asymmetrically rolled microstructure
smaller and it was decreased with a higher factor of asymmetry. Moreover, it is important that the
grains in the asymmetrically deformed samples are, in all three measured sections, more evenly
distributed than for the symmetrically rolled samples. The results for the higher and lower set roll gaps
are presented in Figure 5a,b. In the deformed samples the grain sizes are, for a set roll gap of 4.0 mm,
between 26.4 ± 0.7 µm and 17.7 ± 0.5 µm, and for a set roll gap of 3.1 mm, between 23.3 ± 0.3 µm and
17.5 ± 0.6 µm. The grains in the centre position of the cross-section are, for all three different factors of
asymmetry, larger than those on the top or bottom position. The difference between the grain size on
the top and bottom positions is very small, but at the same time the smaller grains were obtained at the
bottom position, which can be the impact of the higher velocity of the lower roller. This is valid for
the deformed samples as well as for heat-treated samples. After the heat treatment, there is no major
difference in the distribution of the microstructural components for all three sections. The grains in
the microstructure are very even, especially with the asymmetrically rolled samples. Some impacts
of the asymmetry remained after the treatment to the soft condition. For the heat-treated samples
the grain sizes were between 33.9 ± 1 µm and 25.3 ± 0.7 µm for the set roll gap of 4.0 mm and from
25.0 ± 0.5 µm to 19.3 ± 0.6 µm for the set roll gap of 3.1 mm at the top, centre and bottom positions of
the samples’ cross sections. The presented average grain sizes for the heat-treated samples are shown
in Figure 5c,d. A major advantage of the asymmetric rolling is the creation of more homogeneous
material, in our case, confirmed with the average size of the grains and the hardness throughout
the cross-section. 3.4. Hardness Hardness values for all rolling types and both conditions are presented in Table 4. The highest
hardness values were obtained in the centre for all the rolling types. The values for the deformed
condition with the larger set roll gap and factor of asymmetry equal to 1.0 were somehow higher
than in the cases with a factor of asymmetry equal to 1.5 and 2.0. What is more important is that the
difference between the surfaces hardness for the asymmetric types was smaller. The same phenomenon
was observed with deformed samples with a set roll gap of 3.1 mm. The differences in the surface’s
hardness decreased in the case of the set roll gap with a higher factor of asymmetry. All the measured
hardness values at the top, bottom and centre for heat-treated samples were more evenly spread than
with the deformed samples. The differences in the surface hardness were smaller for the heat-treated
samples than for the deformed samples. However, at the same time, it is clear that the hardness
differences were smaller after the heat treatment of the asymmetrically rolled samples than for the
symmetrically rolled samples. 8 of 14 Metals 2020, 10, 156 Table 4. Hardness for all rolling types in deformed and heat-treated condition. Roll gap Set (mm;
Factor of Asymmetry)
Measuring Position
Deformed
Conditio (HB)
Heat-Treated
Condition (HB)
4.0 (1.0)
Top
86 ± 0.5
57 ± 2
Centre
90 ± 2
56 ± 3
Bottom
83 ± 1
57 ± 2
4.0 (1.5)
Top
83 ± 0.5
57 ± 1
Centre
88 ± 1
57 ± 1
Bottom
82 ± 3
56 ± 1
4.0 (2.0)
Top
83 ± 1
57 ± 0.5
Centre
88 ± 2
56 ± 0.5
Bottom
80 ± 3
58 ± 1
3.1 (1.0)
Top
84 ± 1
56 ± 2
Centre
92 ± 0.5
58 ± 1
Bottom
85 ± 2
57 ± 0.5
3.1 (1.5)
Top
87 ± 2
58 ± 3
Centre
92 ± 1
56 ± 1
Bottom
86 ± 2
58 ± 3
3.1 (2.0)
Top
86 ± 1
56 ± 1
Centre
92 ± 1
56 ± 1
Bottom
86 ± 0.5
56 ± 0.5 Table 4. Hardness for all rolling types in deformed and heat-treated condition. 3.5. Microstructure More homogeneous material, i.e., the material with the more evenly distributed
properties throughout the cross-section, is better for further forming processes, heat treatment and
quality of the products [30,31]. 9 of 14
9
f 14 Metals 2020, 10, 156 Figure 4. Microstructure in samples’ cross-sections for symmetrically rolled 1.0 (a), asymmetrically
rolled 1.5 (b), asymmetrically rolled 2.0 (c), symmetrically rolled 1.0 and heat treated (d),
asymmetrically rolled 1. 5 and heat treated (e) and asymmetrically rolled 2.0 and heat treated (f). Figure 4. Microstructure in samples’ cross-sections for symmetrically rolled 1.0 (a), asymmetrically
rolled 1.5 (b), asymmetrically rolled 2.0 (c), symmetrically rolled 1.0 and heat treated (d), asymmetrically
rolled 1. 5 and heat treated (e) and asymmetrically rolled 2.0 and heat treated (f). Figure 4. Microstructure in samples’ cross-sections for symmetrically rolled 1.0 (a), asymmetrically
rolled 1.5 (b), asymmetrically rolled 2.0 (c), symmetrically rolled 1.0 and heat treated (d),
asymmetrically rolled 1. 5 and heat treated (e) and asymmetrically rolled 2.0 and heat treated (f). Figure 4. Microstructure in samples’ cross-sections for symmetrically rolled 1.0 (a), asymmetrically
rolled 1.5 (b), asymmetrically rolled 2.0 (c), symmetrically rolled 1.0 and heat treated (d),
asymmetrically rolled 1. 5 and heat treated (e) and asymmetrically rolled 2.0 and heat treated (f). Figure 4. Microstructure in samples’ cross-sections for symmetrically rolled 1.0 (a), asymmetrically
rolled 1.5 (b), asymmetrically rolled 2.0 (c), symmetrically rolled 1.0 and heat treated (d), asymmetrically
rolled 1. 5 and heat treated (e) and asymmetrically rolled 2.0 and heat treated (f). Figure 4. Microstructure in samples’ cross-sections for symmetrically rolled 1.0 (a), asymmetrically
rolled 1.5 (b), asymmetrically rolled 2.0 (c), symmetrically rolled 1.0 and heat treated (d),
asymmetrically rolled 1. 5 and heat treated (e) and asymmetrically rolled 2.0 and heat treated (f). Figure 5. Average grain size for deformed condition with 4.0 mm set roll gap (a), deformed condition
with 3.1 mm set roll gap (b), heat treated with 4.0 mm set roll gap (c) and heat treated with 3.1 mm
Figure 5. Average grain size for deformed condition with 4.0 mm set roll gap (a), deformed condition
with 3.1 mm set roll gap (b), heat treated with 4.0 mm set roll gap (c) and heat treated with 3.1 mm
ll
(d)
Figure 5. 3.6. Texture 3.6. Texture The texture components were detected with EBSD for the deformed and heat-treated samples. At the same position and for the same asymmetric rolling type, the deformed condition, with its
strongly pronounced texture pattern and higher intensity of the pole figure, and heat-treated condition,
with an even distribution of all the texture components and a very low intensity of the pole figure
were observed. The EBSD mapping confirms the refinement of the crystal grains with asymmetric
rolling, were with the higher factor of asymmetry the smaller grains were produced. The associated
pole figures in Figure 6a–c present a lower intensity and that way also the higher heterogeneity of
texture created with the asymmetric rolling. Observing the central position of the lower deformed
and heat-treated sample, the texture component E at a factor of asymmetry equal to 1.0 and a texture
component R at a factor of asymmetry equal to 2.0 stand out. All the other observed texture components
for all three factors of asymmetry were from 0.0 to 2.5 vol.%. These results are presented in Figure 7a. The same figure shows that the volume fraction of all the rolling texture components in the case of
asymmetric rolling is higher than for the symmetric rolling type. At the same time, a smaller difference
in the volume fractions was observed between the separate texture components in the asymmetric
rolling textures. The same phenomenon appears in Figure 7b, where the centre positions of the more
deformed samples are presented. In the sample that was deformed with a factor of asymmetry equal
to 1.0 and heat-treated higher volume fractions for all the rolling texture components were obtained in
comparison to the asymmetric rolling types with factors of asymmetry equal to 1.5 and 2.0. Moreover,
in the symmetrically rolled sample, the separate texture components were more obvious. This indicates
that the volume fractions of all the texture components with asymmetric rolling were more even than
for the symmetric rolling. In general, the volume fractions of all the observed texture components
were, for the asymmetrically rolled and heat-treated samples with a set roll gap of 3.1 mm, between 0.1
and 1.8 vol.%. 3.5. Microstructure Average grain size for deformed condition with 4.0 mm set roll gap (a), deformed condition
with 3.1 mm set roll gap (b), heat treated with 4.0 mm set roll gap (c) and heat treated with 3.1 mm set
roll gap (d). Figure 5. Average grain size for deformed condition with 4.0 mm set roll gap (a), deformed condition
with 3 1 mm set roll gap (b) heat treated with 4 0 mm set roll gap (c) and heat treated with 3 1 mm
Figure 5. Average grain size for deformed condition with 4.0 mm set roll gap (a), deformed condition
with 3.1 mm set roll gap (b), heat treated with 4.0 mm set roll gap (c) and heat treated with 3.1 mm
Figure 5. Average grain size for deformed condition with 4.0 mm set roll gap (a), deformed condition
with 3.1 mm set roll gap (b), heat treated with 4.0 mm set roll gap (c) and heat treated with 3.1 mm set
roll gap (d). Metals 2020, 10, 156 10 of 14 10 of 14 3.6. Texture 3.6. Texture Closer to the faster roller, i.e., the bottom position of the sample, were with a set roll gap
of 4.0 mm in a symmetrically rolled sample, there was the volume fraction of texture component E,
with 6.3 vol.% and texture component G, with 3.9 vol.%. The highest volume fraction of the rolling
texture components was, in Figure 7c, observed for a factor of asymmetry equal to 2.0. In Figure 7d,
where the bottom section of the more symmetrically deformed and heat-treated sample was presented,
the highest volume fraction of 3.1 vol.% was observed for the texture component R. The higher volume
fractions of the rolling and shear texture components were obtained in this sample with a factor of
asymmetry equal to 1.5. All the compared textures had a very even distribution of texture components,
but at the same time, because the symmetrically rolled sample had more stood out recrystallization
texture components, and the volume fractions of all the textures were, for the asymmetrically rolled
samples with a factor of asymmetry equal to 1.5 and 2.0, significantly equivalent. For comparison of
the observed texture components and the texture evolution the deformation history and the initial
textures of the material are important [35]. The fibers as texture elements connected to different texture
components must be exposed as a possible preference for successful heat treatment [31]. Nevertheless,
a comparison between asymmetrically and symmetrically rolled textures is useful and can explain
some changes in the mechanical properties. This is in addition to the detection of the C, S and B
texture components, which could indicate the formation of β fibers as major texture element for highly
deformed rolling materials with fcc. 11 of 14
11 of 14 11 of 14
11 of 14 Metals 2020, 10, 156
Metals 2020, 10, 156 Metals 2020, 10, 156
11 of
Figure 6. EBSD mapping (IPF Z) and pole figures for rolled and heat-treated samples in centre
position for symmetrically rolled 1.0 (a), asymmetrically rolled 1.5 (b) and asymmetrically rolled 2.0
( )
Figure 6. EBSD mapping (IPF Z) and pole figures for rolled and heat-treated samples in centre position
for symmetrically rolled 1.0 (a), asymmetrically rolled 1.5 (b) and asymmetrically rolled 2.0 (c). Figure 6. EBSD mapping (IPF Z) and pole figures for rolled and heat-treated samples in centre
position for symmetrically rolled 1.0 (a), asymmetrically rolled 1.5 (b) and asymmetrically rolled 2.0
(c). 3.6. Texture Metals 2020, 10, 156
11 of
Figure 6. EBSD mapping (IPF Z) and pole figures for rolled and heat-treated samples in centre
position for symmetrically rolled 1.0 (a), asymmetrically rolled 1.5 (b) and asymmetrically rolled 2.0
(c)
Figure 6. EBSD mapping (IPF Z) and pole figures for rolled and heat-treated samples in centre position
for symmetrically rolled 1.0 (a), asymmetrically rolled 1.5 (b) and asymmetrically rolled 2.0 (c). Figure 6. EBSD mapping (IPF Z) and pole figures for rolled and heat-treated samples in centre
position for symmetrically rolled 1.0 (a), asymmetrically rolled 1.5 (b) and asymmetrically rolled 2.0
(c). Fig Fig Figure 6. EBSD mapping (IPF Z) and pole figures for rolled and heat-treated samples in centre
position for symmetrically rolled 1.0 (a), asymmetrically rolled 1.5 (b) and asymmetrically rolled 2.0
( )
Figure 6. EBSD mapping (IPF Z) and pole figures for rolled and heat-treated samples in centre position
for symmetrically rolled 1.0 (a), asymmetrically rolled 1.5 (b) and asymmetrically rolled 2.0 (c). g
pp
g (
)
p
g
p
position for symmetrically rolled 1.0 (a), asymmetrically rolled 1.5 (b) and asymmetrically rolled 2.0
(c). (c). Figure 7. Volume fraction of texture components in heat-treated samples for 4.0 mm set roll gap and
centre position (a), 3.1 mm set roll gap and centre position (b), 4.0 mm set roll gap and bottom position
Figure 7. Volume fraction of texture components in heat-treated samples for 4.0 mm set roll gap and
centre position (a), 3.1 mm set roll gap and centre position (b), 4.0 mm set roll gap and bottom position
(c) and 3.1 mm set roll gap and centre position (d). Figure 7. Volume fraction of texture components in heat-treated samples for 4.0 mm set roll gap and
centre position (a), 3.1 mm set roll gap and centre position (b), 4.0 mm set roll gap and bottom position
(c) and 3.1 mm set roll gap and centre position (d). (c). Figure 7. Volume fraction of texture components in heat-treated samples for 4.0 mm set roll gap and
centre position (a), 3.1 mm set roll gap and centre position (b), 4.0 mm set roll gap and bottom position
Figure 7. 3.6. Texture Volume fraction of texture components in heat-treated samples for 4.0 mm set roll gap and
centre position (a), 3.1 mm set roll gap and centre position (b), 4.0 mm set roll gap and bottom position
(c) and 3 1 mm set roll gap and centre position (d)
Figure 7. Volume fraction of texture components in heat-treated samples for 4.0 mm set roll gap and
centre position (a), 3.1 mm set roll gap and centre position (b), 4.0 mm set roll gap and bottom position
(c) and 3.1 mm set roll gap and centre position (d). (c) and 3.1 m
( )
4. Conclusions The asymmetric cold rolling of AA 5454 aluminum alloy plates was compared with symmetric
rolling. Due to the uneven distribution of the upper and lower roller pressures and the workpiece
velocity, the mechanisms in the deformation zone were more suitable for producing smaller crystal
grains, which has an impact on improving most mechanical properties. The main advantage of
asymmetric rolling is the presence of significant shear strain, leading to a gradient structure. At the Metals 2020, 10, 156 12 of 14 same time, the asymmetric rolling enables the creation of more shear texture components, in addition
to the rolling and recrystallized texture components. The texture heterogeneity presented with more
different texture components and less stands out volume fraction of a separate texture component will
have lower planar anisotropy. The asymmetric conditions were created by using different roller speeds. The general aim was to
determine the impact of the asymmetry on the rolling process for a specific alloy. At the same time,
metallographic analyses were made to explain some changes to the technological and mechanical
properties. For asymmetric rolling, a lower rolling force was needed to create higher strains at the same
set roll gap. Thickness reductions as well as strains increased and the rolling forces were decreased
with a higher factor of asymmetry. The lengths and the angles of the ski effects were very similar,
nevertheless, it was clear that the ski effect depends much more on the factor of asymmetry than
the strain. The highest values of the tensile and yield strengths were not present in the deformed
and heat-treated condition in the same direction. The elongations after the heat treatment were quite
similar, which indicates an effective heat treatment. The planar anisotropy in the deformed condition of the asymmetrically rolled samples was higher
than with the symmetrically rolled samples. The elimination of the planar anisotropy after the heat
treatment was larger in the asymmetrically rolled samples. The production of a more homogeneous
material is visible in the samples with the more evenly distributed hardness throughout the cross-section,
with smaller deviations in the case of the asymmetrically rolled samples than with the symmetrically
rolled samples. The impact of the asymmetry is through the hardness of the material, also shown after
the heat treatment. In accordance with the homogeneity of the hardness is the average size of the grains,
where with a higher factor of asymmetry grains decreased. (c) and 3.1 m
( )
4. Conclusions The grain size deviations throughout
the cross-section were also lower with the asymmetrically rolled samples. Different crystallographic
textures were detected at the central position, as well as at the bottom position of the same samples. The volume fractions of the detected texture components were, for the asymmetrically rolled samples,
more evenly distributed than for the symmetrically rolled samples, where more separate texture
components stand out. Author Contributions: Conceptualization, J.K. and P.F.; methodology, J.K., P.F. and H.P.; software, J.K., M.G. and
I.P.; validation, J.K., P.F., H.P., M.G. and I.P.; formal analysis, J.K., H.P., M.G. and I.P.; investigation, J.K., P.F. and
H.P.; data curation, G.K.; writing—original draft preparation, J.K., P.F. and I.P.; writing—review and editing, H.P.,
M.G. and G.K.; supervision, P.F., H.P., M.G. and I.P.; funding acquisition, H.P., G.K. and M.G. All authors have
read and agreed to the published version of the manuscript. Funding: The authors acknowledge the financial support from the Slovenian Research Agency, research
corefundings No. P2-0132 and No. P2-0344. This work was also funded by the program MARTINA, supported by
Slovenian Ministry of education, science and sport and European Regional Development Fund. Conflicts of Interest: The authors declare no conflict of interest. s of Interest: The authors declare no conflict of interes Author Contributions: Conceptualization, J.K. and P.F.; methodology, J.K., P.F. and H.P.; software, J.K., M.G. and
I.P.; validation, J.K., P.F., H.P., M.G. and I.P.; formal analysis, J.K., H.P., M.G. and I.P.; investigation, J.K., P.F. and
H.P.; data curation, G.K.; writing—original draft preparation, J.K., P.F. and I.P.; writing—review and editing, H.P.,
M.G. and G.K.; supervision, P.F., H.P., M.G. and I.P.; funding acquisition, H.P., G.K. and M.G. All authors have
read and agreed to the published version of the manuscript. Funding: The authors acknowledge the financial support from the Slovenian Research Agency, research
corefundings No. P2-0132 and No. P2-0344. This work was also funded by the program MARTINA, supported by
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and annealing in AA5754. Mater. Sci. Eng. A 2005, 399, 358–367. [CrossRef] 39. Lee, J.K.; Lee, D.N. Texture control and grain refinement of AA1050 Al alloy sheets by asymmetric rolling. Int. J. Mech. Sci. 2008, 50, 869–887. [CrossRef] © 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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The University of Washington Ice–Liquid Discriminator (UWILD) improves single-particle phase classifications of hydrometeors within Southern Ocean clouds using machine learning
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See next page for additional authors Follow this and additional works at: https://scholarworks.sjsu.edu/faculty_rsca San Jose State University
San Jose State University
SJSU ScholarWorks
SJSU ScholarWorks
Faculty Research, Scholarly, and Creative Activity
11-11-2021
The University of Washington Ice-Liquid Discriminator (UWILD)
The University of Washington Ice-Liquid Discriminator (UWILD)
improves single-particle phase classifications of hydrometeors
improves single-particle phase classifications of hydrometeors
within Southern Ocean clouds using machine learning
within Southern Ocean clouds using machine learning
Rachel Atlas
University of Washington
Johannes Mohrmann
University of Washington
Joseph Finlon
University of Washington
Jeremy Lu
University of Washington
Ian Hsiao
University of Washington
See next page for additional authors
Follow this and additional works at: https://scholarworks.sjsu.edu/faculty_rsca
Recommended Citation
Recommended Citation
Rachel Atlas, Johannes Mohrmann, Joseph Finlon, Jeremy Lu, Ian Hsiao, Robert Wood, and Minghui Dia
"The University of Washington Ice-Liquid Discriminator (UWILD) improves single-particle phase
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San Jose State University
SJSU ScholarWorks
SJSU ScholarWorks Faculty Research, Scholarly, and Creative Activity Faculty Research, Scholarly, and Creative Activity The University of Washington Ice-Liquid Discriminator (UWILD)
The University of Washington Ice-Liquid Discriminator (UWILD)
improves single-particle phase classifications of hydrometeors
improves single-particle phase classifications of hydrometeors
within Southern Ocean clouds using machine learning
within Southern Ocean clouds using machine learning The University of Washington Ice-Liquid Discriminator (UWILD)
The University of Washington Ice-Liquid Discriminator (UWILD)
improves single-particle phase classifications of hydrometeors
improves single-particle phase classifications of hydrometeors
within Southern Ocean clouds using machine learning
within Southern Ocean clouds using machine learning See next page for additional authors Authors
Authors
Rachel Atlas, Johannes Mohrmann, Joseph Finlon, Jeremy Lu, Ian Hsiao, Robert Wood, and Minghui Diao Recommended Citation
Recommended Citation Rachel Atlas, Johannes Mohrmann, Joseph Finlon, Jeremy Lu, Ian Hsiao, Robert Wood, and Minghui Diao. "The University of Washington Ice-Liquid Discriminator (UWILD) improves single-particle phase
classifications of hydrometeors within Southern Ocean clouds using machine learning" Atmospheric
Measurement Techniques (2021): 7079-7101. https://doi.org/10.5194/amt-14-7079-2021 This Article is brought to you for free and open access by SJSU ScholarWorks. It has been accepted for inclusion in
Faculty Research, Scholarly, and Creative Activity by an authorized administrator of SJSU ScholarWorks. For more
information, please contact scholarworks@sjsu.edu. Authors
Authors
Rachel Atlas, Johannes Mohrmann, Joseph Finlon, Jeremy Lu, Ian Hsiao, Robert Wood, and Minghui Diao ohannes Mohrmann, Joseph Finlon, Jeremy Lu, Ian Hsiao, Robert Wood, and Minghui Diao This article is available at SJSU ScholarWorks: https://scholarworks.sjsu.edu/faculty_rsca/2388 Correspondence: Rachel Atlas (ratlas@uw.edu) Correspondence: Rachel Atlas (ratlas@uw.edu) Received: 2 May 2021 – Discussion started: 21 May 2021
Revised: 14 August 2021 – Accepted: 28 August 2021 – Published: 11 November 2021 Received: 2 May 2021 – Discussion started: 21 May 2021 Abstract. Mixed-phase Southern Ocean clouds are challeng-
ing to simulate, and their representation in climate models
is an important control on climate sensitivity. In particular,
the amount of supercooled water and frozen mass that they
contain in the present climate is a predictor of their plan-
etary feedback in a warming climate. The recent Southern
Ocean Clouds, Radiation, Aerosol Transport Experimental
Study (SOCRATES) vastly increased the amount of in situ
data available from mixed-phase Southern Ocean clouds use-
ful for model evaluation. Bulk measurements distinguish-
ing liquid and ice water content are not available from
SOCRATES, so single-particle phase classifications from
the Two-Dimensional Stereo (2D-S) probe are invaluable
for quantifying mixed-phase cloud properties. Motivated by
the presence of large biases in existing phase discrimina-
tion algorithms, we develop a novel technique for single-
particle phase classification of binary 2D-S images using a
random forest algorithm, which we refer to as the University
of Washington Ice–Liquid Discriminator (UWILD). UWILD
uses 14 parameters computed from binary image data, as well
as particle inter-arrival time, to predict phase. We use liquid-
only and ice-dominated time periods within the SOCRATES
dataset as training and testing data. This novel approach to
model training avoids major pitfalls associated with using
manually labeled data, including reduced model generaliz-
ability and high labor costs. We find that UWILD is well
calibrated and has an overall accuracy of 95 % compared
to 72 % and 79 % for two existing phase classification algo- rithms that we compare it with. UWILD improves classifica-
tions of small ice crystals and large liquid drops in particular
and has more flexibility than the other algorithms to iden-
tify both liquid-dominated and ice-dominated regions within
the SOCRATES dataset. UWILD misclassifies a small per-
centage of large liquid drops as ice. Such misclassified par-
ticles are typically associated with model confidence below
75 % and can easily be filtered out of the dataset. UWILD
phase classifications show that particles with area-equivalent
diameter (Deq) < 0.17 mm are mostly liquid at all temper-
atures sampled, down to −40 ◦C. Larger particles (Deq >
0.17 mm) are predominantly frozen at all temperatures be-
low 0 ◦C. Correspondence: Rachel Atlas (ratlas@uw.edu) Between 0 and 5 ◦C, there are roughly equal num-
bers of frozen and liquid mid-sized particles (0.17 < Deq <
0.33 mm), and larger particles (Deq > 0.33 mm) are mostly
frozen. We also use UWILD’s phase classifications to esti-
mate sub-1 Hz phase heterogeneity, and we show examples
of meter-scale cloud phase heterogeneity in the SOCRATES
dataset. Authors
Authors This article is available at SJSU ScholarWorks: https://scholarworks.sjsu.edu/faculty_rsca/2388 Atmos. Meas. Tech., 14, 7079–7101, 2021
https://doi.org/10.5194/amt-14-7079-2021
© Author(s) 2021. This work is distributed under
the Creative Commons Attribution 4.0 License. 1.1
Southern Ocean cloud phase and climate Mixed-phase processes within Southern Ocean clouds mod-
erate cloud radiative effects (Bodas-Salcedo et al., 2016; Mc-
Coy et al., 2014a) and cloud–climate feedbacks associated Rachel Atlas1, Johannes Mohrmann1, Joseph Finlon1, Jeremy Lu1,⋆, Ian Hsiao1,⋆, Robert Wood1, and
Minghui Diao2 1Department of Atmospheric Sciences, University of Washington, Seattle, WA, USA
2Department of Meteorology and Climate Science, San Jose State University, San Jose, CA, USA
⋆These authors contributed equally to this work. 1.2
Existing methods for phase classification Single-particle phase classification techniques for binary
OAP images typically distinguish liquid from frozen hy-
drometeors using particle shape and/or particle roughness. Cober et al. (2001a) used four different ratios computed from
particle length, width, perimeter, and area to estimate particle
sphericity and discriminate between liquid and ice particles. McFarquhar et al. (2013) proposed using area ratio, defined
as the particle area divided by the area of the smallest cir-
cle bounding the particle, to classify the phase of particles
with maximum dimensions between 35 and 60 µm. Using
area ratio alone is the simplest technique for phase classi-
fication and has been implemented in the Earth Observing
Laboratory’s (EOL) OAP processing code. Throughout this
study, we compare UWILD’s performance with this tech-
nique, which we refer to simply as “area ratio”. We include a
schematic of area ratio in Fig. 1a. Given that the strength of the cloud phase feedback in cli-
mate models is related to the base state representation of
cloud phase within Southern Ocean clouds, we can constrain
the cloud phase feedback by assessing how realistic the base
state of each climate model is. Historically, due to a lack
of in situ measurements over the Southern Ocean, satellite
data have been used to evaluate cloud phase within climate
models. Tan et al. (2016) retrieved cloud phase from Cloud–
Aerosol Lidar with Orthogonal Polarization (CALIOP) to
show that climate models with strong cloud phase feedbacks
typically underestimate the fraction of supercooled water in
present-day extra-tropical, mixed-phase clouds. McCoy et al. (2014a) combined retrievals from the International Satel-
lite Cloud Climatology Project (ISCCP), Moderate Resolu-
tion Imaging Spectroradiometer (MODIS), and Multi-angle
Imaging Spectroradiometer (MISR) to connect seasonally
varying cloud radiative fluxes with mixed-phase cloud prop-
erties. Satellite products have been invaluable for constrain-
ing cloud radiative effects and identifying model biases over
the Southern Ocean, but they provide almost no informa-
tion on hydrometeor phase other than at the cloud top. For
this reason and because of low vertical and horizontal reso-
lution and large retrieval uncertainties, satellite products are
not sufficient to support process-oriented studies of mixed-
phase microphysics in models. A limitation of using area ratio alone is that quasi-
spherical frozen particles, such as graupel, may be classified
as liquid. Using particle surface roughness is an attractive
alternative. R. Atlas et al.: University of Washington Ice–Liquid Discriminator (UWILD) R. Atlas et al.: University of Washington Ice–Liquid Discriminator (UWILD) 7080 with the stormy region (McCoy et al., 2014b). The pres-
ence of small amounts of ice within liquid-dominated mixed-
phase clouds can substantially increase precipitation as com-
pared with warm clouds with similar thickness, due to ef-
ficient cold precipitation formation (Bergeron, 1928; Field
and Heymsfield, 2015). Increased precipitation can reduce
cloud lifetime (Albrecht, 1989) and increase aerosol scav-
enging (Radke et al., 1980). particle classification is the most viable way to quantify ice
properties within SOCRATES-sampled clouds. Furthermore,
single-particle phase classifications are useful for making di-
rect comparisons of simulated and observed liquid and frozen
particle size distributions (PSDs). During SOCRATES, opti-
cal array probes (OAPs) such as the Two-Dimensional Stereo
(2D-S) instrument (Lawson et al., 2006) collected binary im-
ages of single particles that can be used for phase classifi-
cation. However, there is no standard procedure for identi-
fying the phase of 2D-S imaged particles, and existing algo-
rithms, which are described in Sect. 1.2, have substantial bi-
ases. Here, we introduce a novel machine-learning-based al-
gorithm, the University of Washington Ice–Liquid Discrimi-
nator (UWILD), and show that it has greater skill in discrim-
inating liquid and ice particles than two pre-existing algo-
rithms. The distribution of liquid and frozen hydrometeors within
Southern Ocean clouds will change as the climate warms
(Mitchell et al., 1989; Storelvmo et al., 2015). Climate mod-
els that simulate a relatively high ice-to-liquid ratio within
Southern Ocean clouds in the present climate, or base state,
exhibit a negative cloud radiative feedback in future climates
(McCoy et al., 2015). One explanation for this is that as the
climate warms, fewer ice particles form, and clouds become
both brighter (Sun and Shine, 1994) and longer lived. This
feedback, known as the cloud phase feedback, has a large
impact on the climate sensitivity, and its weakening has been
suggested as an explanation for an increase in climate sen-
sitivity going from CMIP5 (Coupled Model Intercomparison
Project) to CMIP6 (Zelinka et al., 2020; Bjordal et al., 2020). Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. 1.2
Existing methods for phase classification Czys and Schoen Petersen (1992) fit a fourth-
degree polynomial to quasi-spherical 2D particle images
(with particle circumference represented in polar coordi-
nates) to estimate surface roughness and distinguish between
graupel particles and liquid drops. Other studies (Hunter
et al., 1984; Moss and Johnson, 1994; Bower et al., 1996;
Yang et al., 2016) have used Fourier analysis to quantify the
sphericity and roughness of imaged particles and to classify
them as liquid drops or frozen particles with particular habits. Holroyd (1987) also used a combination of particle shape and
surface roughness to classify particle habit. They quantified
surface roughness using the fine-detail ratio, defined as the
perimeter multiplied by maximum dimension and divided by
particle area, and used that and other particle properties to
classify frozen hydrometeors into 10 habits, which we refer
to as Holroyd habits. McFarquhar et al. (2018) modified this
algorithm for use in mixed-phase clouds by classifying par- Recent in situ measurements of summertime Southern
Ocean clouds from the Southern Ocean Clouds, Radiation,
Aerosol Transport Experimental Study (SOCRATES; Mc-
Farquhar et al., 2020) make it possible to quantify cloud mi-
crophysical properties through the full depth of the cloud
and to create a dataset for evaluating models and remote
sensing retrievals. Since measurements of bulk ice water
content (IWC) are not available during SOCRATES, single- Atmos. Meas. Tech., 14, 7079–7101, 2021 https://doi.org/10.5194/amt-14-7079-2021 7081 R. Atlas et al.: University of Washington Ice–Liquid Discriminator (UWILD) R. Atlas et al.: University of Washington Ice–Liquid Discriminator (UWILD) Heymsfield and Parrish, 1978), is recorded. If particles ex-
hibit this diffraction pattern, they are classified as liquid and
their Holroyd habit is not taken into account. This technique
has been applied to SOCRATES data (Wu et al., 2019), and
Wang et al. (2020) used the resulting phase classifications
to compute number concentrations and water contents of
droplets and frozen hydrometeors in SOCRATES-sampled
clouds. Throughout this study, we compare UWILD’s perfor-
mance with this technique as well, which we refer to as “Hol-
royd” for simplicity, although it employs the Holroyd (1987)
habit classification in conjunction with other techniques. We
include a schematic of Holroyd in Fig. 1b. Figure 1. Schematic comparing the three-phase classification al-
gorithms compared in this study. Using single-particle properties
derived from the 2D-S probe as input, each panel describes the al-
gorithm decision tree used to classify a particle as liquid or ice. 1.2
Existing methods for phase classification Machine learning is attractive for single-particle phase
classification because it allows for the use of multiple particle
parameters without incurring the labor and time costs associ-
ated with handpicking thresholds for those parameters. Ad-
ditionally, many machine learning algorithms produce both a
classification and a model confidence for each particle, which
can be regarded as the uncertainty in the phase classification
for a well-calibrated model. Machine learning has been used
to classify airborne particle probe images in many studies
over the last decade (Lindqvist et al., 2012; Nurzynska et al.,
2012, 2013; O’Shea et al., 2016; Praz et al., 2017, 2018; Xiao
et al., 2019; Wu et al., 2020; Korolev et al., 2020; Touloupas
et al., 2020), but to our knowledge, it has never been used to
directly predict hydrometeor phase for binary OAP images. The remainder of the article is arranged as follows. In
Sect. 2, we describe the SOCRATES dataset and the de-
velopment of our training, testing, and validation dataset. In Sect. 3, we introduce UWILD, and in Sect. 4, we eval-
uate UWILD and compare its performance with two pre-
existing phase classification algorithms. In Sect. 5, we show
how concentrations of liquid and frozen hydrometeors vary
with size and temperature, and we investigate sub-1 Hz cloud
phase heterogeneity in the SOCRATES dataset. A summary
of UWILD and its implications for identifying particle phase
in Southern Ocean clouds is discussed in Sect. 6. 2.1
SOCRATES observations and description of the
2D-S Figure 1. Schematic comparing the three-phase classification al-
gorithms compared in this study. Using single-particle properties
derived from the 2D-S probe as input, each panel describes the al-
gorithm decision tree used to classify a particle as liquid or ice. Between 15 January and 25 February 2018, the US Na-
tional Science Foundation (NSF) supported the SOCRATES
campaign to sample diverse boundary layer clouds between
Hobart, Tasmania, and the Antarctic continent (McFarquhar
et al., 2020). SOCRATES employed the NSF–National Cen-
ter for Atmospheric Research (NCAR) Gulfstream V (G-V)
aircraft outfitted with three OAPs including the 2D-S, the
Two-Dimensional Cloud (2DC) probe, and the Precipitation
Imaging Probe (PIP). The 2DC and the PIP both suffered
from quality control issues (Finlon et al., 2020), so we use ticles labeled as tiny or spherical from the Holroyd (1987)
scheme as liquid and all other particles as ice. Furthermore,
for particles with maximum dimensions < 300 µm, the pres-
ence or absence of a light spot in the center of the particle,
known as the Poisson spot (Arago and Gay-Lussac, 1819; R. Atlas et al.: University of Washington Ice–Liquid Discriminator (UWILD) 7082 the 2D-S particle images to develop single-particle phase
classifications for the SOCRATES dataset. The SOCRATES
campaign included 15 6–8 h flights. We use 14 out of the 15
SOCRATES flights, omitting RF15 due to an anomalously
high occurrence of corrupted 2D-S particle images. Figure 2
shows the SOCRATES flight tracks and the distribution of
1 Hz in-cloud temperatures from the 14 SOCRATES flights
used here. The majority (85.5 %) of the cloudy flight data
occur within the temperature range that can support mixed-
phased clouds, −40 to 0 ◦C, while 15.1 % of the cloudy flight
data are warmer than 0 ◦C, and the remaining 0.4 % of the
flight data are colder than −40 ◦C. set of all of the particle properties that UIOOPS computes. We visually define the particle features using example par-
ticles in Fig. A1, and we show histograms of the particle
features for the SOCRATES dataset in Fig. A2. We use the
common logarithm of inter-arrival time (log10(iat)), which
is the time elapsed since the previous particle was imaged
within the 2D-S sample volume, instead of the absolute inter-
arrival time because machine learning models are optimized
to train on normally distributed variables. Inter-arrival time
is the only feature that is substantially non-normally dis-
tributed and requires normalization. We show distributions
of log10(iat) for liquid and ice-dominated particles from our
training dataset in Fig. 3a. We discuss how we prepare our
training dataset in Sect. 2.2. The clear separation in the peaks
of the two distributions, with liquid particles associated with
smaller inter-arrival times, indicates that inter-arrival time is
useful for phase classification. The distribution for the ice-
dominated particles is bimodal, and the smaller mode in the
smaller inter-arrival times may be a signature of shattered
artifacts (Korolev et al., 2013). These suspected shattered ar-
tifacts with inter-arrival times < 10−5 s represent 7 % of the
ice-dominated particles and are thus expected to minimally
affect how UWILD uses inter-arrival time for phase discrim-
ination. We retrained UWILD with log10(iat) excluded as a
sensitivity test and found that UWILD’s skill in identifying
small ice particles sharply decreased, while its skill at iden-
tifying other particles decreased only slightly. This result is
described in greater detail in Sect. 4. R. Atlas et al.: University of Washington Ice–Liquid Discriminator (UWILD) The 2D-S uses a 128-element photodiode array in conjunc-
tion with high-speed electronics to generate shadowgraphs of
particles with 10 µm pixel resolution as they enter the instru-
ment’s sample volume (Lawson et al., 2006). Particle shad-
owgraphs, or images, are thus composed of 10µm × 10µm
square pixels. Particles with maximum dimensions smaller
than 0.01 mm have areas smaller than that of a single pixel
and cannot be detected by the 2D-S. Particles with maximum
dimensions greater than 1.28 mm (the length of the photo-
diode array) have a higher likelihood of being partially cut
off by the image buffer depending on their habit and orienta-
tion. The 2D-S has a sample volume between 10 and 16 Ls−1
for typical SOCRATES aircraft speeds and can record com-
pressed data at rates associated with particle concentrations
up to about 100 cm−3 (Lawson et al., 2006), as they typ-
ically were during SOCRATES. We use the University of
Illinois/Oklahoma OAP Processing Software (UIOOPS; Mc-
Farquhar et al., 2018) to compute particle properties of in-
dividual particles from the horizontal channel of the 2D-S. Due to optical limitations of the 2D-S, quasi-spherical out-
of-focus particles exhibiting Poisson spots are size-corrected
following Korolev (2007). We apply several criteria to fil-
ter particles for use in this study. First, we only use particles
whose center is within the 2D-S field of view to minimize
uncertainties in determining the reconstructed particle size
(Heymsfield and Baumgardner, 1985; Field, 1999). Due to
uncertainties in defining the probe’s depth of field and sam-
ple area (Heymsfield and Parrish, 1978; Baumgardner and
Korolev, 1997; Jackson et al., 2012) and limited shape infor-
mation to support robust classification for smaller particles
(Korolev et al., 1991; Baumgardner et al., 2017), particles
with fewer than 25 pixels are excluded from this study. Be-
cause of this, there are no particles classified as tiny (defined
as < 25 pixels) using the Holroyd scheme (Fig. 1a), and only
the right subtree of area ratio (Fig. 1b) is relevant. Through-
out this study, we will use area-equivalent diameter (Deq) to
represent particle size. For the 2D-S, Deq in millimeters is
computed from the number of pixels as follows: https://doi.org/10.5194/amt-14-7079-2021 https://doi.org/10.5194/amt-14-7079-2021 Atmos. Meas. Tech., 14, 7079–7101, 2021 R. Atlas et al.: University of Washington Ice–Liquid Discriminator (UWILD) Figure 2. (a) SOCRATES flight tracks from RF01–RF14 (the flights analyzed in this study); (b) 1 Hz in-cloud temperature histograms for
RF01–RF14 (hatched, left y axis), with ice-dominated data from the TTV set (blue, right y axis) and liquid-only data from the TTV set (red,
right y axis). In-cloud data include all samples with at least one particle that satisfy our selection criteria (described in Sect. 2.1). Figure 2. (a) SOCRATES flight tracks from RF01–RF14 (the flights analyzed in this study); (b) 1 Hz in-cloud temperature histograms for
RF01–RF14 (hatched, left y axis), with ice-dominated data from the TTV set (blue, right y axis) and liquid-only data from the TTV set (red,
right y axis). In-cloud data include all samples with at least one particle that satisfy our selection criteria (described in Sect. 2.1). Figure 3. Distributions of the common logarithm of inter-arrival time (a) and particle size (b) for the liquid-only (red) and ice-dominated
(blue) data from the TTV set. Figure 3. Distributions of the common logarithm of inter-arrival time (a) and particle size (b) for the liquid-only (red) and ice-dominated
(blue) data from the TTV set. The RICE is an oscillating probe that is sensitive to mass
buildup from supercooled drops that have frozen onto its
sensing cylinder. When supercooled water is present at tem-
peratures lower than −5 ◦C with a mass concentration above
the theoretical detection limit of 0.025 gm−3 (D’Alessandro
et al., 2021), sufficient ice builds up on the sensing cylinder
to decrease its oscillating frequency, which is converted to a
positive voltage. Thus, a changing RICE voltage indicates the
presence of supercooled water. A near-constant RICE volt-
age indicates that supercooled water is not present in a suffi-
cient quantity to trigger an instrument response. 5 min flight period where the temperature varies between
6 and 12 ◦C as a liquid-only period. We show time series
of 1 Hz temperature, RH, particle count, and liquid fraction
from UWILD for this region in Fig. 4a. There is only RH
data available towards the end of the period, and the RH is
close to 100 % there. UWILD classifies most particles as liq-
uid throughout the flight period, but its accuracy decreases
towards the end of the period. We explain how UWILD clas-
sifies particles in Sect. 3, and we quantify UWILD’s perfor-
mance and identify biases in its classifications in Sect. 4. 2.2
Preparation of training, validation, and test data Classification problems are a type of supervised machine
learning that require a dataset with known classifications to
use for model training, cross-validation during hyperparam-
eter (i.e., model configuration parameter) tuning, and model
testing. Creating this dataset, hereafter referred to as the TTV
(for train–test–validate) set, is the biggest challenge asso-
ciated with this particular machine learning problem. Us-
ing manual inspection to build a TTV set, as most stud-
ies using machine learning for particle image classification
have done, would limit the scope of our TTV set to par-
ticles large enough to identify by eye that have an unam-
biguous phase. This would result in our TTV set being sub-
stantially different from a set of randomly sampled particles
from SOCRATES and would thus reduce the generalizabil-
ity of our machine learning model for the whole SOCRATES
dataset. Instead, we use in situ flight data, including tempera-
ture from the Harco heated total air temperature sensor (EOL,
2019), water vapor mixing ratio from the Vertical-Cavity
Surface-Emitting Laser (VCSEL) hygrometer (Zondlo et al.,
2010; Diao, 2021), and voltage from the Rosemount Icing
Detector (RICE; EOL, 2019), to identify flight periods where
the hydrometeors are most likely to be all or mostly the same
phase. Deq = 2 × 10−2 ×
q
number of pixels × π−1. (1) (1) We use 15 particle features, listed in Table 1, to train our
machine learning model. These 15 features represent a sub- https://doi.org/10.5194/amt-14-7079-2021 Atmos. Meas. Tech., 14, 7079–7101, 2021 7083 al.: University of Washington Ice–Liquid Discriminator (UWILD) R. Atlas et al.: University of Washington Ice–Liquid Discriminator (UWILD) R. Atlas et al.: University of Washington Ice–Liquid Discriminator (UWILD) 7084 Table 1. Description of particle features used in UWILD. Features have units of numbers of pixels except where different units are specified. Particle feature
Description [units]
length
Pixels in time dimension
width
Pixels in photodiode dimension
area
Projected area from the number of shadowed pixels (1 pixel = 0.0001 mm2) [mm2]
perimeter
Perimeter following the particle boundary [mm]
max_dimension
Maximum dimension of the smallest circle bounding the particle [mm]
area_equivalent_diameter
Diameter of a circle with an area equal to the particle area [mm]
area_ratio
Ratio between particle area and the area of the smallest circle bounding the particle
percent_shadow_area
Ratio between particle area and the length × width
touching_edge
Binary depicting whether the particle is entirely within the photodiode array [0] or touches an edge
of the array [1]
max_top_edge_touching
Maximum number of times the top diode is shadowed in succession
max_bottom_edge_touching
Maximum number of times the bottom diode is shadowed in succession
edge_at_max_hole
Number of pixels between the top and bottom edges of the particle for the slice containing the
largest gap inside the particle
max_hole_diameter
Diameter of the largest hole inside the particle
fine_detail_ratio
Ratio between perimeter×maximum dimension and particle area following Holroyd (1987)
log10(iat)
Log of the inter-arrival time between particles [log10(s)] Figure 4. Time series of atmospheric parameters and particle properties for the liquid-only period (a), the two ice-dominated periods (b, c),
and the two example mixed-phase periods (d, e). Voltage from the RICE (row 1), relative humidity with respect to liquid (solid line) and
ice (dashed line) (row 2), the number of particles that satisfy our selection criteria (row 3), and liquid fraction as determined by the UWILD
algorithm (row 4) are shown for each flight period. A RICE response is expected when the supercooled water content exceeds 0.025 gm−3. Missing data are indicated with grey. The temperature range sampled in each period is shown on the plot of RICE voltage in the top row. Figure 4. Time series of atmospheric parameters and particle properties for the liquid-only period (a), the two ice-dominated periods (b, c),
and the two example mixed-phase periods (d, e). R. Atlas et al.: University of Washington Ice–Liquid Discriminator (UWILD) A histogram of temperature for the liquid-only period is
shown in Fig. 2b, and normalized histograms of Deq and
log10(iat) are shown in Fig. 3. Small particles (< 100 pixels
or Deq < 0.1 mm) dominate the liquid-only dataset. Equations of saturation pressure with respect to liquid and
ice (Murphy and Koop, 2005) were used to calculate relative
humidity with respect to liquid (RH) and ice (RHi). Uncer-
tainties in RH and RHi can be derived based on the uncer-
tainties associated with temperature and water vapor. Uncer-
tainties range from 6.4 % to 6.8 % for RH and from 6.5 % to
6.9 % for RHi, from 0 to −40 ◦C, respectively. Identifying
liquid phase regions of clouds is simple because frozen hy-
drometeors rarely persist at temperatures above 5 ◦C (Yuter
et al., 2006; Oraltay and Hallett, 2005). Thus, we select a Supercooled water can persist at all temperatures above
the homogeneous nucleation threshold of −40 ◦C (Korolev
et al., 2017) and below 0 ◦C. Since there are no multi-second
periods of in-cloud data from SOCRATES with temperatures
below −40 ◦C (Fig. 2b), we cannot be certain that all parti-
cles within any given SOCRATES flight period are frozen. However, we can use the aforementioned atmospheric pa- https://doi.org/10.5194/amt-14-7079-2021 Atmos. Meas. Tech., 14, 7079–7101, 2021 R. Atlas et al.: University of Washington Ice–Liquid Discriminator (UWILD) Voltage from the RICE (row 1), relative humidity with respect to liquid (solid line) and
ice (dashed line) (row 2), the number of particles that satisfy our selection criteria (row 3), and liquid fraction as determined by the UWILD
algorithm (row 4) are shown for each flight period. A RICE response is expected when the supercooled water content exceeds 0.025 gm−3. Missing data are indicated with grey. The temperature range sampled in each period is shown on the plot of RICE voltage in the top row. Atmos. Meas. Tech., 14, 7079–7101, 2021 https://doi.org/10.5194/amt-14-7079-2021 R. Atlas et al.: University of Washington Ice–Liquid Discriminator (UWILD) 7085 spherical during our ice-dominated period from RF04. Man-
ual classifications of 320 images are universally frozen. rameters and particle probe images to identify periods where
we have very high confidence that over 99 % of the parti-
cles are frozen. We refer to these as ice-dominated periods. We use temperature, RH with respect to ice and liquid water,
voltage from the RICE, and particle images from the 2D-S
to identify two ice-dominated periods, from flights RF01 and
RF04, which we show in Fig. 4b and c, respectively. The ice-
dominated periods are defined as having no RICE response,
are subsaturated with respect to liquid, and are supersatu-
rated with respect to ice. A RICE response, which consists
of the 1 Hz voltage oscillating over a 20 s period, is expected
when the supercooled water content exceeds 0.025 gm−3. RF04 is the source of 70 % of particles in the combined ice-
dominated dataset, and RF01 is the source of the remain-
ing particles. A histogram of temperature for the combined
ice-dominated dataset is shown in Fig. 2b, and normalized
histograms of Deq and log10(iat) for the same dataset are
shown in Fig. 3. The ice-dominated dataset is composed pri-
marily of medium-sized and large particles (≥100 pixels or
Deq ≥0.1 mm). We compare the liquid-only period and two ice-dominated
periods with two examples of mixed-phase periods in Fig. 4. The first mixed-phase period (Fig. 4d) samples a stratocumu-
lus cloud within the boundary layer. The RICE voltage oscil-
lates throughout the period, and the liquid fraction is greater
than 75 % most of the time. The cloud is saturated with re-
spect to liquid. The second mixed-phase period (Fig. 4e)
sampled near the top of altostratus cloud. R. Atlas et al.: University of Washington Ice–Liquid Discriminator (UWILD) This period is
colder and, on average, subsaturated with respect to liquid
water and saturated with respect to ice. The RH is subsatu-
rated even though the cloud is liquid-dominated in the sam-
pled region because the aircraft is skirting a horizontally vari-
able cloud top. The liquid fraction is close to 1.0, and the
RICE voltage oscillates until 00:23:00 UTC (∼0.4 UTC), af-
ter which the liquid fraction decreases abruptly, and the RICE
voltage stabilizes. The change in phase occurs because the
aircraft is initially sampling the cloud top and transitions to
sampling below the cloud top. The liquid-only period includes 90 000 particles that pass
our size threshold, while the ice-dominated periods include
55 000 particles in total. All particles drawn from the liquid-
only period are labeled liquid, and all particles drawn from
the ice-dominated periods are labeled ice. These labels are
taken as truth for the purposes of model training and eval-
uation (Sect. 4). We partition the particles into three size
categories: small (corresponding to 25–99 pixels or Deq of
approximately 0.056–0.1 mm), medium (100–699 pixels or
0.1–0.3 mm), and large (> 700 pixels or > 0.3 mm). In the
remainder of this study, all references to particle size will
be in terms of Deq. We then randomly subsample the liq-
uid particles down to have an equal total number of ice and
liquid particles, preserving the ratio between the three size
categories for each phase separately. These particles are then
partitioned into training (60 %), test (20 %), and validation
(20 %) sets, again preserving the original ratios between the
three size categories for each phase separately and including
an equal number of particles from each phase, in each set. We refer to the combined training, test, and validation sets
as the TTV set. We explicitly preserve these rough size dis-
tributions to ensure that the test set has a reasonable number
of small ice crystals and large liquid drops for evaluation, as
these particles are rare enough in the full TTV dataset that
a completely random partition risks having them undersam-
pled in the test set. Having the same number of liquid and ice
particles in the test set simplifies interpretation of model per-
formance summary statistics, as discussed at the beginning
of Sect. 4. The composition of the TTV set is broken down
in Table 2. Atmos. Meas. Tech., 14, 7079–7101, 2021 3
UWILD: description and interpretation A key consideration for all machine learning applications
is the choice of machine learning model. One approach
to analyzing particle probe images is to apply deep learn-
ing directly to the captured image (e.g., Xiao et al., 2019;
Touloupas et al., 2020; Wu et al., 2020; Korolev et al., 2020). Here, we take a simpler approach and employ a random for-
est model (Breiman, 2001; Pedregosa et al., 2011), which
requires a preprocessing step to extract relevant image fea-
tures (e.g., particle area or perimeter; full list in Table 1). Classification is then carried out using these features as in-
puts (Lindqvist et al., 2012; Nurzynska et al., 2012, 2013;
O’Shea et al., 2016; Praz et al., 2018, 2017). An advantage
of this approach is that it simplifies the inclusion of features
not directly related to particle appearance; in particular, we
show that inter-arrival time is a valuable discriminator of liq-
uid and ice particles. Random forests can also provide more
interpretable results, as the trained model can be analyzed
to investigate relative feature importance. Another advantage
(shared by many machine learning approaches) is the deter-
mination of classification confidence, which can be useful in
filtering out more uncertain classifications, or estimating un-
certainties in calculated bulk properties such as liquid water
content. Figure 5. UWILD confidence is plotted against UWILD accuracy
with a black solid line (this is referred to as the calibration curve)
in the top row, and a histogram of model confidence is shown in
the bottom row on a shared x axis. This analysis is done on the test
set. The proximity between the calibration curve and the one-to-one
blue dashed line implies that UWILD confidence can be used as a
proxy for the uncertainty in UWILD’s classifications. For a decision tree trained using a supervised learning ap-
proach, the training set is split by thresholding features (e.g.,
whether area ratio is more or less than 0.8); precisely which
feature and which value is determined by whatever “best”
splits the dataset into distinct categories (for UWILD the max
Gini impurity reduction criterion is used). This process is re-
peated on each data subset until the data are entirely parti-
tioned into distinct categories. In a random forest, multiple
such trees (100 for UWILD) are trained using random sub-
sets of data features. R. Atlas et al.: University of Washington Ice–Liquid Discriminator (UWILD) The proximity between the calibration curve and the one-to-one
blue dashed line implies that UWILD confidence can be used as a
proxy for the uncertainty in UWILD’s classifications. Figure 5 shows this relationship between model confidence
(from the random forest votes) and model accuracy (how
likely the model was to correctly classify particles), evalu-
ated on the test set. A one-to-one relationship is ideal be- R. Atlas et al.: University of Washington Ice–Liquid Discriminator (UWILD) R. Atlas et al.: University of Washington Ice–Liquid Discriminator (UWILD) 7086 Table 2. Number of particles by size class in the training, validation, and test sets. Phase
Particle size class
Training
Validation
Test
Ice
Small (0.056mm ≤Deq < 0.1 mm)
1.7 k
0.59 k
0.6 k
Medium (0.1mm ≤Deq < 0.3 mm)
12.6 k
4.2 k
4.2 k
Large (Deq ≥0.3 mm)
18.6 k
6.2 k
6.2 k
Liquid
Small (0.056mm ≤Deq < 0.1 mm)
20.1 k
6.7 k
6.7 k
Medium (0.1mm ≤Deq < 0.3 mm)
11.4 k
3.8 k
3.8 k
Large (Deq ≥0.3 mm)
1.5 k
0.5 k
0.5 k able 2. Number of particles by size class in the training, validation, and test sets. Table 2. Number of particles by size class in the training, validation, and test sets. mm)
20.1 k
6.7 k
6.7 k
mm)
11.4 k
3.8 k
3.8 k
1.5 k
0.5 k
0.5 k
Figure 5. UWILD confidence is plotted against UWILD accuracy
with a black solid line (this is referred to as the calibration curve)
in the top row, and a histogram of model confidence is shown in
the bottom row on a shared x axis. This analysis is done on the test
set. The proximity between the calibration curve and the one-to-one
blue dashed line implies that UWILD confidence can be used as a
proxy for the uncertainty in UWILD’s classifications. R. Atlas et al.: University of Washington Ice–Liquid Discriminator (UWILD) We show histograms of particle features from the
TTV set and from the whole SOCRATES dataset (14 flights)
in Fig. A2, and we discuss out of sample particles in Ap-
pendix A. We manually inspected 1000 2D-S particle images from
the combined ice-dominated dataset with 0.2mm < Deq <
0.8 mm, which account for 87 % of the particles in the ice-
dominated dataset, and found that 0.6 % of the particles
are likely liquid. Thus, if UWILD classified all particles in
the ice-dominated region correctly, it would have a slightly
higher performance than what is reported here (Sect. 4) be-
cause we compute performance metrics assuming that all
particles in the ice-dominated region are frozen. p
g
The SOCRATES payload also included the Particle Habit
Imaging and Polar Scattering (PHIPS) probe, which records
high-quality images of particles with a maximum imaging
rate set at 3 Hz for SOCRATES and measures particle scat-
tering phase functions at a maximum rate of 3.5 kHz (Ab-
delmonem et al., 2016; Schnaiter et al., 2018). The PHIPS
dataset (Schnaiter, 2018b) includes manual classifications of
particle phase based on particle images and automated clas-
sifications of particle sphericity based on particle scattering
phase functions (Waitz et al., 2021). Because the maximum
scattering phase function data acquisition rate is greater than
the maximum imaging rate, there are more automated classi-
fications than manual classifications. Particle sphericity is a
good indicator of particle phase for small and medium-sized
particles. However, large liquid drops are typically aspherical
or elongated because they are distorted due to pressure differ-
ences in the instrument’s inlet, as discussed in Supplement 4
of Waitz et al. (2021). We examine manual and automated classifications for the
PHIPS to further evaluate our liquid-only and ice-dominated
periods. The PHIPS automated classification algorithm iden-
tified 132 particles as spherical and 45 particles as aspherical
during our liquid-only period, while manual classifications
of 106 images are universally liquid. While the PHIPS was
not available for RF01, the PHIPS algorithm automatically
classified 3905 particles as aspherical and only 4 particles as Atmos. Meas. Tech., 14, 7079–7101, 2021 https://doi.org/10.5194/amt-14-7079-2021 7086
R. Atlas et al.: University of Washington Ice–Liquid Discriminator (UWILD)
Table 2. Number of particles by size class in the training, validation, and test sets. R. Atlas et al.: University of Washington Ice–Liquid Discriminator (UWILD) Phase
Particle size class
Training
Validation
Test
Ice
Small (0.056mm ≤Deq < 0.1 mm)
1.7 k
0.59 k
0.6 k
Medium (0.1mm ≤Deq < 0.3 mm)
12.6 k
4.2 k
4.2 k
Large (Deq ≥0.3 mm)
18.6 k
6.2 k
6.2 k
Liquid
Small (0.056mm ≤Deq < 0.1 mm)
20.1 k
6.7 k
6.7 k
Medium (0.1mm ≤Deq < 0.3 mm)
11.4 k
3.8 k
3.8 k
Large (Deq ≥0.3 mm)
1.5 k
0.5 k
0.5 k
3
UWILD: description and interpretation
A key consideration for all machine learning applications
is the choice of machine learning model. One approach
to analyzing particle probe images is to apply deep learn-
ing directly to the captured image (e.g., Xiao et al., 2019;
Touloupas et al., 2020; Wu et al., 2020; Korolev et al., 2020). Here, we take a simpler approach and employ a random for-
est model (Breiman, 2001; Pedregosa et al., 2011), which
requires a preprocessing step to extract relevant image fea-
tures (e.g., particle area or perimeter; full list in Table 1). Classification is then carried out using these features as in-
puts (Lindqvist et al., 2012; Nurzynska et al., 2012, 2013;
O’Shea et al., 2016; Praz et al., 2018, 2017). An advantage
of this approach is that it simplifies the inclusion of features
not directly related to particle appearance; in particular, we
show that inter-arrival time is a valuable discriminator of liq-
uid and ice particles. Random forests can also provide more
interpretable results, as the trained model can be analyzed
to investigate relative feature importance. Another advantage
(shared by many machine learning approaches) is the deter-
mination of classification confidence, which can be useful in
filtering out more uncertain classifications, or estimating un-
certainties in calculated bulk properties such as liquid water
content. For a decision tree trained using a supervised learning ap-
proach, the training set is split by thresholding features (e.g.,
whether area ratio is more or less than 0.8); precisely which
feature and which value is determined by whatever “best”
Figure 5. UWILD confidence is plotted against UWILD accuracy
with a black solid line (this is referred to as the calibration curve)
in the top row, and a histogram of model confidence is shown in
the bottom row on a shared x axis. This analysis is done on the test
set. 4
Comparison between phase classification schemes For quantitative evaluation of a classification model, an in-
tuitive summary metric is model accuracy (the ratio of cor-
rect classifications to total classifications). The overall ac-
curacy of UWILD, Holroyd, and area ratio on our test set
is 94.9 %, 78.5 %, and 71.8 %, respectively, indicating that
UWILD is performing quite well. However, accuracy is most
suitable for balanced classification problems (i.e., when data
are spread evenly across categories). In the case of highly
unbalanced problems, high accuracy can be achieved by sys-
tematically erring in favor of the dominant category. For ex-
ample, small particles in the test set are overwhelmingly liq-
uid (Fig. 3b), so high accuracy can be achieved by predicting
that all small particles are liquid at the expense of correctly
classifying small ice particles. Figure 6. Permutation feature importance for the 10 most important
features is shown for all particles (black squares) and for the 3 size
classes separately. Features are shuffled 10 times, and the mean fea-
ture importance from the 10 trials is shown here. ture renders that feature useless to the model classification,
and the model accuracy will decrease substantially for a very
significant feature. This analysis can be rapidly performed
multiple times for each feature. Another advantage of per-
mutation feature importance is that it is a function of the
dataset being used for evaluation as well as the model, so
it can be calculated separately for different subsets of the
data or for entirely new test datasets. Other measures of fea-
ture importance (such as impurity-based feature importance)
are functions only of the model and do not share this advan-
tage. A relevant drawback to all measures of feature impor-
tance is that they are affected by correlations between fea-
tures. As correlated features share information, model per-
formance may not decrease as much when a particular fea-
ture is shuffled, as a (previously) correlated different feature
may still encode the relevant information. However, decor-
relating variables prior to use, which would address this is-
sue, complicates model interpretation (while not significantly
affecting model performance), and so we chose to preserve
original features and caution against too minute a dissection
of the permutation feature importance. R. Atlas et al.: University of Washington Ice–Liquid Discriminator (UWILD) 7087 cles, log10(iat) is most important, and for large particles, area
ratio is most important (likely because small and medium
particles are mostly quasi-spherical irrespective of phase). Regarding correlated features, the results in Fig. 6 should not
be taken to mean that width in particular is a key discrimi-
nator as opposed to length or max dimension but rather that
width is a good estimator of particle size, which is the par-
ticle characteristic that matters in determining its phase. If
particle width were removed from the feature set, then an-
other size-encoding feature would appear more important. Atmos. Meas. Tech., 14, 7079–7101, 2021 3
UWILD: description and interpretation Randomness is introduced here to re-
duce overfitting to the training set and improve model gen-
eralizability. For a given test data point, each tree provides a
classification, and the plurality vote of all trees is the over-
all category assigned to the data point, with the proportion
of trees voting for that category as the model confidence. We
include a simple schematic of UWILD in Fig. 1c. Figure 5 shows this relationship between model confidence
(from the random forest votes) and model accuracy (how
likely the model was to correctly classify particles), evalu-
ated on the test set. A one-to-one relationship is ideal be-
cause it indicates that we can directly use model confidence
as an estimate of prediction uncertainty. For example, a par-
ticle classified as ice with a model confidence of 75 % should
be seen as 75 % likely to be ice, and 25 % likely to be liquid. Figure 5 also shows that UWILD has a confidence of 95 %
or higher for 73 % of the particles in the TTV set. To better understand how the UWILD classifier deter-
mines particle phase, we quantify how much it relies on each
of the 15 different features (listed in Table 1) using permu-
tation feature importance analysis. This technique measures
how much a model relies on the information encoded in a
particular feature by calculating model accuracy on a test
set, randomly shuffling a given feature, and measuring how
much the accuracy decreases. The random shuffling of a fea- A model is well-calibrated if its model confidence (internal
prediction probability) accurately reflects its performance. Atmos. Meas. Tech., 14, 7079–7101, 2021 https://doi.org/10.5194/amt-14-7079-2021 R. Atlas et al.: University of Washington Ice–Liquid Discr
Figure 6. Permutation feature importance for the 10 most important
features is shown for all particles (black squares) and for the 3 size
classes separately. Features are shuffled 10 times, and the mean fea-
ture importance from the 10 trials is shown here. R. Atlas et al.: University of Washington Ice–Liquid Discriminator (UWILD) R. Atlas et al.: University of Washington Ice–Liquid Discriminator (UWILD) R. Atlas et al.: University of Washington Ice–Liquid Discriminator (UWILD) In other words, we can use
these atmospheric parameters to make broad predictions of
hydrometeor phase and determine which algorithm is most
consistent with these predictions. We expect that small parti-
cles will be entirely liquid above 0 ◦C and that large particles
will be primarily liquid above 0 ◦C and entirely liquid above
5 ◦C due to having longer melting timescales (Oraltay and
Hallett, 2005). Furthermore, we expect that if the liquid frac-
tion is not already 1.0 at 0 ◦C, then it will increase as temper-
ature increases above 0 ◦C, for any particle size. We note that
the abrupt disappearance of particles with Deq > 0.5 mm at
0 ◦C is a signature of ice particles melting. Ice and liquid precipitation formation mechanisms have
been observed to operate simultaneously at temperatures as
low as −28 ◦C (Huffman and Norman, 1988; Cober et al.,
2001b; Kajikawa et al., 2000; Korolev et al., 2002; Silber
et al., 2019), so we cannot use temperature alone to make
a prediction for the liquid fraction of particles at tempera-
tures below 0 ◦C. However, we do expect to see a size depen-
dence in the liquid fraction. To our knowledge, the largest
liquid particle associated with supercooled drizzle forma-
tion (as opposed to melting frozen hydrometeors) that has
been noted in the literature has a maximum dimension of
0.625 mm (Cober et al., 2001b). Most SOCRATES data were
collected in conditions that could not support the relofting of
melted frozen hydrometeors. Furthermore, melted frozen hy-
drometeors are rarely lofted to temperatures below −5 ◦C in
environments that do support relofting (Oraltay and Hallett,
2005). Thus, we expect that medium-sized and large droplets
at temperatures below −5 ◦C are primarily formed via super-
cooled drizzle formation and will not be present at the largest
sizes (0.625–1 mm). Table 3 shows how many particles each algorithm clas-
sified as liquid and ice for each size class and in total. In
general, Holroyd classifies the most particles as ice, and area
ratio classifies the most particles as liquid. UWILD and area
ratio both classify over 90 % of the small particles as liq-
uid, whereas Holroyd classifies only 70 % of them as liquid. Area ratio classifies three-quarters of the medium particles
and half of the large particles as liquid. R. Atlas et al.: University of Washington Ice–Liquid Discriminator (UWILD) 7088 We tested the sensitivity of UWILD’s performance to the
inclusion of log10(iat) by retraining UWILD without it. We
found that the F1 score for small ice particles dropped from
0.765 to 0.475, whereas the F1 scores for the other phase and
size classes dropped only slightly. We tested the sensitivity of UWILD’s performance to the
inclusion of log10(iat) by retraining UWILD without it. We
found that the F1 score for small ice particles dropped from
0.765 to 0.475, whereas the F1 scores for the other phase and
size classes dropped only slightly. increase in the liquid fraction at temperatures below −30 ◦C
(Fig. 8c–e). This behavior is a consequence of small sample
size, as the liquid-dominated data below −30 ◦C come from
just one flight that sampled the top of an altostratus cloud,
whereas the ice-dominated data at higher temperatures come
from several flights that sampled the middle of altostratus
clouds. pp
y
g
y
The TTV set only includes ice particles occurring
at the lowest temperatures (< −23 ◦C) sampled during
SOCRATES, which may have different average ice proper-
ties than ice crystals occurring at higher temperatures. To ac-
count for this, we generated a hand-labeled dataset to eval-
uate UWILD’s ability to detect ice crystals at higher tem-
peratures. We manually classified 1000 randomly sampled
images with 0.2 < Deq < 0.8 mm, occurring at temperatures
higher than −23 ◦C. The resulting dataset contained 861 ice
particles, 78 liquid particles, and 61 ambiguous particles
(which were discarded). We found that UWILD, Holroyd,
and area ratio had F1 scores of 0.63, 0.44, and 0.26, respec-
tively. For UWILD, broken down by phase, the F1 scores
were 0.97 (ice) and 0.63 (liquid). Thus, UWILD still outper-
forms the other two algorithms despite all three exhibiting
lower F1 scores for the hand-labeled set. This lower perfor-
mance is consistent with lower model confidences for this
size and temperature range, as discussed below. In the rest of
this section, we identify differences between the algorithms
and biases within each algorithm to explain the discrepancies
in their performances. We use the whole SOCRATES dataset
(14 flights), which includes 5.76 million classified 2D-S im-
ages, for our analysis from hereon. Since temperature and RH are not inputs to any of the al-
gorithms, we can use them to gauge whether the particle clas-
sifications make physical sense. 4
Comparison between phase classification schemes Model performance, especially for unbalanced classifica-
tion problems, can be better measured by calculating preci-
sion (the ratio of all particles correctly classified as liquid to
all particles classified as liquid) and recall (the ratio of all
particles classified as liquid to all true liquid particles). Both
scores range from 0–1, and they penalize false positives and
false negatives, respectively, for a particular category. These
scores are unified in the F1 score, which is their harmonic
mean: F1 = 2 × precision × recall
precision + recall
. (2) (2) The F1 score is a conservative measure of model perfor-
mance because the lesser of recall and precision will dom-
inate the harmonic mean, and it can be calculated for various
data subsets. We show the F1 scores for Holroyd, area ratio,
and UWILD in Fig. 7 as a function of phase and size class. This analysis is performed on our test set. UWILD outper-
forms Holroyd and area ratio for all phases and size classes. It has the best performance for small liquid (F1 = 0.982) and
large ice (F1 = 0.992) particles and performs less well with
small ice (F1 = 0.765) and large liquid (F1 = 0.893) parti-
cles. While UWILD performs least well when classifying
small ice and large liquid, it nevertheless has a particularly
large performance advantage over Holroyd and area ratio for
those categories. Holroyd outperforms area ratio for medium
and large ice particles, whereas area ratio outperforms Hol-
royd for small ice particles and liquid particles of all sizes. Figure 6 shows the permutation feature importance of the
top 10 features, split by particle size and evaluated on the
model test set. Overall, we see that width, area ratio, and
log10(iat) are the most important. The next two features (max
dimension and length) both encode size and correlate well
with width, while the remaining features have low impact
on model accuracy. Put another way, the model primarily re-
lies on these first three features for classification. Consider-
ing differences between size classes, we note that width and
all other size-related features are most important for medium
particles, which is to be expected, as larger particles are pre-
dominantly ice, and smaller particles are predominantly liq-
uid, with medium particles varying the most. For small parti- https://doi.org/10.5194/amt-14-7079-2021 Atmos. Meas. Tech., 14, 7079–7101, 2021 https://doi.org/10.5194/amt-14-7079-2021 R. Atlas et al.: University of Washington Ice–Liquid Discriminator (UWILD) 7089 Figure 7. F1 scores are shown for UWILD (green), Holroyd (red), and area ratio (blue) for different size classes. F1 scores for small and
medium particles may be slightly underestimated due to the presence of liquid drops in the ice-dominated TTV set. Figure 7. F1 scores are shown for UWILD (green), Holroyd (red), and area ratio (blue) for different size classes. F1 scores for small and
medium particles may be slightly underestimated due to the presence of liquid drops in the ice-dominated TTV set. Table 3. Total particles and number of particles classified as liquid from the three-phase discrimination algorithms for each size class. Numbers in parentheses are the percentage of total particles classified as liquid. SOCRATES flights RF01–RF14 are used. Table 3. Total particles and number of particles classified as liquid from the three-phase discrimination algorithms for each size class. Numbers in parentheses are the percentage of total particles classified as liquid. SOCRATES flights RF01–RF14 are used. Particle size class
Total
UWILD
Holroyd
Area ratio
Small (0.056mm ≤Deq < 0.1 mm)
2 788 219
2 641 062 (94.7)
1 867 682 (70.0)
2 563 437 (91.9)
Medium (0.1mm ≤Deq < 0.3 mm)
1 710 833
674 648 (39.4)
636 655 (37.2)
1 252 175 (73.2)
Large (Deq ≥0.3 mm)
1 265 670
34 040 (2.7)
0 (0.0)
614 144 (48.5)
All particles
5 764 722
3 349 750 (58.1)
2 504 337 (43.4)
4 429 756 (76.8) UWILD’s liquid fraction for particles with Deq between 0.2
and 0.5 mm at temperatures between 0 and 5 ◦C is approxi-
mately 0.5 (Fig. 8c). This is unrealistically low particularly
for the warmer end of this temperature range, where most
frozen particles would have melted. We note that UWILD,
unlike Holroyd, achieves a liquid fraction near 1.0 for tem-
peratures above 5 ◦C. There are also clear differences between the three algo-
rithms’ classifications in RH vs. particle size space (Fig. 8h–
j). UWILD and area ratio both have liquid fractions near
1.0 for small particles (Deq < 0.1 mm) near liquid saturation
(RH = 100 %), whereas Holroyd has a liquid fraction closer
to 0.75 for the same region. Uncertainty in RH is around
7 %, so while high liquid fractions are most common at liq-
uid saturation, they occur at a wide range of RH values. R. Atlas et al.: University of Washington Ice–Liquid Discriminator (UWILD) In contrast, the other
two algorithms classify about 40 % of the medium particles
and 0 % (Holroyd) to 2.7 % (UWILD) of the large particles
as liquid. In Fig. 8, we show the fraction of particles classified as liq-
uid, from the three-phase discrimination algorithms, in the
phase spaces of temperature vs. particle size (left column)
and RH vs. particle size (right column). In the first row, we
show a 2D histogram of the number of classified particles;
in the second row, we show a 2D histogram of the confi-
dence from UWILD; in the third, fourth, and fifth rows, we
show 2D histograms of the fraction of particles classified as
liquid by the three-phase discrimination algorithms. At tem-
peratures greater than −20 ◦C, UWILD confidence is lowest
in areas where UWILD transitions between having a high
liquid fraction and a low liquid fraction (Fig. 8b). UWILD
confidence is also low for small particles at temperatures be-
low −20 ◦C, which can have high or low liquid fractions. All
three algorithms show a decrease in liquid fraction for small
particles at temperatures between −20 and −30 ◦C and an Holroyd
and
UWILD
classify
many
medium-sized
(0.1 mm < Deq < 0.3 mm) and large (Deq > 0.3 mm) parti-
cles as ice at high temperatures (> 0 ◦C). Liquid fractions for
Holroyd sharply decrease to near 0.0 for particles with Deq
between 0.2 and 0.3 mm at all temperatures (Fig. 8e). This
strong size dependence arises from the fact that Holroyd only
considers the presence or absence of a Poisson spot for par-
ticles with maximum dimensions less than 0.3 mm (Fig. 1b). Particles exceeding that maximum dimension threshold must
be nearly spherical in shape to be classified as liquid be-
cause the presence of a Poisson spot is not factored into the
phase classification. A near-0.0 liquid fraction for Holroyd
for Deq > 0.3 mm is unrealistic for temperatures above 0 ◦C,
where the liquid fraction should increase with temperature. https://doi.org/10.5194/amt-14-7079-2021 Atmos. Meas. Tech., 14, 7079–7101, 2021 R. Atlas et al.: University of Washington Ice–Liquid Discriminator (UWILD) Ad-
ditionally, fluctuations in RH from dry-air entrainment and
in-cloud circulation can lead to deviations from liquid satu-
ration at 1 Hz resolution. UWILD classifies fewer particles
as liquid in subsaturated air than either area ratio or Holroyd. In the mid-sized particle range (0.1 < Deq < 0.2 mm), which
includes drizzle, the liquid fraction is near 1.0 when the RH
is close to 100 %, and it drops down to 0.2 when the RH de-
creases to 50 %. Holroyd also classifies many small particles (Deq <
0.1 mm) as ice (Fig. 8e) at all temperatures. Its liquid fraction
never exceeds 0.86 for small particles at temperatures above
−20 ◦C, whereas area ratio and UWILD have a liquid frac-
tion near 1.0 (Fig. 8c and d). Holroyd’s relatively low liquid
fractions for small particles are unrealistic for temperatures
above 0 ◦C. Area ratio classifies many large particles (Deq > 0.3 mm)
as liquid at low temperatures (Fig. 8d). Area ratio’s liq-
uid fraction rarely drops below 0.8 for particles with Deq >
0.2 mm and temperatures below −5 ◦C, whereas Holroyd
and UWILD have liquid fractions near 0.0 (Fig. 8c and e). While a liquid fraction between 0.5 and 0.8 for these par-
ticles is not physically impossible, the fact that there is no
decrease in the liquid fraction with increasing particle size
for particles with Deq between 0.5 and 1 mm, where parti-
cle sizes and temperatures are inconsistent with supercooled
drizzle formation, implies that area ratio’s higher liquid frac-
tions may be unrealistic. Liquid particles can persist in subsaturated air at or below
the cloud base, and these regions were purposefully sampled
within the boundary layer during SOCRATES. Drizzle drops
falling below liquid clouds evaporate in the subsaturated en-
vironment, reducing their size. Subsaturated air can also be
associated with ice-dominated clouds, as RHi is higher than
RH throughout the mixed-phase temperature range. In ice-
dominated clouds, cloud droplets are produced at the tur-
bulent cloud top and tend to freeze before forming drizzle
drops. For both of these reasons, we expect the average size
of liquid particles to decrease as the RH decreases below liq- https://doi.org/10.5194/amt-14-7079-2021 Atmos. Meas. Tech., 14, 7079–7101, 2021 7090 R. https://doi.org/10.5194/amt-14-7079-2021 R. Atlas et al.: University of Washington Ice–Liquid Discriminator (UWILD) Atlas et al.: University of Washington Ice–Liquid Discriminator (UWILD) images, area ratio misclassifies 8 quasi-spherical ice crystals
as liquid for box c and at least 13 quasi-spherical ice crys-
tals as liquid for box d. Particles in box c primarily come
from boundary layer clouds, and most have columnar habits
with small area ratios, although there are a smaller number
of large quasi-spherical frozen particles with large area ratios
present as well. Many of the particles in box d come from the
atmospheric-river case described in Finlon et al. (2020) and,
thus, are more likely to have quasi-spherical heavily rimed
habits with large area ratios. The different particle habits ex-
plain the discrepancy in area ratio’s performance in the two
different regions. gorithms, there are more misclassifications in these regions
than in the dataset as a whole. Box a highlights a region where area ratio has a liquid frac-
tion near 1.0 across all size categories, Holroyd has a liquid
fraction of about 0.75 for small particles and 0.0 for large par-
ticles, and UWILD has a liquid fraction of 1.0 for the highest
temperatures and 0.5 for temperatures near 0 ◦C. Since tem-
perature ranges from 0 to 10 ◦C here, we expect that the par-
ticles are mainly liquid, although large ice particles can per-
sist at temperatures above 0 ◦C. Furthermore, quasi-spherical
frozen particles can have a close resemblance to large liquid
drops, and the two cannot necessary be distinguished by eye
from 2D-S images. In both boxes c and d, UWILD and Holroyd likely mis-
classify several large particles as ice, and those particles are
largely associated with low confidence in UWILD. It is dif-
ficult to quantify this bias because there are several particles
that could be either quasi-spherical frozen particles or large
droplets and cannot be distinguished by eye. This does not
mean that UWILD cannot classify those particles because it
uses inter-arrival time in addition to image-derived parame-
ters to make classifications. Randomly sampled particles from this region appear to be
predominantly liquid due to the absence of rough edges along
the perimeter and the high prevalence of quasi-spherical
habits (Fig. 9a). Out of 50 randomly sampled images, area
ratio classifies 1 particle as ice, Holroyd classifies 21 parti-
cles as ice, and UWILD classifies 12 particles as ice. R. Atlas et al.: University of Washington Ice–Liquid Discriminator (UWILD) Atlas et al.: University of Washington Ice–Liquid Discriminator (UWILD) A 2D histogram of the number of particles meeting our criteria (row 1), UWILD confidence (row 2), and phase classifications
ree algorithms (rows 3–5) are shown in temperature–particle size phase space in the left column and relative-humidity–particle-
e space in the right column. A threshold of 100 total particles per 2D histogram bin is used for both plots. SOCRATES flights
14 are included. Figure 8. A 2D histogram of the number of particles meeting our criteria (row 1), UWILD confidence (row 2), and phase classifications
for the three algorithms (rows 3–5) are shown in temperature–particle size phase space in the left column and relative-humidity–particle-
size phase space in the right column. A threshold of 100 total particles per 2D histogram bin is used for both plots. SOCRATES flights
RF01–RF14 are included. that we expect at the lowest temperatures and the largest par-
ticles sizes, and in subsaturated regions. uid saturation. UWILD is the only algorithm for which the
50 % liquid fraction (in white) shifts to smaller sizes as RH
decreases below 100 %. Thus, UWILD’s lower liquid frac-
tions in regions with RH < 100 %, for particles in the mid-
sized particle range (0.1 < Deq < 0.2 mm), are more realistic
than Holroyd’s and area ratio’s higher liquid fractions. The dashed boxes labeled a–d on the 2D histograms in
the left column of Fig. 8 highlight areas of disagreement
between the models, whereas box e highlights agreement
between the models regarding the presence of supercooled
water at −35 ◦C. Figure 9 shows randomly sampled images
from each of the five regions within the dashed boxes. Each
particle image has the UWILD confidence displayed above
the particle and the phase classifications from all three algo-
rithms displayed below the particle. Since we have chosen
to primarily focus on areas of disagreement between the al- UWILD is the only algorithm of the three that can achieve
liquid fractions near 0.0 and near 1.0, in both temperature vs. particle size space (Fig. 8c) and RH vs. particle size space
(Fig. 8h). Thus, it has the flexibility to represent both (i) the
liquid-only regions that we expect at the highest temperatures
and near-liquid saturation and (ii) the ice-dominated regions https://doi.org/10.5194/amt-14-7079-2021 Atmos. Meas. Tech., 14, 7079–7101, 2021 7091 R. R. Atlas et al.: University of Washington Ice–Liquid Discriminator (UWILD) Of the
sampled particles classified by UWILD with a confidence
≥75 %, all but one of them appear to be properly identi-
fied as liquid. The only exception is one particle with a con-
fidence of 81 %, which is likely misclassified due to being
truncated at the end of the image buffer and having a lower
area ratio as a result. A greater proportion of particles with a
confidence below 75 % are classified as ice by UWILD but
are likely liquid, comprising about 55 % of the sampled par-
ticles for these lower confidences. The misclassifications can
be removed from UWILD, if desired, by filtering out parti-
cles that have a confidence of less than 75 % and/or are touch-
ing the edge of the image buffer. Holroyd misclassifies nine
more liquid particles as ice than UWILD but does not pro-
vide a measure of confidence that can be used to assess the
likelihood of misclassification. Of the particles that UWILD
likely misclassifies as ice, many have particularly large Pois-
son spots. Area is computed from shadowed diodes exclu-
sively, so Poisson spots are not included. Additionally, parti-
cles with Poisson spots are resized following Korolev (2007). Both of these factors affect the calculation of area ratio and,
thus, the phase classification of the particle. Box e (Fig. 9e) highlights a region where all three-
phase discrimination algorithms have liquid fractions greater
than 0.5 despite sampling very low temperatures (−33 to
−36 ◦C). Randomly sampled images with high confidence in
UWILD have spherical habits, and most have Poisson spots
as well, suggesting that the particles in this region are pri-
marily liquid. UWILD and area ratio classify all 20 randomly
sampled images as liquid, whereas Holroyd classifies 4 par-
ticles in the sample as ice. These particles are particularly
small and lack Poisson spots, so we cannot identify their
phase by eye. Nevertheless, it is clear that all three algorithms
correctly identify this region as liquid-dominated. These par-
ticles were sampled during a period from RF03 that is plotted
in the second half of Fig. 4e, where the aircraft skirted the top
of an altostratus layer. 5
Applications Box b highlights a temperature and size range where
UWILD and area ratio are in agreement that the liquid frac-
tion is near 1.0 but where Holroyd has a lower liquid fraction
of about 0.75. The 50 randomly sampled images shown in
Fig. 9b appear to be mostly liquid with some irregular small
ice crystals also present. UWILD performs better for these
temperatures and particle size ranges than in Box a; it clas-
sifies four particles as ice, of which one is clearly liquid and
three are unidentifiable by eye. Holroyd classifies 20 parti-
cles as ice, of which most are likely misclassifications. Atmos. Meas. Tech., 14, 7079–7101, 2021 5.1
Particle size distributions We use UWILD’s classifications and confidences to com-
pute median 1 Hz liquid and frozen particle size distri-
butions (PSDs) and uncertainties for all SOCRATES data
(14 flights). We show average PSDs for five different temper-
ature ranges and for the whole dataset, in Fig. 10. The x axis
is Deq (consistent with other figures) and the y axis is the
particle concentration normalized by the common logarithm
of the bin width. Both axes are plotted on a log scale. The
dashed lines are deterministic distributions, which means
they are generated using the UWILD classifications without
taking the model confidence into account. All classified par-
ticles are used for this analysis regardless of model confi-
dence. The solid lines and shaded areas around them are the Boxes c and d (Fig. 9c and d) both highlight regions where
UWILD and Holroyd are in agreement that the liquid fraction
is near 0.0 but where area ratio has a higher liquid fraction. The discrepancy is larger for box d, where area ratio has a
liquid fraction of about 0.75. Out of 45 randomly sampled https://doi.org/10.5194/amt-14-7079-2021 Atmos. Meas. Tech., 14, 7079–7101, 2021 R. Atlas et al.: University of Washington Ice–Liquid Discriminator (UWILD) 7092 Randomly sampled images are shown for the five regions overlaid on the temperature–particle-size phase space in Fig. 8. U
is displayed above each particle, and, from left to right, UWILD phase (green), area ratio phase (blue), and Holroyd phas
ed below each particle. The time dimension is vertical, and the photodiode dimension is horizontal. Figure 9. Randomly sampled images are shown for the five regions overlaid on the temperature–particle-size phase space in Fig. 8. UWILD
confidence is displayed above each particle, and, from left to right, UWILD phase (green), area ratio phase (blue), and Holroyd phase (red)
are displayed below each particle. The time dimension is vertical, and the photodiode dimension is horizontal. median and interquartile range of 30 bootstrapped samples
which are generated using the model confidence. For exam-
ple, if a particle is classified as ice with 75 % confidence then
it is considered an ice particle for the deterministic distribu-
tion, but, on average, it will be considered an ice (liquid) par-
ticle in 75 % (25 %) of the bootstrapped samples. https://doi.org/10.5194/amt-14-7079-2021 5.1
Particle size distributions The concen-
trations of medium-sized (0.1 mm < Deq < 0.3 mm) and
large (Deq > 0.3 mm) particles decrease as temperature in-
creases. Medium-sized particles are ice-dominated between
−40 and −20 ◦C (Fig. 10a) and liquid-dominated above
−5 ◦C (Fig. 10c–e). Large particles are liquid-dominated
above 5 ◦C (Fig. 10e), where drizzle formation becomes
the dominant mode of precipitation. The largest particles
(Deq > 1 mm) are ice-dominated at all temperatures but
have small concentrations (< 2 × 10−4 cm−3) above 5 ◦C
(Fig. 10e). The crossover point, or the Deq at which the
PSDs transition from being liquid-dominated to being ice-
dominated, is 0.1 mm for temperatures between −40 and
−20 ◦C (Fig. 10a), 0.17–0.33 mm for temperatures between
−20 and 5 ◦C (Fig. 10b–d) and 0.7 mm at higher tempera-
tures (Fig. 10e). The crossover point for the whole dataset is
0.17 mm (Fig. 10f), which is in agreement with the phase
classifications from the PHIPS for all SOCRATES flights
(Waitz et al., 2021, figure 8). Small ice crystals and large
liquid drops are associated with the most uncertainty at all
temperature ranges. We derive an estimate of sub-1 Hz heterogeneity by con-
sidering whether adjacent particles in the 2D-S image buffer
are of the same phase or of different phases, which we de-
note as a phase “flip” (from ice to liquid or from liquid to
ice). If, for a 1 s period, there are many phase flips given the
number of particles, that sample is more heterogeneous than
one where there are few (or no) phase flips for a population
of particles. We leverage the fact that our classifications are
probabilistic in determining phase flips and create a proba-
bilistic phase flip prediction as well. Given two adjacent par-
ticles p1 and p2, In Sect. 4, we showed that UWILD misclassifies some
large liquid particles as ice. We examined 200 randomly sam-
pled images of particles with Deq > 0.16 mm from temper-
atures between 0 and 5 ◦C and found that 16 % of parti-
cles classified as ice are actually liquid (not shown). These
misclassified particles universally lack Poisson spots. Thus,
large particles within that temperature range are indeed ice-
dominated but to a lesser extent than what the PSDs sug-
gest. 5.1
Particle size distributions Note that
due to the log scale on the y axis, the effect of bootstrapping
is mainly noticeable where concentrations are small and is
strongest where the differences between ice and liquid con-
centrations span orders of magnitude. Note also that the boot-
strapped distributions fall entirely between the determinis- tic distributions. This can be understood by considering, for
example, the smallest particles just below 0 ◦C, where there
are approximately 100 times as many liquid particles as ice. If, when taking into consideration model confidence, 2 % of
the liquid particles are reclassified as ice, this is barely no-
ticeable in the liquid particle concentration but results in a
tripling of the ice particle concentration (Fig. 10c). The boot-
strapped PSDs are better representations of the true PSDs, so
considering model confidence is most essential in the areas
in Fig. 10 where there are large discrepancies between the https://doi.org/10.5194/amt-14-7079-2021 Atmos. Meas. Tech., 14, 7079–7101, 2021 7093 R. Atlas et al.: University of Washington Ice–Liquid Discriminator (UWILD) geneity. Cloud phase heterogeneity, or the degree to which
liquid and ice particles are evenly mixed within mixed-phase
clouds, can influence cloud radiative (Sun and Shine, 1994)
and thermodynamic properties (Korolev et al., 2017). It may
also modulate the rates of certain mixed-phase processes
such as the Wegener–Bergeron–Findeisen process (Tan and
Storelvmo, 2016) and has implications for how those pro-
cesses should be parameterized in microphysics models. Most studies of cloud phase heterogeneity from in situ ob-
servations have computed heterogeneity metrics using 1 Hz
phase data (Cober et al., 2001a; Korolev et al., 2003; Field
et al., 2004; D’Alessandro et al., 2019, 2021). Field et al. (2004) used PSDs from the Small Ice Detector in combina-
tion with other in situ measurements to identify cloud phase
and found that segments as short as 100 m could contain both
liquid and ice. They used 1 Hz data but could investigate rel-
atively small length scales due to low aircraft speeds (100–
120 ms−1). Here, we use single-particle phase classifications
to investigate sub-1 Hz cloud phase heterogeneity, and we
identify mixed-phase periods on the meter-scale. deterministic and bootstrapped distributions (e.g., estimating
sub-millimeter ice particle concentrations around 0 ◦C). Within the SOCRATES dataset, small particles (Deq <
0.1 mm) are more likely to be liquid at all temperature
ranges but have much higher concentrations and are more
liquid-dominated above −20 ◦C (Fig. 10b–e). 5.1
Particle size distributions We also examined 200 randomly sampled 2D-S images
of particles with Deq > 1 mm from temperatures between 5
and 40 ◦C and found that all particles are classified as ice but
are actually liquid (not shown). These particles have an elon-
gated shape due to being distorted by the instrument inlet,
lack a Poisson spot, and often touch the edge of the image
buffer so that they are partially cut off. We note that classi-
fication skill decreases in general for particles approaching
1 mm because particles of this size are less likely to be fully
imaged by the instrument. Thus, we caution that phase clas-
sifications for the largest particles may be less certain and
that the 5–40 ◦C temperature range is particularly affected
by misclassifications of large drops as ice. We note that there
are so few of these large misclassified liquid particles at high
temperatures that they do not show up in Fig. 8c, which uses
a threshold of 100 particles for each 2D histogram bin. P(flip) = P(p1 = ice) × P(p2 = liquid)
+ P(p1 = liquid) × P(p2 = ice). (3) (3) We estimate the most likely number of flips over all parti-
cles within a given sample by adding these probabilities to-
gether. Thus a hypothetical sample containing 100 particles
may have between 0 flips (completely homogeneous sam-
ple with 100 % classification confidence on all particles) and
99 flips (particles are alternating 100 % likely ice and 100 %
likely liquid), although both of these extremes are unlikely
with our probabilistic estimate. A limitation of this hetero-
geneity estimate is that it implicitly assumes that phase flip
probabilities are independent. An advantage of this metric is
that it avoids using particle mass to compute phase hetero-
geneity. Ice particle mass estimated from 2D-S images can
vary over an order of magnitude depending on the assumed
mass–dimensional relationship (Wu et al., 2019), and more
reliable measurements from a Nevzorov instrument with a
deep cone (Korolev et al., 2003) were not available during
SOCRATES. Atmos. Meas. Tech., 14, 7079–7101, 2021 5.2
Cloud phase heterogeneity We create a 1 Hz heterogeneity measure, which we refer
to as the phase flip fraction, by dividing the number of prob-
abilistic phase flips described above by the total number of We also use UWILD’s classifications and confidences to
compute a 1 Hz estimate of sub-1 Hz cloud phase hetero- https://doi.org/10.5194/amt-14-7079-2021 Atmos. Meas. Tech., 14, 7079–7101, 2021 7094 R. Atlas et al.: University of Washington Ice–Liquid Discriminator (UWILD) 7094
R. Atlas et al.: University of Washington Ice–Liquid Discriminator (UWILD)
Figure 10. Particle size distributions are averaged over 1 Hz data from SOCRATES flights RF01–RF14 and shown for six different temper-
ature ranges. Dashed lines are deterministic predictions from UWILD. Solid lines and shaded areas are medians and interquartile ranges,
respectively, for 30 bootstrapped samples generated from UWILD confidences. Red dashed lines separate the three different size classes. Figure 10. Particle size distributions are averaged over 1 Hz data from SOCRATES flights RF01–RF14 and shown for six different temper-
ature ranges. Dashed lines are deterministic predictions from UWILD. Solid lines and shaded areas are medians and interquartile ranges,
respectively, for 30 bootstrapped samples generated from UWILD confidences. Red dashed lines separate the three different size classes. particles imaged by the 2D-S within 1 s. We implicitly as-
sume that all unclassified particles, which are mainly par-
ticles with fewer than 25 pixels and Deq < 0.056 mm, are
liquid, which is a reasonable extrapolation from our PSDs
(Fig. 10f), as only 1 % of the smallest particles are classified
as ice. However, this assumption may lead to an underesti-
mate in phase flips for the coldest samples (−40 ◦C < T <
−20 ◦C), where there are similar numbers of small droplets
and small ice crystals (Fig. 10a). Figure 11. A 2D histogram of 1 Hz particle phase flips (y axis) and
1 Hz total particles (including both successfully classified particles
and unclassifiable particles) is shown in the large plot. A 1D his-
togram of 1 Hz total particles is shown on top, on a shared x axis,
and a 1D histogram of 1 Hz phase flips per second is shown on the
right, on a shared y axis. A value closer to the upper left of the plot
indicates a higher degree of particle heterogeneity. White lines in-
dicate lines of constant heterogeneity for varying particle counts. SOCRATES flights RF01–RF14 are included. 5.2
Cloud phase heterogeneity Red indicates ice classifications with confidences greater than 75 %; blue indicates liquid
classifications with confidences greater than 75 %; and purple indicates ice or liquid classifications with confidences less than 75 % Figure 12. Short periods from two different cloud types highlight instances of meter-scale heterogeneity in the SOCRATES dataset. The
photodiode dimension is vertical, and the time dimension is horizontal, which is the reverse of Fig. 9. Particle labels show UWILD classi-
fications and UWILD confidences in parentheses. Red indicates ice classifications with confidences greater than 75 %; blue indicates liquid
classifications with confidences greater than 75 %; and purple indicates ice or liquid classifications with confidences less than 75 % cations with confidence ≥75 %; and purple labels for ice or
liquid classifications with confidence < 75 %. paign in January–February of 2018. The SOCRATES dataset
includes nearly 6 million Two-Dimensional Stereo (2D-S)
shadow images of particles with 25 or more pixels and
with area equivalent diameters (Deq) greater than 0.056 mm,
which are good candidates for single-particle phase classi-
fication. Here, we introduce the University of Washington
Ice–Liquid Discriminator (UWILD), a phase classification
algorithm that takes a random forest approach, and show that
it outperforms two existing phase classification algorithms
which have been applied to 2D-S images from SOCRATES. In particular, UWILD has the flexibility to identify both
liquid-dominated and ice-dominated regions in the dataset,
whereas the other two algorithms both demonstrate strong
biases in favor of one phase. UWILD also returns a model
confidence for each classification, which is invaluable for
computing uncertainties on variables derived from its clas-
sifications and for filtering out particles that have a higher
likelihood of being misclassified. We believe that the perfor-
mance of UWILD is limited largely by the SOCRATES 2D-S
data quality (many particle images are out of focus) and the
challenges of building a train–test–validation (TTV) set and
not the choice of machine learning model. Thus, we would
expect to see only modest gains in performance from em-
ploying more sophisticated machine learning models. q
In the example in the first row, there is a pocket of small
droplets surrounded by large ice crystals within a 3.4 m seg-
ment of cloud. There are three particles within the pocket that
have low model confidence and thus may be small droplets or
small ice crystals. This example resembles the conditionally
mixed-phase condition described by Korolev et al. 5.2
Cloud phase heterogeneity Figure 11 shows a 2D histogram with phase flips on the
x axis and the total number of 2D-S imaged particles on the
y axis, with white lines indicating lines of constant phase
flip fraction. We see two distinct modes in this phase space:
1 s samples with total particle counts below 1000 typically
have between 0.02 and 0.5 flips per particle, whereas 1 s sam-
ples with total particle counts between 3000 and 30 000 typi-
cally have between 0.002 and 0.0005 flips per particle. In the
SOCRATES dataset, high total particle counts generally in-
dicate the presence of many small droplets. Since these sam-
ples are dominated in number by the liquid phase, they have
low phase heterogeneity. Figure 11. A 2D histogram of 1 Hz particle phase flips (y axis) and Figure 11. A 2D histogram of 1 Hz particle phase flips (y axis) and
1 Hz total particles (including both successfully classified particles
and unclassifiable particles) is shown in the large plot. A 1D his-
togram of 1 Hz total particles is shown on top, on a shared x axis,
and a 1D histogram of 1 Hz phase flips per second is shown on the
right, on a shared y axis. A value closer to the upper left of the plot
indicates a higher degree of particle heterogeneity. White lines in-
dicate lines of constant heterogeneity for varying particle counts. SOCRATES flights RF01–RF14 are included. Figure 12 shows two examples of meter-scale phase het-
erogeneity. The statistics of the 1 s periods containing the
plotted segments are included to the right of the image strips. Grey lines bound each particle, and all particles that are large
enough to be classified and are not suspected artifacts are la-
beled with their UWILD classifications and the model confi-
dence in parentheses. Red labels are used for ice classifica-
tions with confidence ≥75 %; blue labels for liquid classifi- https://doi.org/10.5194/amt-14-7079-2021 Atmos. Meas. Tech., 14, 7079–7101, 2021 7095 R. Atlas et al.: University of Washington Ice–Liquid Discriminator (UWILD) Figure 12. Short periods from two different cloud types highlight instances of meter-scale heterogeneity in the SOCRATES dataset. The
photodiode dimension is vertical, and the time dimension is horizontal, which is the reverse of Fig. 9. Particle labels show UWILD classi-
fications and UWILD confidences in parentheses. 5.2
Cloud phase heterogeneity (2017),
where the cloud is single phase if you look at a small enough
length scale. For the segment of cloud shown, that length
scale is approximately 1 m. The second row shows alternat-
ing liquid and ice particles of similar size within a 2.8 m
segment of cloud. Here, there is a larger proportion of low-
confidence particles that could be either liquid or ice. This is
a result of UWILD’s tendency, described in Sect. 4, to clas-
sify some large liquid drops as ice. There is a sequence of
four particles classified as I–L–L–I, all with high confidence,
towards the center of the image strip. This example more
closely resembles the genuinely mixed-phase condition de-
scribed by Korolev et al. (2017) because the phase changes
every one or two particles. The difference in the heterogene-
ity between the two regions is captured in the 1 s flips per
particle, which is about twice as high for the period in the
bottom row than it is for the period in the top row. However,
there may be fewer or greater flips per particle in the periods
shown than in the 1 s periods if the periods shown are not
representative of the entire 1 s periods. Since many hydrometeors within mixed-phase clouds are
unidentifiable by eye, we use in situ observations of atmo-
spheric parameters to select liquid-only and ice-dominated
periods within SOCRATES to build a TTV set. If we had
limited the TTV set to particles identifiable by eye, as most
studies applying machine learning to airborne probe images
have done, the TTV set would be less representative of the
SOCRATES dataset. UWILD’s most prominent bias is the
misclassification of larger liquid particles (Deq > 0.2 mm)
as ice. A small percentage (∼0.6 %) of particles in the ice-
dominated training data are likely liquid, and this may con- Appendix A: Particle features used for phase
classification tribute to this bias. UWILD’s lower skill in distinguishing
larger liquid drops from ice particles is shared by Holroyd
and by the automated Particle Habit Imaging and Polar Scat-
tering (PHIPS) algorithm which uses scattering phase func-
tions to identify particle sphericity. Thus, we posit that dis-
tinguishing drizzle droplets and raindrops from ice crystals is
a major outstanding problem in hydrometeor phase discrim-
ination within mixed-phase clouds. We also note that larger
liquid drops that are misclassified by UWILD typically have
model confidence below 75 % or are touching the edge of the
image buffer and can be easily filtered out of the dataset if
desired. Throughout this section, we use the term “particle property”
to refer to any quantity that is computed from the 2D-S
dataset. We use the term “particle feature” to refer to particle
properties that we input into UWILD. Figure A1 shows how
particle features are computed from 2D-S images. Particle
properties that can be represented visually are shown in the
top two boxes, using two example particles. Particle proper-
ties in the bottom box are functions of the particle properties
shown in the top two boxes and cannot directly be visual-
ized. All of the particle properties shown in Fig. A1 are parti-
cle features except “smallest bounding circle”, which is used
to compute max_dimension and area_ratio, and “perimeter”,
which is used to compute fine_detail_ratio. Underscores are
left out of the particle names in Fig. A1. We use classifications and confidence from UWILD to
generate particle size distributions (PSDs) for the whole
SOCRATES dataset and find that hydrometeors with Deq
greater than 0.15 mm are ice-dominated and that smaller par-
ticles are liquid-dominated, which is in agreement with phase
classifications from the PHIPS for the SOCRATES dataset. We also develop a novel estimate of sub-1 Hz phase hetero-
geneity by tallying the number of probabilistic phase flips
per particle within 1 s periods, which we refer to as the phase
flip fraction. This particle-number-based approach to esti-
mating heterogeneity avoids uncertainties in estimating par-
ticle mass. We use the phase flip fraction to identify two pe-
riods in the SOCRATES dataset exhibiting meter-scale phase
heterogeneity. We show histograms of 14 particle features for the TTV
set and the 14 SOCRATES flights analyzed in this study, in
Fig. A2. We do not include the parameter touching_edge in
Figs. A1 or A2 because it is binary. Appendix A: Particle features used for phase
classification The histograms are plot-
ted on a log scale so that the tails of their distributions are
visible. About 0.6 % of particle images from the SOCRATES
dataset that we analyzed here are out of sample. This means
that at least one particle parameter has a value that is out-
side of the range of the TTV set. Such particles are usually
out of sample because they are larger than all of the particles
in the TTV set. We examined 45 randomly sampled images
from the SOCRATES dataset that have an area-equivalent di-
ameter (Deq) greater than all particles in the TTV set. These
particles are mainly heavily rimed aggregates. All of them
are clearly frozen, and UWILD classifies them as such. Thus,
we do not believe that the small percentage of out-of-sample
particles present in the SOCRATES dataset reduces the per-
formance of UWILD. g
y
The SOCRATES campaign sampled mixed-phase South-
ern Ocean clouds with the goal of improving their represen-
tation in climate models. Here, we process SOCRATES in
situ observations from the 2D-S to create datasets that can
be more effectively compared with atmospheric models and
remote sensing datasets. Liquid and ice PSDs can be used to
evaluate microphysical model output. Single-particle phase
classifications can be coarsened and compared with cloud
top phase products from Himawari and MODIS. Single-
particle classifications from UWILD, as well as the 1 Hz
variables that we derive from them, are also useful for in-
forming the development of microphysics parameterizations. Large droplets are necessary for Hallett–Mossop rime splin-
tering and droplet freezing, and estimating their concentra-
tions from liquid classifications of 2D-S images is useful for
determining an upper bound on the rate of these ice pro-
duction processes. Additionally, mixed-phase processes such
as the Wegener–Bergeron–Findeisen mechanism for rapid
ice growth and Hallett–Mossop rime splintering may oper-
ate more slowly in conditionally mixed-phase clouds, which
have a small number of flips per particle, than genuinely
mixed-phase clouds, which have a large number of flips per
particle. UWILD is publicly available, and we encourage
readers to adapt it for other in situ datasets to examine pro-
cesses controlling phase partitioning in mixed-phase clouds
globally. Area ratio is greater than 1.0 about 25 % of the time. R. Atlas et al.: University of Washington Ice–Liquid Discriminator (UWILD) R. Atlas et al.: University of Washington Ice–Liquid Discriminator (UWILD) 7096 Atmos. Meas. Tech., 14, 7079–7101, 2021 6
Conclusions In situ observations of Southern Ocean cloud phases, vi-
tal for evaluating simulations and remote sensing products,
were sparse prior to the Southern Ocean Clouds, Radiation,
Aerosol Transport Experimental Study (SOCRATES) cam- https://doi.org/10.5194/amt-14-7079-2021 Atmos. Meas. Tech., 14, 7079–7101, 2021 R. Atlas et al.: University of Washington Ice–Liquid Discriminator (UWILD) References Abdelmonem, A., Järvinen, E., Duft, D., Hirst, E., Vogt, S.,
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2D-S
(https://data.eol.ucar.edu/
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https://dash.harvard.edu/bitstream/1/3355830/1/Huybers_AntarcticSummerDuration.pdf
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English
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Antarctic temperature at orbital timescales controlled by local summer duration
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Nature geoscience
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Published Version
http://dx.doi.org/10.1038/ngeo311 http://nrs.harvard.edu/urn-3:HUL.InstRepos:3355830 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available
under the terms and conditions applicable to Other Posted Material, as set forth at http://
nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#LAA Antarctic temperature at orbital timescales
controlled by local summer duration PETER HUYBERS1* AND GEORGE DENTON2
1Harvard University, Cambridge, Massachusetts 02138, USA
2University of Maine, Orono, Maine 04469, USA
*e-mail: phuybers@fas.harvard.edu PETER HUYBERS1* AND GEORGE DENTON2 Published online: 21 September 2008; doi:10.1038/ngeo311 During the late Pleistocene epoch, proxies for Southern Hemisphere climate from the Antarctic ice cores vary nearly in phase with
Northern Hemisphere insolation intensity at the precession and obliquity timescales. This coherence has led to the suggestion that
Northern Hemisphere insolation controls Antarctic climate. However, it is unclear what physical mechanisms would tie southern
climate to northern insolation. Here we call on radiative equilibrium estimates to show that Antarctic climate could instead respond to
changes in the duration of local summer. Simple radiative equilibrium dictates that warmer annual average atmospheric temperatures
occur as a result of a longer summer, as opposed to a more intense one, because temperature is more sensitive to insolation when the
atmosphere is cooler. Furthermore, we show that a single-column atmospheric model reproduces this radiative equilibrium effect
when forced exclusively by local Antarctic insolation, generating temperature variations that are coherent and in phase with proxies
of Antarctic atmospheric temperature and surface conditions. We conclude that the duration of Southern Hemisphere summer is more
likely to control Antarctic climate than the intensity of Northern Hemisphere summer with which it (often misleadingly) covaries. In
our view, near interhemispheric climate symmetry at the obliquity and precession timescales arises from a northern response to local
summer intensity and a southern response to local summer duration. Southern Hemisphere climate proxies follow northern summer
insolation intensity1–7, leading to the common interpretation
that northern insolation controls southern climate, at least at
the obliquity and precession timescales2,5,7. Proposed mechanisms
include northern insolation influencing atmospheric CO2(refs 8,9),
North Atlantic Deep Water affecting the Southern Ocean10,11,
and variations in the extent of northern glaciation influencing
the south7. However, such explanations are problematic because
conventional statistical techniques indicate that southern changes
are in phase with or lead those in the north1,2,4,8,12–15—a situation
Mercer12 called ‘a fly in the ointment of the Milankovitch theory’. intensity values.) This covariation arises because the precession
of the equinoxes results in opposite effects on the intensity
and duration of summer seasons within a single hemisphere17,
and opposite effects on intensity and duration of summer
seasons between the two hemispheres. Increases in obliquity cause
symmetric increases in intensity and duration of summer at high
latitudes in both hemispheres. Share Your Story The Harvard community has made this article openly available. Please share how this access benefits you. Submit a story . The Harvard community has made this article openly available. Please share how this access benefits you. Submit a story . Accessibility Antarctic temperature at orbital timescales
controlled by local summer duration Therefore, although the signature
of orbital variations is discernible within high-latitude southern
climate records, signal processing alone cannot distinguish between
northern or southern insolation forcing. Determining the origins
of orbital-period variability in southern climate proxies requires
identification of physical mechanisms that connect insolation at
the top of the atmosphere with proxy signals in ice and in
marine sediments. y
y
Although northern forcing of Antarctic climate cannot be ruled
out, particularly if small or otherwise difficult-to-detect northern
perturbations precede those in the south8, the lack of an obvious,
operative mechanism to produce symmetric interhemispheric
changes at the precession period prompts further examination of
the connection between Earth’s orbit and southern climate. Our
focus is on the obliquity and precession bands in the climate record,
largely to the exclusion of more rapid variations and of ∼100 kyr
glacial cycles. Nonetheless, the climate spectrum is coupled across
timescales16, and we expect investigation of the orbital response to
improve our understanding of the continuum of climate variability. Perhaps
the
most
fundamental
relationship
between
temperature and insolation is that of radiative equilibrium,
F = σT4. F is the absorbed incoming solar radiation, T is
the temperature in Kelvin and σ is the Stefan–Boltzmann
constant. The precession of the equinoxes redistributes insolation
throughout the seasons but has no influence on the total annual
insolation18. Therefore, there must be a nonlinear response to
insolation if precession is to exert any control on mean annual
temperature. Radiative equilibrium furnishes such a nonlinearity. The radiative equilibrium temperature is less sensitive to insolation
at higher temperatures, dT/dF = 1/(4σT3), with a value of
0.41 K/(W m−2) for typical winter atmospheric temperatures
in Antarctica (T = 220 K) and 0.25 K/(W m−2) for summer
conditions (T = 260 K). Thus, the annual average radiative
equilibrium temperature will be greatest when insolation is spread
away from peak summer intensity—as occurs when precession nature geoscience ADVANCE ONLINE PUBLICATION www.nature.com/naturegeoscience COMPARISON OF MODEL RESULTS AGAINST ICE-CORE PROXIES The SCAM results are compared against the Dome F (ref. 7)
ice-core record of δ18Oice (Fig. 3). A full interpretation of this proxy
requires an understanding of the tracer’s time-variable evaporation,
transport, precipitation, and ultimate densification into ice26,27, but
we adopt the simple and common interpretation of mean annual
atmospheric temperature above the deposition site. The variability
in SCAM’s annual average atmospheric temperature is coherent
and consistent with an in-phase relationship with the Dome F
δ18Oice record at both the obliquity and the precession bands (see
Fig. 4). SCAM’s temperature results are likewise coherent and in
phase with the temperature proxy record from Dome C, which is
also located on the high East Antarctic plateau (see Supplementary
Information). Similar orbital-band variability is generated when
SCAM is specified to have zero heat-flux convergence, double
or half the modern CO2 concentration, or is run in a clear-sky
mode—indicating that the orbital response is robust to changes
in the model parametrization and to changes in climate. These
model results support our hypothesis that local variations in
the duration of summer control the orbital-period variability
in Antarctic atmospheric temperature. In this view, similarities
between southern proxies and northern summer insolation
intensity are a red herring, pointing to the wrong hemisphere. p
g q
Is simple radiative balance a reasonable first-order description
of the atmospheric temperature variability? To explore this question
in more detail, we examine the response of the Single Column
Atmospheric Model (SCAM, see Methods section)23,24 to variations
in insolation. When run over the course of the past 350,000 years at
Dome F, SCAM produces a mean annual tropospheric temperature
that corresponds to the changes in duration of the Antarctic
summer. Subtracting the seasonal cycle of temperature associated
with perihelion occurring at summer solstice from that associated
with aphelion at summer solstice reveals a pattern of anomalous
spring and early summer warming that extends from the lower
troposphere (∼5 ◦C) up through the stratosphere (∼10 ◦C, Fig. 2). An increase in obliquity also raises the atmospheric temperature
in SCAM, as well as in the simple energy-balance model, but here
we focus on precession because the obliquity-induced changes are
symmetric between the hemispheres and, thus, do not enable us
to distinguish between southern3,6 or northern2,7,8 control. Note δO2/N2 ice-core records from Dome F (ref. RADIATIVE EQUILIBRIUM AND THE DURATION OF SUMMER We build on the fact that the duration of southern summer covaries
almost identically with northern summer insolation intensity
(Fig. 1). (Here summer duration is defined as the number of days in
which the diurnal average insolation intensity exceeds 250 W m−2,
a threshold which gives good agreement with northern solstice nature geoscience ADVANCE ONLINE PUBLICATION www.nature.com/naturegeoscience 1 © 2008 Macmillan Publishers Limited. All rights reserved. © 2008 Macmillan Publishers Limited. All rights reserved. ARTICLES
425
450
475
500
525
550
Insolation intensity (W m–2),
June 21, 65° N
Time (kyr)
120
125
130
135
Summer duration (d), 77° S
Winter duration (d), 77° S
230
235
240
245
Summer energy (GJ m–2), 65° N
0
–50
–100
–150
–200
–250
–300
–350
2.50
2.75
3.00
3.25
3.50
3.75
4.00
Figure 1 Different measures of insolation covary with one another. Examples are the summer energy17 at 65◦N using a threshold of 400Wm−2 (yellow), the diurnal
average insolation intensity at the top of the atmosphere for the summer solstice at 65◦N (black), the duration of the summer at 77◦S (red, measured as the number of days
whose diurnal average insolation exceeds 250Wm−2) and the number of winter days (hidden beneath the summer duration curve, measured as days below 250Wm−2; note
the reversed y axis). Figure 1 Different measures of insolation covary with one another. Examples are the summer energy17 at 65◦N using a threshold of 400Wm−2 (yellow), the diurnal
average insolation intensity at the top of the atmosphere for the summer solstice at 65◦N (black), the duration of the summer at 77◦S (red, measured as the number of days
whose diurnal average insolation exceeds 250Wm−2) and the number of winter days (hidden beneath the summer duration curve, measured as days below 250Wm−2; note
the reversed y axis). that the peculiarities of the Antarctic climate represented in SCAM
seem to make local radiative balance an important control on
temperature. A strong thermal inversion layer exists over Antarctica
for most of the year25 because the ice surface emits radiation more
efficiently than the atmosphere. As a result, convection and the
exchange of heat between the troposphere and the ice surface is
inhibited. Furthermore, Antarctic temperatures rarely exceed the
freezing point, and such low temperatures are associated with
consistently small amounts of water vapour in the atmosphere,
hence muting changes in emissivity and absorptivity. aligns aphelion with summer solstice. RADIATIVE EQUILIBRIUM AND THE DURATION OF SUMMER We note that Milankovitch19
also considered the influence of orbital variations on the radiative
equilibrium temperature but used a fixed half-year interval, thereby
excluding effects associated with changes in the duration of the
seasons. Rubincam20 determined that annual average radiative
equilibrium temperature would be highest for an orbit that aligns
aphelion with summer solstice but considered only cases with
neither heat capacity nor an atmosphere. The influence of orbital variations on radiative equilibrium
temperature can be more fully demonstrated using a simple
energy balance model (see Methods section). The seasonal cycle
of insolation at 77◦S, the latitude of the Dome F ice core on
the high plateau of the East Antarctic Ice Sheet, is considered
under two scenarios: one when Earth is at perihelion during
southern summer solstice, averaged over the 15 occurrences in
the past 350 kyr, and another averaged over the times when
aphelion coincides with summer solstice (Fig. 2). Although the
total insolation is equivalent in the two cases of precessional
alignment, the second forcing scenario leads to warmer annual
average temperatures because increased insolation in spring or early
summer (when atmospheric temperatures are lower) causes more
warming relative to the magnitude of cooling caused by decreased
insolation during mid-to-late summer (when temperatures are
higher). Some studies call on related changes in autumn18 or
spring21,22 insolation intensity to influence climate. Here we attempt
to address the full seasonal distribution of insolation, but note
that spring insolation intensity at high southern latitudes varies
closely with the duration of the summer (assuming the calendar
is referenced to northern spring equinox). COMPARISON OF MODEL RESULTS AGAINST ICE-CORE PROXIES 7) and another
site on the high East Antarctic Plateau at Vostok28 affords an
additional constraint regarding the influence of orbital variations
on Antarctica’s climate. δO2/N2 seems to follow the intensity
of local summer insolation, possibly because maximum seasonal
temperatures at the surface alter ice crystals and gas close-off
depths, although the exact mechanism remains uncertain28. The
maximum seasonal surface temperatures obtained in SCAM follow
summer solstice insolation and are coherent and in phase with
the δO2/N2 signal at both the obliquity and the precession
periods (Fig. 3 and Supplementary Information). Therefore, SCAM nature geoscience ADVANCE ONLINE PUBLICATION www.nature.com/naturegeoscience nature geoscience ADVANCE ONLINE PUBLICATION www.nature.com/naturegeoscience 2 © 2008 Macmillan Publishers Limited. All rights reserved. © 2008 Macmillan Publishers Limited. All rights reserved. © 2008 Macmillan Publishers Limited. All rights reserved.
© 2008 Macmillan Publishers Limited. All rights reserved. ARTICLES ARTICL
Insolation (W m–2)
Anomaly (W m–2)
Temperature anomaly (°C)
Temperature (°C)
–90
–80
–80
–70
–70
–60
–60
–60
–50
–40
–50
–50
–50
–40
–40
–40
–40
–30
–30
–30
–30
–20
–20
–10
–10
0
Pressure (hPa)
Month
Month
Month
Month
Month
Month
100
200
300
400
500
600
Pressure (hPa)
100
200
300
400
500
600
–2
–1
–1
0
0
0
0
0
0
0
0
0
0
0
1
1
1
1
2
2
2
2
2
3
3
3
3
4
4
4
5
5
6
7
8
9
0
200
400
J
J
A
S
O
N
D
J
F
M
A
M
J
J
A
S
O
N
D
J
F
M
A
M
–50
0
50
–80
–60
–40
–20
0
J
J
A
S
O
N
D
J
F
M
A
M
J
J
A
S
O
N
D
J
F
M
A
M
–10
0
10
J
J
A
S
O
N
D
J
F
M
A
M
J
J
A
S
O
N
D
J
F
M
A
M
–110
–100
a
b
c
d
e
f
e 2 The seasonal cycle in Antarctic insolation and atmospheric temperature. a, The diurnal average insolation in SCAM averaged over runs in which aphelio
and perihelion (dashed) coincide with southern summer solstice; b, the difference, which averages to zero. c, Runs of the energy-balance model forced with sum
on insolation (solid) and summer perihelion insolation (dashed); d, their differences, which show more warming than cooling for summer aphelion. Runs in c and
capacity of 2,500kJ ◦C−1 (black) and half (blue) and twice (red) this value. e, Contour plot of the average seasonal cycle in temperature (◦C) in SCAM averaged
when perihelion coincides with summer solstice; f, the relative amount of warming for average conditions when aphelion coincides with summer solstice. Time i
d from 1 June to 31 May. © 2008 Macmillan Publishers Limited. All rights reserved.
© 2008 Macmillan Publishers Limited. All rights reserved. Anomaly (W m–2)
Month
J
J
A
S
O
N
D
J
F
M
A
M
–50
0
50
b Insolation (W m–2)
Month
0
200
400
J
J
A
S
O
N
D
J
F
M
A
M
a a b Temperature (°C)
Month
–80
–60
–40
–20
0
J
J
A
S
O
N
D
J
F
M
A
M
c Temperature anomaly (°C)
Month
J
J
A
S
O
N
D
J
F
M
A
M
–10
0
10
d d c Month
Pressure (hPa)
100
200
300
400
500
600
–2
–1
–1
0
0
0
0
0
0
0
0
0
0
0
1
1
1
1
2
2
2
2
2
3
3
3
3
4
4
4
5
5
6
7
8
9
J
J
A
S
O
N
D
J
F
M
A
M
f –90
–80
–80
–70
–70
–60
–60
–60
–50
–40
–50
–50
–50
–40
–40
–40
–40
–30
–30
–30
–30
–20
–20
–10
–10
0
Pressure (hPa)
Month
100
200
300
400
500
600
J
J
A
S
O
N
D
J
F
M
A
M
–110
–100
e e f f Figure 2 The seasonal cycle in Antarctic insolation and atmospheric temperature. a, The diurnal average insolation in SCAM averaged over runs in which aphelion
(solid) and perihelion (dashed) coincide with southern summer solstice; b, the difference, which averages to zero. c, Runs of the energy-balance model forced with summer
aphelion insolation (solid) and summer perihelion insolation (dashed); d, their differences, which show more warming than cooling for summer aphelion. Runs in c and d have
a heat capacity of 2,500kJ ◦C−1 (black) and half (blue) and twice (red) this value. e, Contour plot of the average seasonal cycle in temperature (◦C) in SCAM averaged over
years when perihelion coincides with summer solstice; f, the relative amount of warming for average conditions when aphelion coincides with summer solstice. Time is
plotted from 1 June to 31 May. responds to insolation at Dome F in a manner consistent with the
δO2/N2 proxy for surface conditions and the δ18Oice proxy for mean
annual atmospheric temperature—in particular, reproducing their
antiphased precession variability. © 2008 Macmillan Publishers Limited. All rights reserved.
© 2008 Macmillan Publishers Limited. All rights reserved. and temperature, and render the observations consistent with
SCAM’s prediction of an in-phase relationship (see Supplementary
Information), particularly given additional sources of uncertainty
related to seasonality in the deposition of snow29 and the physical
relationship between δO2/N2 and insolation28. Alternatively, the
phase lag of temperature with respect to insolation could be real,
possibly resulting from slow feedbacks within the climate system
that involve processes such as atmospheric CO2. There do exist two important discrepancies between the
atmospheric temperatures derived from SCAM and the orbital-
period variations in temperature estimated from the Dome F
ice core. First, according to the age model of ref. 7, Dome F
temperatures slightly lag northern summer solstice insolation
in the obliquity (by 46◦± 63◦, 2σ uncertainty) and precession
(by 18◦± 38◦) bands, whereas SCAM predicts that insolation
and temperature are in phase. Phase uncertainties arise both
because of uncertainty in the ice-core chronology and because of
imperfect coherence between northern summer insolation intensity Second,
the
average
peak-to-trough
amplitude
of
the
temperature
change
in
SCAM
is
just
over
1 ◦C,
whereas
reconstructed temperatures from ice-core proxies6,7 have an
amplitude of about 3 ◦C at the orbital bands. The proxy
reconstruction may exaggerate the variability in annual mean
temperature because of seasonality in the deposition of snow or 3 nature geoscience ADVANCE ONLINE PUBLICATION www.nature.com/naturegeoscience ARTICLES ARTICLES δ18O temperature anomaly (°C)
115
120
125
130
135
Summer duration
(d), 77° S
Summer duration
(d), 77° S
–34
–33
–32
Annual average atm. temperature (°C)
δO2/N2
475
525
575
Insolation (W m–2)
December 21, 77° S
Insolation (W m–2)
December 21, 77° S
–15
–10
Maximum summer surface
temperature (°C)
Time (kyr)
475
525
575
–350
–300
–250
–200
–150
–100
–50
0
–8
–6
–4
–2
0
2
4
–12
–8
–4
115
120
125
130
135
a
b
c
d
Figure 3 Temperature variability in the Antarctic over the past 350kyr. a, The atmospheric temperature anomaly estimated from Dome F (ref. 7) (red), the orbital period
temperature variability (dashed brown, band-pass filtered between 1/45kyr and 1/17kyr) and the southern summer duration (black). b, The annual average tropospheric
temperature from SCAM (blue) and the southern summer duration (black). c, The Dome F δO2/N2 record7 (red) and the southern summer solstice insolation intensity (black). d, The maximum summer surface temperature in SCAM (blue, daily average) and the southern solstice insolation intensity (black). b –34
A
δO2/N2
475
525
575
Insolation (W m–2)
December 21, 77° S
Insolation (W m–2)
December 21, 77° S
–15
–10
Maximum summer surface
temperature (°C)
Time (kyr)
475
525
575
–350
–300
–250
–200
–150
–100
–50
0
–12
–8
–4
115
c
d A
c c d Figure 3 Temperature variability in the Antarctic over the past 350kyr. a, The atmospheric temperature anomaly estimated from Dome F (ref. 7) (red), the orbital period
temperature variability (dashed brown, band-pass filtered between 1/45kyr and 1/17kyr) and the southern summer duration (black). b, The annual average tropospheric
temperature from SCAM (blue) and the southern summer duration (black). c, The Dome F δO2/N2 record7 (red) and the southern summer solstice insolation intensity (black). d, The maximum summer surface temperature in SCAM (blue, daily average) and the southern solstice insolation intensity (black). symmetry of interhemispheric changes during the late Pleistocene
depends on northern climate responding primarily to summer
intensity (Milankovitch’s hypothesis2,19,35,36) and southern climate
responding primarily to the duration of summer and winter seasons
(a version of Adh´emar’s hypothesis37). These contrasting northern
and southern responses may reflect the contrasting distributions of
land and ocean—and hence ice sheets—in the two hemispheres. ARTICLES The large northern ice sheets maintained extensive melting margins
on land, reached equatorward as far south as 37◦N in central
North America and lost much of their ice through surface melting
atop land. Increased melting along a land margin will tend to
decrease the area and volume of such ice sheets and thereby increase
atmospheric temperatures through albedo and elevation effects. Thus, insomuch as the alignment of perihelion with northern
summer serves to increase ablation, it will also tend to increase
northern temperature. In contrast, the polar-centric Antarctic Ice
Sheet covers nearly the entire continent, even in today’s interglacial
climate. Most of its ice loss is pinned at the periphery of the
continent, occurring through calving and basal melting of ice
shelves. As a consequence, the area and volume of the Antarctic ice
sheet is relatively stable38, affording little leverage for the albedo and
elevation feedbacks to move atmospheric temperature. We suggest
that the relative lack of ice-temperature feedbacks in Antarctica
leaves atmospheric radiation balance, along with the possible
effects of winter duration, as major determinants of the southern
temperature response to orbitally driven changes in insolation. because of changes in the relationship between temperature and
δ18Oice (ref. 27), but a more likely explanation involves climate
feedbacks. Antarctic sea ice seems to covary with changes in
atmospheric temperature30. The complement of a long summer is
a short winter, and we suggest that a short winter may decrease the
production and extent of Antarctica’s apron of sea ice. (A related
suggestion is for spring insolation intensity, which covaries with
summer duration, to influence Antarctic sea ice22.) Furthermore,
atmospheric CO2 has also covaried with Antarctic temperature
over the past 800,000 years4,31,32. CO2 variation may itself be linked
with changes in Antarctic sea ice33,34, and would tend to cause
symmetric changes between the poles because it is well mixed in
the atmosphere9. Thus we speculate that the increasing summer
and decreasing winter durations caused by the alignment of
aphelion with southern summer solstice coordinates the effects
of summer radiation balance, winter sea ice and atmospheric CO2
so as to increase Antarctic temperature. Variations in sea ice and
CO2 may also explain why climate variations similar to those
in Antarctica are observed in mid-latitude southern marine and
continental environments9. nature geoscience ADVANCE ONLINE PUBLICATION www.nature.com/naturegeoscience 4 © 2008 Macmillan Publishers Limited. All rights reserved. © 2008 Macmillan Publishers Limited. All rights reserved. nature geoscience ADVANCE ONLINE PUBLICATION www.nature.com/naturegeoscience © 2008 Macmillan Publishers Limited. All rights reserved.
© 2008 Macmillan Publishers Limited. All rights reserved. ARTICLES
δ18O temperature anomaly (°C)
115
120
125
130
135
Summer duration
(d), 77° S
Summer duration
(d), 77° S
–34
–33
–32
Annual average atm. temperature (°C)
δO2/N2
475
525
575
Insolation (W m–2)
December 21, 77° S
Insolation (W m–2)
December 21, 77° S
–15
–10
Maximum summer surface
temperature (°C)
Time (kyr)
475
525
575
–350
–300
–250
–200
–150
–100
–50
0
–8
–6
–4
–2
0
2
4
–12
–8
–4
115
120
125
130
135
a
b
c
d
Figure 3 Temperature variability in the Antarctic over the past 350kyr. a, The atmospheric temperature anomaly estimated from Dome F (ref. 7) (red), the orbita
temperature variability (dashed brown band-pass filtered between 1/45kyr and 1/17kyr) and the southern summer duration (black) b The annual average tropos IMPLICATIONS FOR NORTHERN AND SOUTHERN CLIMATE Local control of Antarctic temperature conceptually frees southern
climate from northern forcing at the precession and obliquity
timescales. It enables an alternative hypothesis in which the near nature geoscience ADVANCE ONLINE PUBLICATION www.nature.com/naturegeoscience © 2008 Macmillan Publishers Limited. All rights reserved.
© 2008 Macmillan Publishers Limited. All rights reserved. Furthermore, the reanalysis indicates that the seasonal cycle in heat flux at
Dome F is only 20 W m−2 about a mean consistent with the Antarctic average,
suggesting that a constant heat convergence is a reasonable approximation for
this site, though the annual cycle in the reanalysis is larger at other sites (for
example 140 W m−2 at Dome C). The seasonal cycle in temperature produced
by SCAM is similar to observations, including reproduction of a thermal
inversion layer with an average magnitude of ∼15 ◦C and a seasonal amplitude
of ∼50 ◦C within the troposphere25. Frequency (kyr–1) Figure 4 Coherence and phase between proxy and model estimates of mean
annual atmospheric temperature at Dome F. A multitaper method is used to Figure 4 Coherence and phase between proxy and model estimates of mean
annual atmospheric temperature at Dome F. A multitaper method is used to
estimate the coherence (red line) and phase (black line). A Monte Carlo method is
used to estimate the 95% confidence level for the coherence (red dashed line,
indicating that the orbital bands are significantly coherent) and the 95% confidence
interval for the phase (shading, including an age model uncertainty of ±0.75kyr,
1σ). Positive phases indicate that the proxies lag behind the model output, and at
the orbital bands (vertical dotted lines) results are consistent within uncertainties
with an in-phase relationship. Figure 4 Coherence and phase between proxy and model estimates of mean
annual atmospheric temperature at Dome F. A multitaper method is used to
estimate the coherence (red line) and phase (black line). A Monte Carlo method is
used to estimate the 95% confidence level for the coherence (red dashed line,
indicating that the orbital bands are significantly coherent) and the 95% confidence
interval for the phase (shading, including an age model uncertainty of ±0.75kyr,
1σ). Positive phases indicate that the proxies lag behind the model output, and at
the orbital bands (vertical dotted lines) results are consistent within uncertainties
with an in-phase relationship. One implication of independent hemispheric responses is that
the lead–lag relationships between the hemispheres at orbital
timescales need not be indicative of causality. The lead of
southern changes relative to northern climate at the orbital
bands2 can instead be understood from the Antarctic climate
remaining in near radiative equilibrium with local insolation
whereas northern changes are mediated through the slow response
of the northern ice sheets35,36. © 2008 Macmillan Publishers Limited. All rights reserved.
© 2008 Macmillan Publishers Limited. All rights reserved. ARTICLES –180
–90
0
90
180
Phase (°)
Obliquity
Precession
Coherence
Frequency (kyr–1)
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
0.10
0
0.5
1.0
Figure 4 Coherence and phase between proxy and model estimates of mean
annual atmospheric temperature at Dome F. A multitaper method is used to
estimate the coherence (red line) and phase (black line). A Monte Carlo method is
used to estimate the 95% confidence level for the coherence (red dashed line,
indicating that the orbital bands are significantly coherent) and the 95% confidence
interval for the phase (shading, including an age model uncertainty of ±0.75kyr,
1σ). Positive phases indicate that the proxies lag behind the model output, and at
the orbital bands (vertical dotted lines) results are consistent within uncertainties
with an in-phase relationship. –180
–90
0
90
180
Phase (°)
Obliquity
Precession
Coherence
Frequency (kyr–1)
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
0.10
0
0.5
1.0 complicated versions of the energy-balance model, including constant, periodic,
and temperature-dependent parametrization of heat-flux convergence, yield
modifications of the seasonal cycle but inevitably give the same effect, namely
that a greater summer duration leads to a positive perturbation in annual
average temperature. complicated versions of the energy-balance model, including constant, periodic,
and temperature-dependent parametrization of heat-flux convergence, yield
modifications of the seasonal cycle but inevitably give the same effect, namely
that a greater summer duration leads to a positive perturbation in annual
average temperature. Coherence Coherence Phase (°) Temperature is essentially assured to follow simple radiative equilibrium
in the energy-balance model, making it useful to also analyse the response
of SCAM23,24 to orbitally induced changes in insolation. SCAM contains the
vertical radiation, cloud and surface-process physics of the NCAR Community
Atmospheric Model but spans only a single lateral grid box, here made
to coincide with the Dome F ice-core site. We specify a sensible heat-flux
convergence of 100 W m−2 and a latent heat-flux convergence of 50 W m−2,
consistent with the annual average convergences over Antarctica found in the
NCEP/NCAR reanalysis43 (but which are uncertain44). The latent and sensible
heat convergences are distributed so as to generate uniform warming within the
troposphere. The simplification of a constant heat convergence avoids building
in a particular seasonal cycle45 and distinguishes our hypothesis of local
insolation control from hypotheses calling on remote variations in climate46. © 2008 Macmillan Publishers Limited. All rights reserved.
© 2008 Macmillan Publishers Limited. All rights reserved. (Note that meltwater pulses enable
a rapid northern-ice-sheet response to insolation or other forcing,
but that such threshold-like processes are probably more important
at timescales shorter than those associated with orbital periods.)
The relatively small changes in the glacial–interglacial dimensions
of the Antarctic Ice Sheet may also account for the apparently
consistent response of Antarctic temperature to orbital variations
between interglacial5 and glacial times6,7. We integrate SCAM forward using a 20 min time step, and thus resolve the
full diurnal and seasonal cycle of insolation as well as the modulation of these
quantities by shifts in Earth’s orbital configuration47 (Fig. 3). The integration is
started 350,000 years ago, and the model is run to equilibrium over the course
of 25 years. Then, to accelerate the model integrations, the calendar is stepped
forward 975 years, and the equilibration process is repeated. Results are averaged
over the last 5 years of each of the 350 equilibration runs. SCAM’s annual
average atmospheric temperature is computed from the pressure-weighted
average of the four lowest atmospheric layers (centred at ∼580, 620, 650
and 660 mb), but essentially the same variability is present throughout the
entire vertical column. Longer runs of the model over the past 800 kyr yield
atmospheric and surface variability that, respectively, remain consistent with
the duration and insolation intensity of summer as well as with the Dome C
atmospheric-temperature proxy record6. SCAM also generates results consistent
with those reported here when run at the Vostok and Dome C ice-core sites, as
well as when run in configurations with a clear sky, no atmospheric heat-flux
convergence or lower atmospheric CO2 concentration (180 p.p.m.). Finally,
we
note
that
the
obliquity-and-precession-band
variability in Antarctic atmospheric temperature represents only
∼20% of the variance in the 800 kyr δ18Oice Dome C record, but that
the summer-duration hypothesis may also afford insight into the
origins of the stronger ∼100 kyr variability, which represents ∼50%
of the variance. An Antarctic response to local changes in insolation
is consistent with hypotheses calling on terminations to be triggered
by changes in southern insolation15,39–42. If a long summer and
a short winter lead to a decrease in production and extent of
Antarctic sea ice, they may also increase the outgassing of CO2 from
the Southern Ocean by decreasing near-surface stratification34. Hence, the coincidence of aphelion with southern summer solstice
may coordinate a powerful combination of southern warming
agents. References Linear responses to
Milankovitch forcing. Paleoceanography 7, 701–738 (1992). 3. Lorius, C. et al. A 150, 000-year climatic record from Antarctic ice. Nature 316, 591–596 (1985). 4. Petit, J. et al. Climate and atmospheric history of the past 420,000 years from the Vostok ice core,
Antarctica. Nature 399, 429–436 (1999). 5. Masson, V. et al. Holocene climate variability in Antarctica based on 11 ice-core isotopic records. Quat. Res. 54, 348–358 (2000). 6. Jouzel, J. et al. Orbital and millennial Antarctic climate variability over the past 800,000 years. Science
317, 793–796 (2007). 6. Jouzel, J. et al. Orbital and millennial Antarctic climate variability over the past 800,000 years. Science
317, 793–796 (2007). 7. Kawamura, K. et al. Northern Hemisphere forcing of climatic cycles in Antarctica over the past
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360,000 years. Nature 448, 912–916 (2007). 8. Alley, R., Brook, E. & Anandakrishnan, S. A northern lead in the orbital band: North–south phasing
of Ice-Age events. Quat. Sci. Rev. 21, 431–441 (2002). 9. Barrows, T., Juggins, S., De Deckker, P., Calvo, E. & Pelejero, C. Long-term sea surface temperature
and climate change in the Australian–New Zealand region. Paleoceanography 22, 1–17 (2007). © 2008 Macmillan Publishers Limited. All rights reserved.
© 2008 Macmillan Publishers Limited. All rights reserved. Once the northern ice sheets are sufficiently large to become
unstable, the combination of a long southern summer and an
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simple energy-balance model, dT/dt = (F(t)−σT(t)4)ϵ/C, where t is time. For simplicity, the heat capacity is taken as a constant (C = 2,500 kJ ◦C−1),
and the short-wave absorptivity and long-wave emissivity are assumed equal
to each other (ϵ = 0.05). When forced by insolation changes at 77◦S, the
selected parameters give a seasonal cycle of temperatures ranging between −60◦
and −10 ◦C, in agreement with the 700 mb temperatures in the NCEP/NCAR
reanalysis43 at the Dome Fuji site (77◦S and 40◦E). Experiments with more 13. Charles, C., Lynch-Stieglitz, J., Ninnemann, U. & Fairbanks, R. Climate connections between the
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variability. Nature 441, 329–332 (2006). 15. Broecker, W. & Henderson, G. The sequence of events surrounding Termination II and their
implications for the cause of glacial–interglacial CO2 changes. Paleoceanography 13, 352–364 (1998). Broecker, W. & Henderson, G. The sequence of events surrounding Termination II implications for the cause of glacial–interglacial CO2 changes. Paleoceanography 13, 352–364 (1998). 16. Huybers, P. & Curry, W. Links between the annual, Milankovitch, and continuum of climate
variability. Nature 441, 329–332 (2006). 16. Huybers, P. & Curry, W. Links between the annual, Milankovitch, and continuum of climate
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Monograph, American Geophysical Union, 1984). nature geoscience ADVANCE ONLINE PUBLICATION www.nature.com/naturegeoscience References References
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© 2008 Macmillan Publishers Limited. All rights reserved. © 2008 Macmillan Publishers Limited. All rights reserved. © 2008 Macmillan Publishers Limited. All rights reserved. nature geoscience ADVANCE ONLINE PUBLICATION www.nature.com/naturegeoscience 6 © 2008 Macmillan Publishers Limited. All rights reserved. © 2008 Macmillan Publishers Limited. All rights reserved.
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Hydrogeological Conditions and Natural Factors Forming the Regime of Groundwater Levels in the Ivano-Frankivsk Region (Ukraine)
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Journal of Ecological Engineering
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Journal of Ecological Engineering
Volume 19, Issue 6, November 2018, pages 34–44
https://doi.org/10.12911/22998993/91883 Received: 2018.04.13
Accepted: 2018.08.15
Published: 2018.11.01 ABSTRACT The purpose of the article is to carry out a complex analysis of conditions and natural factors forming the hydrogeo
logical regime as well as to integrate the existing information and groundwater monitoring data in Ivano-Frankivsk
region for further creating the unified automated system to collect, process, analyze and store the monitoring ob
servation data. The authors offer methodology, which is based on the geoinformation approach, for organizing and
monitoring of the groundwater. The structure of spatial and attributive data that afford to systemize the existing
schemes of the hydrogeological zoning as well as the results of long-term observations over variability of the
groundwater levels and factors forming the hydrogeological regime were developed. The new approaches for im
proving the techniques to forecast the groundwater level with due consideration of temporal patterns for changes in
water level, the Moon phases and seismic activity, are proposed. The obtained results are considered as an informa
tional basis for reorganization of the state hydrogeological monitoring on the example of Ivano-Frankivsk region. Keywords: groundwater, hydrogeological zoning, geodatabase, the Moon phases, earthquakes Hydrogeological Conditions and Natural Factors Forming the
Regime of Groundwater Levels in the Ivano-Frankivsk Region
(Ukraine) Lidiia Davybida1*, Dmytro Kasiyanchuk1, Liudmyla Shtohryn1,
Eduard Kuzmenko1, Mariia Tymkiv1 1 Department of Geotechnogenic Safety and Geoinformatics, Ivano-Frankivsk National Technical University of
Oil and Gas, 15 Karpatska Street, Ivano-Frankivsk, 76019, Ukraine * Corresponding author’s e-mail: davybida61085@gmail.com * Corresponding author’s e-mail: davybida61085@gmail.com INTRODUCTION problematic because of purely economic rea
sons, we have to develop the new approaches to
obtain and process information on the ground
water conditions, as well as on the dynamics
of their levels first of all. The groundwater, as the most dynamic com
ponent of the geologic environment, responds
quickly to changes in their forming conditions
and, hence, to the impact of anthropogenic fac
tors. It means that the condition peculiar to the
underground hydrosphere, and primarily, to the
groundwater, as the first constant water-bearing
formation, may serve as an indicator of the eco
logical state of the geological environment as a
whole. Nowadays, it is believed that the function
ing of a scientifically grounded stationary system
of hydro-geological monitoring supplies the most
complete information about the groundwater con
dition [Koshliakov et al. 2016, Shestopalov et al. 2016]. However, since such system is practically
absent in the most territories of Ukraine, and in
the studied Ivano-Frankivsk region in particu
lar, and its creation and effective functioning is Upon analyzing the current experience in
the application of the geoinformation approach
to optimization of groundwater monitoring [Hu
dak et al. 1992, Ben-Jema et al. 1994, Berezko et
al. 2012, Brovko et al. 2015, Pivovarova 2016,
Tymkiv et al. 2016], the authors identify the pri
ority tasks to renovate the state network of hy
drogeological monitoring and to adapt it to the
European standards in the shortest possible pe
riod. These tasks are as follows: inventory of the
observation points, evaluation of their represen
tativeness, and development of the concept for
monitoring system reformation as well as forma
tion of a single database with the involvement of
GIS-technologies. 34 Journal of Ecological Engineering Vol. 19(6), 2018 It is well known that the regime of underwater
is affected by the exogenous factors (meteorolog
ical, hydrological characteristics, lunar tides), the
endogenous factors (tectonic movements, volca
nism, and earthquakes) and the technological fac
tors (water intake). It is clear that the climatic fac
tors (atmospheric precipitations, air temperature)
and the hydrological factors (a regime of surface
stream flows) have effect on the hydrogeological
regime formation [Ruban et al. 2005] and, in par
ticular, on the examined area, which is character
ized by natural or slightly degraded regime un
dergoing the influence of local technological fac
tors. First, the earthquakes should be enumerated
among the geological factors, the effect of which
may be fixed by hydrogeological observations. INTRODUCTION The impact of earthquakes on the groundwater
regime is usually episodic and it is the most of
ten seen in sharp changes in the level of pressure
water. The study of peculiarities in changes of the
natural regime of groundwater, in turn, gives the
possibility to monitor the process of earthquake
emerging. It is noted in scientific papers [Adu
shkin et al. 2017; Maréchal et al 2002] that the
lunar and solar tides are clearly distinguished in
the variations of level of underground water. It is
confirmed by the spectral analysis. It is believed
that the monthly lunar tides within the pressure
water have an influence on the water-bearing ho
rizon by periodically increasing the load, chang
ing the elastic state of the water-bearing horizon. The influence of tidal force is fixed in water-ta
ble wells, because the gravity forces stretch the Earth’s crust, thus increasing the level of ground
water. These, so called ground, tidal oscillations
are measured only by few centimeters and they
have clear periodicity in 12 and 24 hours, and less
clear periodicity in 14 and 28 days. It should be noted that there has been no pur
poseful study of the influence produced by the
seismic activity and lunar cycles on the hydrogeo
logical regime formation in the Ivano-Frankivsk
region. At the same time, it has been studied for
nowadays that in the Western Ukraine, abnormal
rains (one of the direct factors of influence over
variability of the groundwater levels) usually oc
cur in the Last or the First Moon Quarter. As a
rule, the maximum precipitations are recorded on
the first – third day of the Moon Phase [Shtohryn
et al. 2015]. The cycles of extreme precipitations
in 5.5–6.9.4 and 11 years are present in the spec
tra of all specified phases and in the whole period,
as well as they are multiple of the solar activity
periods and the Moon cycle (Fig. 1). The purpose of the study is to integrate the
existing information and groundwater monitoring
data as well as the factors forming the hydrogeo
logical regime in the Ivano-Frankivsk region for
further creation of the unified automated system
to collect, process, analyse and store the monitor
ing observation data. MATERIALS AND METHODS The smallest taxonomic units
of typing, which characterize the stratigraphic
features of the conditions for the groundwater
formation in the profile, are the landscape hy
drogeological complexes (LHC). They include
separate areas, where the first water bearing hori
zon is deposited having the defined stratigraphic
sections, which reflect the structure of the aerated
zone, water-bearing and waterproof layers and,
to a certain extent, stipulate the peculiarities of
the groundwater recharge and discharge as well
as the reservoir properties of the upper layer of
rocks, where their formation takes place. LHC are
azonal units of zoning and they are characterized
by common features of the most hydrogeological
areas, mainly within the groundwater basins. In
general, 26 varieties of plain LHC and 6 varieties
of mountainous LHC are identified in Ukraine. There are 10 varieties (7 plain LHC and 3 moun
tainous LHC) in the Ivano-Frankivsk region. The
indexation of LHC is specified in accordance with
the hydrogeological zoning scheme of Ukraine
[Ruban et al 2005] According to the zoning of Ukraine under the
conditions for the groundwater formation (Fig. 2),
Ivano-Frankivsk region is located in the area of
such hydrogeological regions as Volyn-Podolian
Artesian Basin and Precarpathian Artesian Basin
as well as the Carpathian folded zone [Shesto
palov et al. 2010]. The complex geological struc
ture of the area has caused a great variety of its
hydrogeological conditions, which are unequal
for the groundwater using and its vulnerability
to the industrial pollution. It should be stressed
that the basic current network of the state hydro
geological monitoring, which in 2016 included
only 5 observation wells and 2 wells within the
water intake area in Ivano-Frankivsk region, did
not fulfil the assigned functions and it requires
serious reorganization and further development
[Shestopalov et al. 2016]. p
The materials by ‘Geoinform of Ukraine’,
State Research-and-Production Enterprise, Ukrai
nian State Geological Prospecting Institute, Insti
tute of Geological Sciences of National Academy
of Science of Ukraine, concerning the hydrologi
cal zoning pursuant to the water exchange prin
ciple, location of hydrogeological observation
points as well as the results of long-term observa
tions have been used for creating the geodatabase
to provide the functioning of the hydrogeological
monitoring system within the Ivano-Frankivsk
region. MATERIALS AND METHODS as projecting and improving the current network
of observation wells. In this paper, the zoning
scheme based on groundwater formation condi
tions as well as the scheme of functional zoning
pursuant to the basin principle are chosen as the
initial cartographic materials that characterize
conditions creating the regime on the area. The studied Ivano-Frankivsk region is char
acterised by the well-developed river network and
belongs to the area, which is one of the richest ar
eas in Ukraine in terms of surface water resources. However, this area takes one of last places with
regard to the reserves of the drinking groundwa
ter. Despite this fact, the individual water supply
to the population and enterprises of the region is
mainly carried out by the groundwater. The first
water-bearing horizon is used the most often. The studied Ivano-Frankivsk region is char
acterised by the well-developed river network and
belongs to the area, which is one of the richest ar
eas in Ukraine in terms of surface water resources. However, this area takes one of last places with
regard to the reserves of the drinking groundwa
ter. Despite this fact, the individual water supply
to the population and enterprises of the region is
mainly carried out by the groundwater. The first
water-bearing horizon is used the most often. Zoning scheme (Fig. 2) has been developed
pursuant to the conditions of the groundwater for
mation in the Ukrainian State Geological Pros
pecting Institute, which is detailed in the paper
[Ruban et al. 2005]. The zoning scheme is formed
in accordance with the geological and structural
principle of hydrogeological zoning, which takes
into account the heterogeneity of the Earth’s crust
composition and structure. The regions, which
are covering the largest geo-structural units –
the hydrogeological massifs (such as basins of
produced-block water and basins of fracture-vein
water) as well as artesian basins (produced water
basins), are allocated at the core of the scheme. There are smaller taxonomic units that include
the hydrogeological areas representing a part of
the groundwater basin expressed on the certain
territory, which is characterised by a specific
combination of geological structure, relief, cli
matic, hydrological and, eventually, hydrogeo
logical conditions. One may distinguish the sub
areas, which differ by some features of geological
structure, relief and climatic conditions, within a
majority of areas. INTRODUCTION The Ivano-Frankivsk region
was chosen due to three reasons: 1) its location on
the border of mountainous areas and flat terrains;
2) the availability of output data; 3) possibility to
spread the findings obtained to other territories of
Ukraine and abroad regions. Figure 1. Diagram of interrelations between the Moon phases and extreme levels of groundwater igure 1. Diagram of interrelations between the Moon phases and extreme levels of groundwater Figure 1. Diagram of interrelations between the Moon phases and extreme levels of groundwater 35 Journal of Ecological Engineering Vol. 19(6), 2018 MATERIALS AND METHODS The spatial and attributive information
is supplemented by the topographical data and
brought to a single scale (1:100 000) and format
of digital cartographic models in GIS MapInfo
Professional (Gauss-Krüger projection, Pulkovo,
1942, zone 6). The results of the monitoring ob
servations are stored in the external relational
database and they may be adjoined to the spatial
objects (hydrogeological monitoring stations) us
ing unique codes of the observations points. i The hydrogeological areas identified by vari
ous principles are considered as a territorial basis
to conduct the groundwater monitoring as well 36 Journal of Ecological Engineering Vol. 19(6), 2018 Figure 2. Zoning of Ivano-Frankivsk region according to the formation conditions and groundwater occur
rence: І – Volyn-Podolian Artesian Basin (4 – Podolian area (4а – Roztochya-Opilian suberea), 5 – Prut-Dniester
area); VI – Precarpathian Artesian Basin (26 – Precarpathian area); XIV – Сarpathian Groundwater Basin of
(36 – Mountain Carpathian area) Figure 2. Zoning of Ivano-Frankivsk region according to the formation conditions and groundwater occur
rence: І – Volyn-Podolian Artesian Basin (4 – Podolian area (4а – Roztochya-Opilian suberea), 5 – Prut-Dniester
area); VI – Precarpathian Artesian Basin (26 – Precarpathian area); XIV – Сarpathian Groundwater Basin of
(36 – Mountain Carpathian area) The scholars of the Institute of Geological Sci
ences of National Academy of Science of Ukraine
have developed a functional zoning scheme, ac
cording to the basin principle (under the condi
tions of water exchange in the upper hydrogeo
logical layer) [Shestopalov et al. 2010] (Fig. 3). The given scheme may be considered as the basic
one for reorganization of the state system for the
groundwater monitoring, in accordance with the
principles of the EU Water Framework Directive, and for transition from the administrative-territo
rial approach to creation of the integrated basin
model to control the water resources. This model
envisages coordination of water protection mea
sures as to the surface water and groundwater,
which belong to the common ecologic, hydrolog
ic, and hydrogeological system. The system for
studying the groundwater drainage is based on
the dynamic features of objects allocated accord
ing to the principle of groundwater runoff unity 37 Journal of Ecological Engineering Vol. 19(6), 2018 united within the bounds of the single zoning
units (provinces), where the subareas correspond
ing to water exchange basins of different main
rivers tributaries are allocated. MATERIALS AND METHODS The studied territory of Ivano-Frankivsk region
belongs to the hydrogeological provinces of the
Dniester River and the Dunabe River, which are
divided within the studied region in 7 hydrologi
cal districts (V-2 – the Strypa River, V-3 – the
Svich River and the Limnytsia River, V-4 – the
Bystrytsia Nadvirnianska River and the Bystryt
sia Solotvynska River, V-5 – the right bank of the
Dniester River from the mouth of the Bystrytsia
River to the mouth of the Liadova River, V-6 –
the Stryvigora River and the Vereshchytsia River,
V-7 – the Davydivka River and Svira River, V-8 –
the Gnyla Lypa River and the Zolota Lypa River)
and 2 hydrogeological districts (B-1 – the Tysa
River, B-2 – the Prut River). for both surface and underground water is also
confirmed by the synchronicity of the long-term
variability of the mid-annual surface water flow
and the annual average depth of the groundwa
ter level. The synchronicity is observed for both
the actual values of the regime elements and for
long-term trends established for the hydrological
stations and hydrogeological wells located in the
same hydrogeological district [Davybida 2017]. The studied territory of Ivano-Frankivsk region
belongs to the hydrogeological provinces of the
Dniester River and the Dunabe River, which are
divided within the studied region in 7 hydrologi
cal districts (V-2 – the Strypa River, V-3 – the
Svich River and the Limnytsia River, V-4 – the
Bystrytsia Nadvirnianska River and the Bystryt
sia Solotvynska River, V-5 – the right bank of the
Dniester River from the mouth of the Bystrytsia
River to the mouth of the Liadova River, V-6 –
the Stryvigora River and the Vereshchytsia River,
V-7 – the Davydivka River and Svira River, V-8 –
the Gnyla Lypa River and the Zolota Lypa River)
and 2 hydrogeological districts (B-1 – the Tysa
River, B-2 – the Prut River). sition of waters contained in the ground water-
bearing horizon, limit values peculiar to the
filtration coefficient of water-bearing horizon,
age of watercourse rocks, filtration coefficient
of watercourse rocks, limit values of mineral
ization, water hardness and pH index; 9. 9. vector layers of the basic information includ
ing administrative boundaries, settlements,
roads, rivers, vegetation, relief. MATERIALS AND METHODS The water ex
change basins, sizes and boundaries of which are
determined by sizes and configuration of the river
basins as a rule, should be considered as charac
teristic systems with homogenous regime in fore
casting the changes in level of the ground water,
which is a part of the underground hydrosphere
that is most closely related to the external surface
factors forming the groundwater recharge and
discharge. The expediency of monitoring study from the areas of drainage formation to its main
discharge, which is controlled by a single closed
balance of the groundwater. Within the hydro
geological structures, one may allocate the water
exchange basins of the first order, which are char
acterized by single or close trends of the regional
water exchange. The hierarchy of this zoning is as
follows: there are basins of sea drainage (regions)
and main rivers that flow into the seas (provinc
es). The next zoning level takes into account the
peculiarities of the hydrogeological conditions as
well as the close trends of lateral water exchange;
thus, the basins of rivers’ groundwater flow are Figure 3. Zoning of the Ivano-Frankivsk region under the conditions for water exchange formation in the hyd
geological structures Figure 3. Zoning of the Ivano-Frankivsk region under the conditions for water exchange formation in the hydro
geological structures Figure 3. Zoning of the Ivano-Frankivsk region under the conditions for water exchange formation in the hydro
geological structures Figure 3. Zoning of the Ivano-Frankivsk region under the conditions for water exchange formation in the hydro
geological structures 38 Journal of Ecological Engineering Vol. 19(6), 2018 for both surface and underground water is also
confirmed by the synchronicity of the long-term
variability of the mid-annual surface water flow
and the annual average depth of the groundwa
ter level. The synchronicity is observed for both
the actual values of the regime elements and for
long-term trends established for the hydrological
stations and hydrogeological wells located in the
same hydrogeological district [Davybida 2017]. MATERIALS AND METHODS The factual input database contains the in
formation on the structure of the state hydrogeo
logical monitoring network (spatial coordinates
and attributive data), location of hydrogeologi
cal points, meteorological points as well as the
results of long-term regime observations over
the groundwater levels, presented in the form of
average monthly values of groundwater depth
levels calculated under the data of timing obser
vations, results of observations on the variability
of weather and climate characteristics (average
annual air temperature, amount of precipitation),
long-term change of numerical characteristics
peculiar to the geological features (the energy of
earthquakes) and the cosmogenic features (the
Moon phases, solar activity) required to form the
hydrogeological regime. In order to integrate the spatial data, the spa
tial binding of raster maps was conducted and they
were transferred into the vector format by digitali
zation. A geodatabase was created that includes the
following layers with the relevant attributes: It should be noted that due to the absence
of observation wells for intermediate waters as
well as of the results of observations over the
hydrochemical and hydrothermal regime in the
Ivano-Frankivsk region, the present informa
tive elements were not entered into the devel
oped database and they are considered as an
object of separate study. 1. hydrological monitoring stations (code, num
ber, type, depth, coordinates X and Y, type of
water-bearing horizon, regime type, distur
bance of regime); 2. hydrological provinces (code, index, water ex
change basin); 3. hydrological districts (code, index, water ex
change basin); RESULTS AND DISCUSSION The lowest groundwater
levels are Н1, Н5, which mostly coincide with the
extreme level Нmin, and respectively, the highest
groundwater levels are H3 or Hmax. 3
max
In Table 1 shows the coordinates (geographic
latitude jо N and longitude lо Е) of the hydro
geological points, for which were conducted the
following measurements of the groundwater lev
els are summarized as well as coordinates of the
registered earthquakes, their energies (E) and en
ergy classes (K) and the Moon Phases (NL – the
New Moon, PQ – the First Quarter of the Moon,
PL – the Full-Moon, DQ – the Last Quarter of
the Moon) observed at the fixation extremums
and earthquakes. An outcome analysis of long-termed obser
vations over the groundwater level, the weather
and climate characteristics as well as over the hy
drological regime creating factors typical for the
observation territory (Fig. 4) has established the
presence of synchronically changed rhythm and
significant relation, first of all, for the time series
of the average annual groundwater levels and
the series of average annual water consumption,
the value of which is connected with the modu
lus of flow, including such factors as surface and
groundwater flow. The regime of the surface wa
ter, in its turn, is also determined by climatic fac
tor having an influence on the groundwater. While analyzing the Table 1, one may notice
that the average value of the groundwater level
equals 2.3–3.6 meters. The extreme levels, as a
rule, were observed in the same months when
the earthquake was registered. When the wa
ter levels are recorded before the earthquakes,
the water rises closer to the surface in relation
to the average value. Synchronicity of the long-term variability of
the annual average discharge of surface flows and
the average annual depths of groundwater lev
els confirms the feasibility of joint monitoring
studies for surface water and groundwater in the
studied region, as provided by Water Framework
Directive [Directive 2000]. The established regu
larities may be used for renovating the time se
ries of observations over the groundwater levels,
for bringing them to a single timeframe, and for
drawing up the medium and long-term hydrogeo
logical forecasts. The authors may give the data under observa
tion well No. RESULTS AND DISCUSSION 4. water-resource regions (code, index, depart
mental subordination); The spatial analysis concerning the subject
layers of hydrogeological monitoring stations
and hydrogeological zoning taxon shows the in
efficient location of the observation wells within
the studied territory. In addition, all observations
are conducted over the groundwater regime only. However, given the characteristic features of the
conditions peculiar to the groundwater regime
formation, number, configuration and location of
hydrogeological districts in Ivano-Frankivsk re
gion, it is necessary to increase the number of hy
drogeological monitoring stations up to 18–20 (3
wells per each hydrogeological district allocated
under the water exchange principle; they should 5. hydrological basins, allocated under the condi
tions of formation and groundwater occurrence
(code, index, basin); 6. hydrogeological district (code, index, district); 7. hydrogeological sub-district (code, index,
sub-district); 8. landscape hydrological complexes (code, in
dex, age of rocks of the aeration zone, rocks
of the aeration zone, chemical composition of
the aeration zone, limit values of aeration zone
depth, limit values peculiar to the coefficient of
the aeration zone filtration, age of rocks con
tained in the water-bearing formation; rocks of
the water-bearing formation; chemical compo 39 Journal of Ecological Engineering Vol. 19(6), 2018 25Ο54’ – 25Ο57’, North Latitude 48Ο44’ – 48Ο48’. The considered earthquakes were registered with
in the region and adjacent areas with coordinates
East Longitude 24Ο – 24 Ο6’, North Latitude 47Ο7’
– 48Ο8’9 (Table 1). be located in such a way so as to be representative
for riverside, terraced and water-dividing types of
the groundwater regimes). Another criterion for
choosing the location of the observation point is
the LHC occurrence (Fig. 2). The location of the
monitoring stations with due allowance for con
ditions of the hydrogeological regime formation
will permit to obtain more complete information
and to organize a random placement of wells to
provide the uniformity and independence of tests. The next stage of the study is determination of op
timal coordinates to locate the monitoring wells
on the basis of a formal statistical estimation of
the points’ density, taking into account the given
extrapolation error of the investigated parameters
in a GIS environment. (
)
The extreme values of groundwater levels,
which were calculated according to the periodic
measurements including season levels (pre-spring
minimum level (Н1), spring maximum level (Н3),
summer-autumn minimum level (Н5)) and annual
levels (high level (Нmax), low level (Нmin)), were
considered in particular. RESULTS AND DISCUSSION 95p, as an ex ample, where the av
erage level Hmin equals 3.94 m, and the registered
index that is fixed a day before the earthquake of
28.05.96 with the energy class K=7.9 amounts to
3.16 m. This is closer to the surface by 0.78 m
in comparison with the average value. It should
be noted that in the Ivano-Frankivsk region, the
abnormal quantity of the extreme precipitations
was not observed in May of 1996. Such infor
mation may indicate the series of the Earth’s
break ups and rifts, along which the water rises
before the earthquakes. In order to improve the reliability of forecast
ing the annual regime of variability peculiar to
the groundwater levels as well as the examination
of its interconnection with the seismic activity in
the studied Ivano-Frankivsk region, the extreme
values of groundwater levels in five wells from
1973 to 2003 were analyzed. These wells were
drilled into the water-bearing low upper quaterna
ry alluvial horizon AQ1–3 within Volyn-Podolian
Artesian Basin. The location of the wells may be
set by the boundary coordinates East Longitude While analyzing the graphs of the extreme
groundwater levels (Fig. 5), we can see that the
minimum and maximum groundwater levels are
in-phase during the second part of the New Moon
Phase (NL), from the first to the third day within
the First Quarter of the Moon Phase (PQ) and the 40 Journal of Ecological Engineering Vol. 19(6), 2018 Figure 4. Estimation of interconnections peculiar to changing in groundwater levels and numerical characteris
tics of the regime creating factors applied for the monitoring stations in the Ivano-Frankivsk region: a) graphs
and cross-correlation function for the groundwater level and surface water discharge; b) graphs and cross-cor
relation function for the groundwater level and annual amount of precipitation; c) graphs and cross-correlation
function for the level of groundwater and average annual air temperature Figure 4. Estimation of interconnections peculiar to changing in groundwater levels and numerical characteris
tics of the regime creating factors applied for the monitoring stations in the Ivano-Frankivsk region: a) graphs
and cross-correlation function for the groundwater level and surface water discharge; b) graphs and cross-cor
relation function for the groundwater level and annual amount of precipitation; c) graphs and cross-correlation
function for the level of groundwater and average annual air temperature Full Moon (PL) of the Moon cycle. RESULTS AND DISCUSSION Interconnection between the Moon Phases, earthquakes, and groundwater level
Earthquaekes
Date of
earthquake
registration
K
Е×108,
J
Well’s
cadastral
number
jо N
of well
lо Е
of well
Date of level
registration
Day of the Lunar Cycle
and the Moon Phase
Level of
ground-
water (Н)
Нregistere,
m
Нaverage, m
jо N
lо Е
47.90
24.60
14.11.74
9.5
31.60
113p/e
48.44
25.56
06.11.74
23
/DQ
Н5
3.63
3.69
47.97
24.51
14.11.74
7.2
0.16
113p/e
48.44
25.56
06.11.74
23
/DQ
Н5
3.63
3.69
49.03
24.00
18.11.74
7.6
0.40
113p/e
48.44
25.56
06.11.74
23
/DQ
Н5
3.63
3.69
49.05
24.02
14.01.76
11.0
1000.0
114p
48.44
25.57
03.01.76
3
NL//
Н1
2.98
3.16
49.05
24.02
14.01.76
11.0
1000.0
3
48.48
25.54
03.01.76
3
NL//
Нmin
3.61
4.53
49.05
24.02
14.01.76
11.0
1000.0
95p
48.47
25.54
03.01.76
3
NL//
Нmin
3.48
3.94
47.83
24.75
11.06.76
7.0
0.10
113p/e
48.44
25.56
06.06.76
9
PQ/
Нmax
2.81
3.08
47.83
24.75
11.06.76
7.0
0.10
114p
48.44
25.57
06.06.76
9
PQ/
Нmax
1.93
2.25
47.83
24.75
11.06.76
7.0
0.10
95p
48.47
25.54
06.06.76
9
PQ/
Нmax
2.58
2.92
48.95
24.07
31.10.79
9.5
31.60
114p
48.44
25.57
30.10.79
13
//PL
Н5
2.80
3.24
48.05
25.00
13.07.85
7.7
0.50
113p/e
48.44
25.56
27.07.85
11
Нmin
4.56
4.06
48.05
25.00
13.07.85
7.7
0.50
114p
48.44
25.57
06.07.85
19
Нmax
1.90
2.25
47.70
24.50
28.08.90
7.8
0.63
102p/e
27.08.90
8
/PQ
Н5
1.80
2.02
48.20
24.70
08.01.93
7.6
0.40
102p/e
03.01.93
11
PQ//
Нmin
1.68
2.09
48.80
24.29
16.08.95
8.2
1.58
114p
48.44
25.57
09.08.95
14
/PL
Н5
2.97
3.24
48.69
24.40
28.05.96
7.9
0.79
95p
48.47
25.54
27.05.96
11
PQ//
Нmin
3.16
3.94
Figure 5. Dependency graph for a number of the registered extreme groundwater levels (by the lunar calendar Table 1. RESULTS AND DISCUSSION Interconnection between the Moon Phases, earthquakes, and groundwater level
Earthquaekes
Date of
earthquake
registration
K
Е×108,
J
Well’s
cadastral
number
jо N
of well
lо Е
of well
Date of level
registration
Day of the Lunar Cycle
and the Moon Phase
Level of
ground-
water (Н)
Нregistere,
m
Нaverage, m
jо N
lо Е
47.90
24.60
14.11.74
9.5
31.60
113p/e
48.44
25.56
06.11.74
23
/DQ
Н5
3.63
3.69
47.97
24.51
14.11.74
7.2
0.16
113p/e
48.44
25.56
06.11.74
23
/DQ
Н5
3.63
3.69
49.03
24.00
18.11.74
7.6
0.40
113p/e
48.44
25.56
06.11.74
23
/DQ
Н5
3.63
3.69
49.05
24.02
14.01.76
11.0
1000.0
114p
48.44
25.57
03.01.76
3
NL//
Н1
2.98
3.16
49.05
24.02
14.01.76
11.0
1000.0
3
48.48
25.54
03.01.76
3
NL//
Нmin
3.61
4.53
49.05
24.02
14.01.76
11.0
1000.0
95p
48.47
25.54
03.01.76
3
NL//
Нmin
3.48
3.94
47.83
24.75
11.06.76
7.0
0.10
113p/e
48.44
25.56
06.06.76
9
PQ/
Нmax
2.81
3.08
47.83
24.75
11.06.76
7.0
0.10
114p
48.44
25.57
06.06.76
9
PQ/
Нmax
1.93
2.25
47.83
24.75
11.06.76
7.0
0.10
95p
48.47
25.54
06.06.76
9
PQ/
Нmax
2.58
2.92
48.95
24.07
31.10.79
9.5
31.60
114p
48.44
25.57
30.10.79
13
//PL
Н5
2.80
3.24
48.05
25.00
13.07.85
7.7
0.50
113p/e
48.44
25.56
27.07.85
11
Нmin
4.56
4.06
48.05
25.00
13.07.85
7.7
0.50
114p
48.44
25.57
06.07.85
19
Нmax
1.90
2.25
47.70
24.50
28.08.90
7.8
0.63
102p/e
27.08.90
8
/PQ
Н5
1.80
2.02
48.20
24.70
08.01.93
7.6
0.40
102p/e
03.01.93
11
PQ//
Нmin
1.68
2.09
48.80
24.29
16.08.95
8.2
1.58
114p
48.44
25.57
09.08.95
14
/PL
Н5
2.97
3.24
48.69
24.40
28.05.96
7.9
0.79
95p
48.47
25.54
27.05.96
11
PQ//
Нmin
3.16
3.94 Table 1. Interconnection between the Moon Phases, earthquakes, and groundwater level Figure 5. Dependency graph for a number of the registered extreme groundwater levels (by the lunar calendar Figure 5. Dependency graph for a number of the registered extreme groundwater levels (by the lunar calendar While considering the graphs of the extreme
groundwater levels and number of the earth
quakes both in the Ivano-Frankivsk region and in
the adjacent territories with the energy class K> 6
(Fig. 6) for 1962–2003, we may notice an inverse
relation. It is especially traced for maximum
groundwater levels. The same trend is observed
for the minimum groundwater levels from the
first to third day of New Moon Phase (NL), dur
ing the First Quarter (PQ) and the full Moon (PL). RESULTS AND DISCUSSION The ground
water levels are in the antiphase during the sec
ond part of the Last Quarter Phase (DQ) during
the twenty-fourth-twenty-sixth days as well as
from the beginning of the New Moon Phase (NL)
from the twenty-eighth to the thirties day. First Quarter of the Moon Phase (PQ) (7 cases of
21 values registered within the phase), that con
stitutes 36% and 33%, respectively. There is a characteristic feature as to the regis
tration of the minimum groundwater levels, which
is conducted the next day of the phase. Such cases
are registered during the New Moon (NL), the Last
Quarter of the Moon Phase (DQ), the Full Moon
(PL) (6–7 cases), which constitutes from 28% to
37 % of the registered values within the phase. As it seen from Table 2, the maximum
groundwater levels are registered during the New
Moon Phase (NL) (8 cases of 22 values were reg
istered within the phase) and 2 days before the 41 Journal of Ecological Engineering Vol. 19(6), 2018 Table 1. RESULTS AND DISCUSSION Interconnection between the Moon Phases, earthquakes, and groundwater levels
The Moon Phase
Day of the Lunar
Cycle
Number of registered extreme groundwater levels
Number of
earthquakes with К>6
Hmax
Hmin
Total number
//NL
28, 29
3
4
7
2
/NL
29, 30
5
5
10
1
NL
1
8
2
10
5
NL/
2
2
6
8
3
NL//
3
4
4
8
1
//PQ
6, 7
7
3
10
2
/PQ
7, 8
4
3
7
6
PQ
9
2
3
5
3
PQ/
8, 9
5
4
9
1
PQ//
11, 12
2
5
7
4
//PL
13, 14
4
1
5
5
/PL
14, 15, 16
4
5
9
2
PL
15, 16
4
3
7
6
PL/
16, 17, 18
5
7
12
3
PL//
18, 19
3
3
6
1
//DQ
21, 22
5
4
9
2
/DQ
22, 23
5
5
10
3
DQ
23
2
5
7
3
DQ/
23, 25
2
6
8
3
DQ//
25, 26
4
3
7
4
Total number of registered extreme
groundwater levels during the Moon Phases
/ Total number
80/118
81/131
161/249
60/94 Table 2. Interconnection between the Moon Phases, earthquakes, and groundwater levels
The Moon Phase
Day of the Lunar
Cycle
Number of registered extreme groundwater levels
Number of
earthquakes with К>6
Hmax
Hmin
Total number
//NL
28, 29
3
4
7
2
/NL
29, 30
5
5
10
1
NL
1
8
2
10
5
NL/
2
2
6
8
3
NL//
3
4
4
8
1
//PQ
6, 7
7
3
10
2
/PQ
7, 8
4
3
7
6
PQ
9
2
3
5
3
PQ/
8, 9
5
4
9
1
PQ//
11, 12
2
5
7
4
//PL
13, 14
4
1
5
5
/PL
14, 15, 16
4
5
9
2
PL
15, 16
4
3
7
6
PL/
16, 17, 18
5
7
12
3
PL//
18, 19
3
3
6
1
//DQ
21, 22
5
4
9
2
/DQ
22, 23
5
5
10
3
DQ
23
2
5
7
3
DQ/
23, 25
2
6
8
3
DQ//
25, 26
4
3
7
4
Total number of registered extreme
groundwater levels during the Moon Phases
/ Total number
80/118
81/131
161/249
60/94 Figure 6. Graph of the earthquake numbers in the Ivano-Frankivsk region and the extreme groundwater levels
(according to the lunar calendar) Figure 6. RESULTS AND DISCUSSION However, since the second half of Full Moon
Phase (PL) and during the Last Quarter Phase
(DQ) we can notice a significant correlation be
tween changing of minimum groundwater levels
and registration of strong earthquakes with ener
gy class K> 6 (correlation class from the fifteenth
to thirties day of the Moon cycle equals 0.47). it coincided with Perigee (the distance between
the Moon and the Earth is the closest). Thus,
eight cases of the registered earthquakes with
energy class К=7.1–9.1 occurred during Perigee
or within it (≈ 2 days). In the days of the New Moon Phase (NL)
(from the first to seventh day) and the Full Moon
(from the thirteenth to seventeenth day), we may
observe the inverse relation, which is primarily
agreed with Hmax, between the number of mea
surements of the extreme groundwater levels and
number of the registered earthquakes. Only a
graph of changing measurement of Hmin repeats
the trend of the earthquake registration. The maximum values peculiar to occurrence
of the groundwater levels (Hmax) are most of
ten registered during the First and the Last Moon
Quarter (PQ and DQ). The coefficients of corre
lation equal 0.98 and 0.8, respectively. It should We must also note the increase in seismic
activity during the Full Moon Phase (PL), when 42 Journal of Ecological Engineering Vol. 19(6), 2018
Table 2. RESULTS AND DISCUSSION Interconnection between the Moon Phases, earthquakes, and groundwater levels
The Moon Phase
Day of the Lunar
Cycle
Number of registered extreme groundwater levels
Number of
earthquakes with К>6
Hmax
Hmin
Total number
//NL
28, 29
3
4
7
2
/NL
29, 30
5
5
10
1
NL
1
8
2
10
5
NL/
2
2
6
8
3
NL//
3
4
4
8
1
//PQ
6, 7
7
3
10
2
/PQ
7, 8
4
3
7
6
PQ
9
2
3
5
3
PQ/
8, 9
5
4
9
1
PQ//
11, 12
2
5
7
4
//PL
13, 14
4
1
5
5
/PL
14, 15, 16
4
5
9
2
PL
15, 16
4
3
7
6
PL/
16, 17, 18
5
7
12
3
PL//
18, 19
3
3
6
1
//DQ
21, 22
5
4
9
2
/DQ
22, 23
5
5
10
3
DQ
23
2
5
7
3
DQ/
23, 25
2
6
8
3
DQ//
25, 26
4
3
7
4
Total number of registered extreme
groundwater levels during the Moon Phases
/ Total number
80/118
81/131
161/249
60/94
Figure 6. Graph of the earthquake numbers in the Ivano-Frankivsk region and the extreme groundwater levels
(according to the lunar calendar) Journal of Ecological Engineering Vol. 19(6), 2018 Table 2. CONCLUSION odological approach for determination of the
technogenic impact on groundwater (case study
of quaternary aquifer on the territory of Rivne
NPP). Visnyk of Taras Shevchenko National
University of Kyiv: Geology, 69, 75–78. DOI:
10.17721/1728–2713.69.12.75–78. odological approach for determination of the
technogenic impact on groundwater (case study
of quaternary aquifer on the territory of Rivne
NPP). Visnyk of Taras Shevchenko National
University of Kyiv: Geology, 69, 75–78. DOI:
10.17721/1728–2713.69.12.75–78. It is necessary to review and expand the net
work of the observation wells as well as to pro
vide the development and improvement of the
automated database on the grounds of contem
porary geo-informative technologies in order to
ensure the functioning of the groundwater moni
toring system. The geo-informative approach to
decision of the hydrogeological monitoring tasks
envisages the systematic use of geo-informative
analysis for collection, process and storage of in
formation. In addition, the approach aims at the
creation of permanent hydrogeological models
based on GIS and allows the efficient analysis
of the spatially organized information obtained
because of the regime observations. Moreover,
the introduction of GIS-technologies permits to
implement the basin principle for processing of
the groundwater monitoring data while the single
geodatabase within the groundwater monitoring
system will allow the maximum preservation of
the existing hydrogeological survey results, en
sure the integration of heterogeneous data and
their operational analysis. A complex analysis
of the periodical observation data in the Ivano-
Frankivsk region, in particular, confirms the inter
connections of the recording time of the seasonal
and annual extremes peculiar to the groundwater
levels with seismic activity and the lunar calen
dar. The results obtained may be used not only
for clarification of the hydrogeological forecasts
but also for the risk assessment of the earthquake
arising in the adjacent territories. 5. 5. Directive 2000/60/EC of the European Parliament
and of the Council of 23 October 2000 establish
ing a framework for Community action in the field
of water policy [official website]. http://eur-lex. europa.eu/resource.html?uri=cellar:5c835afb-
2 e c 6 4 5 7 7 b d f 8 7 5 6 d 3 d 6 9 4 e e b . 0 0 0 4 . 0 2 /
DOC_1&format=PDF [access: 29.01.2018]. 6. Hudak P., Loaiciga H. 1992. A location modeling
approach for groundwater monitoring network
augmentation. Water Resources Research, 28,
643–649. DOI: 10.1029/91WR02851 7. Koshliakov O., Dyniak O., Koshliakova I. 2016. RESULTS AND DISCUSSION Graph of the earthquake numbers in the Ivano-Frankivsk region and the extreme groundwater levels
(according to the lunar calendar) The registration of the extreme underground
water levels Hmax and Hmin is in-phase in the
days of the New Moon (NL), the coefficient
of correlation equals 0.8–0.9. During the Full
Moon (PL), the registration of Hmin decreases
with the increasing of the earthquake num
ber (the coefficient of correlation is negative
(-0.64). This shows a rise of the water level up
to the surface. Such regularities may indicate
an increase in probability of the earthquake
arising in the days of the First Quarter (PQ),
the Full Moon (PL), and the New Moon (NL)
in seismic-active territories. be noted that 35 earthquakes were registered
during PQ Phase; it constitutes 37% of the total
number of the studied earthquakes. The mini
mal extreme values Hmin were also registered
at that time and the coefficient of correlation is
positive and equals 0.51. Thus, one may state that the tides occurring
on the Earth during the Full Moon (PL) are one of
the factors, which not only influence the changing
in the occurrence of the groundwater levels but
also cause the earthquakes (at that time 17 cases
were registered, which equaled to 28% of the to
tal number of earthquakes registered during the
Moon Phases). 43 Journal of Ecological Engineering Vol. 19(6), 2018 CONCLUSION Hydrogeological and geoinformatical as
pects of European standards implementation
in Ukraine in the area of natural waters qual
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details/?publication=84608 [access: 18.02.2018]. DOI: 10.3997/2214–4609.201600507. 8. Maréchal J.-C. et al. 2002. Establishment of earth
tides effect on water level fluctuations in an un
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ing into account international experience and the re
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Directive. Mineral Resources of Ukraine, 2, 3–4. 2. Berezko O., Vasneva O. 2012. Groundwater
monitoring in Belarus: implication and future
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tivariate geostatistical design of groundwater mon
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Impact of High-Cut-Off Dialysis on Renal Recovery in Dialysis-Dependent Multiple Myeloma Patients: Results from a Case-Control Study
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Neuropathic Pain Develops Normally in Mice Lacking both Na<sub>v</sub>1.7 and Na<sub>v</sub>1.8
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Molecular pain
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BioMed Central BioMed Central BioMed Central Open Ac
Research
Neuropathic pain develops normally in mice lacking both Nav1.7
and Nav1.8
Mohammed A Nassar, Alessandra Levato, L Caroline Stirling and
John N Wood* Open Access Address: Molecular Nociception Group, and London Pain Consortium, Department of Biology, University College London, Gower Street, WC1E
6BT London UK Address: Molecular Nociception Group, and London Pain Consortium, Department of Biology, University College London, Gower Street, WC1E
6BT, London, UK Email: Mohammed A Nassar - M.nassar@ucl.ac.uk; Alessandra Levato - Alessandra_Levato@yahoo.it; L Caroline Stirling - C.Stirling@ucl.ac.uk;
John N Wood* - J.wood@ucl.ac.uk * Corresponding author * Corresponding author Published: 22 August 2005
Molecular Pain 2005, 1:24
doi:10.1186/1744-8069-1-24
Received: 04 August 2005
Accepted: 22 August 2005
This article is available from: http://www.molecularpain.com/content/1/1/24
© 2005 Nassar 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. Received: 04 August 2005
Accepted: 22 August 2005 This article is available from: http://www.molecularpain.com/content/1/1/24
© 2005 Nassar 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. © 2005 Nassar 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. Abstract Two voltage gated sodium channel α-subunits, Nav1.7 and Nav1.8, are expressed at high levels in
nociceptor terminals and have been implicated in the development of inflammatory pain. Mis-
expression of voltage-gated sodium channels by damaged sensory neurons has also been implicated
in the development of neuropathic pain, but the role of Nav1.7 and Nav1.8 is uncertain. Here we
show that deleting Nav1.7 has no effect on the development of neuropathic pain. Double knockouts
of both Nav1.7 and Nav1.8 also develop normal levels of neuropathic pain, despite a lack of
inflammatory pain symptoms and altered mechanical and thermal acute pain thresholds. These
studies demonstrate that, in contrast to the highly significant role for Nav1.7 in determining
inflammatory pain thresholds, the development of neuropathic pain does not require the presence
of either Nav1.7 or Nav1.8 alone or in combination. of VGSC in humans are significant factors in aetiology of
neuronal diseases [4]. Background g
Voltage gated sodium channels (VGSC) underlie the elec-
trical excitability of nerve and muscle. VGSCs consist of
pore forming α-subunits and auxiliary β-subunits. There
are ten cloned α-subunits and 4 β-subunits. The β-subu-
nits modulate the localisation, expression and functional
properties of α-subunits [1]. Different α-subunits have
distinct electrophysiological and pharmacological proper-
ties [2]. The complex pattern of expression of α-subunits
may imply special roles for particular subunits in different
cell types [3]. Many loss- as well as gain-of-function muta-
tions of α-subunits have been identified in human condi-
tions characterised with epilepsy, seizures, ataxia and
increased sensitivity to pain. This suggests that mutations Nociceptors are a subset of sensory neurons that respond
to noxious thermal, mechanical and chemical stimuli. Nociceptors express multiple subtypes of α-subunits [3]. Tissue and nerve damage leads to changes in expression
and function of α-subunits that in turn can lead to change
in the excitability of sensory neurons. Changes in the
excitability of sensory neurons are thought to underlie
some chronic pain conditions [5,6]. Nav1.8 and Nav1.7
are two α-subunits that are abundant in nociceptive sen-
sory neurons [3,7,8]. Nav1.8 is expressed exclusively in
sensory neurons and is not found in the CNS [9]. Func-
tional characterization of Nav1.8 positive neurons Page 1 of 9
(page number not for citation purposes) Page 1 of 9
(page number not for citation purposes) Molecular Pain 2005, 1:24 http://www.molecularpain.com/content/1/1/24 Generation and genotyping of Nav1.7 and Nav1.8 double
knockout mice
Figure 1
Generation and genotyping of Nav1.7 and Nav1.8
double knockout mice. PCR genotyping of mice gener-
ated by breeding homozygous Nav1.8Cre/heterozygous
floxed Nav1.7 mice with each other (lanes 2, 3, and 4). All
the three groups are Nav1.8 Knockouts, i.e. positive for the
Cre band (249 bp) and negative for the wildtype Nav1.8 band
(460 bp). Mice homozygous for the floxed Nav1.7 allele (461
vs. 317 bp band) are nociceptor-specific Nav1.7 knockout as
well (lane 4). C57BL6 wildtype control is shown in lane 1 and
no DNA negative control is in lane 5. Floxed Nav1.7 band
Wildtype Nav1.7 band
Wildtype Nav1.8 band
Cre band
All Nav1.8 KO
C57BL6
Nav1.7 KO
Negative control
Nav1.7 hetero. Nav1.7 Wildtype Floxed Nav1.7 band
Wildtype Nav1.7 band
Wildtype Nav1.8 band
Cre band
All Nav1.8 KO
C57BL6
Nav1.7 KO
Negative control
Nav1.7 hetero. Nav1.7 Wildtype revealed that more than 85% are nociceptors [8]. Cre band Peripheral nerve injury leads to lowered pain thresholds,
enhanced responsiveness and/or ectopic activity in sen-
sory neurons that ultimately leads to hyperalgesia and
allodynia [5,6]. Changes in expression of α-subunits of
VGSCs have been documented in models of peripheral
nerve injury [5,6]. This has lead to the hypothesis that
modulation of α-subunits in sensory neurons may under-
lie the increased neuronal excitability of sensory neurons
following peripheral nerve injury. Pharmacological block-
ade of sodium channel activity has been shown to attenu-
ate ectopic activity [14,15] and reverse hyperalgesia
following nerve injury [16]. While the role of Nav1.7 in
neuropathic pain remains to be investigated, analysis of a
Nav1.8 knockout mouse indicated that it is not involved
in alteration of pain threshold following peripheral nerve
injury [17]. This is in contrast to the finding of Lai et al
who reported that antisense oligonucleotides directed
against Nav1.8 administered intrathecally completely
reverse neuropathic pain behavior [18]. It is possible that
this discrepancy could be due to the up-regulation of the
Nav1.7 channel seen in the Nav1.8 knockout mouse [12]
which might mask an otherwise important role for Nav1.8
in neuropathic pain. Generation and genotyping of Nav1.7 and Nav1.8 double
knockout mice
Figure 1
Generation and genotyping of Nav1.7 and Nav1.8
double knockout mice. PCR genotyping of mice gener-
ated by breeding homozygous Nav1.8Cre/heterozygous
floxed Nav1.7 mice with each other (lanes 2, 3, and 4). All
the three groups are Nav1.8 Knockouts, i.e. positive for the
Cre band (249 bp) and negative for the wildtype Nav1.8 band
(460 bp). Mice homozygous for the floxed Nav1.7 allele (461
vs. 317 bp band) are nociceptor-specific Nav1.7 knockout as
well (lane 4). C57BL6 wildtype control is shown in lane 1 and
no DNA negative control is in lane 5. Generation and genotyping of Nav1.7 and Nav1.8 double
knockout mice
Figure 1
Generation and genotyping of Nav1.7 and Nav1.8
double knockout mice. PCR genotyping of mice gener-
ated by breeding homozygous Nav1.8Cre/heterozygous
floxed Nav1.7 mice with each other (lanes 2, 3, and 4). All
the three groups are Nav1.8 Knockouts, i.e. positive for the
Cre band (249 bp) and negative for the wildtype Nav1.8 band
(460 bp). Mice homozygous for the floxed Nav1.7 allele (461
vs. 317 bp band) are nociceptor-specific Nav1.7 knockout as
well (lane 4). C57BL6 wildtype control is shown in lane 1 and
no DNA negative control is in lane 5. lethal at P0 [13]. Cre band Therefore, we generated Nav1.7 and
Nav1.8 double-knockout (DKO) mice by exploiting the
fact that the Nav1.8Cre allele has Cre sequence inserted in
exon 1 followed by transcriptional stop signals [19]. Con-
sequently homozygous Nav1.8Cre mice are Nav1.8 global-
knockouts (Nav18 KO) and show no Nav1.8 currents in
sensory neurons [19]. lethal at P0 [13]. Therefore, we generated Nav1.7 and
Nav1.8 double-knockout (DKO) mice by exploiting the
fact that the Nav1.8Cre allele has Cre sequence inserted in
exon 1 followed by transcriptional stop signals [19]. Con-
sequently homozygous Nav1.8Cre mice are Nav1.8 global-
knockouts (Nav18 KO) and show no Nav1.8 currents in
sensory neurons [19]. We compared the DKO strain to the Nav1.8 KO
(homozygous Nav1.8Cre). To obtain the desired strains
we mated mice homozygous for the Nav1.8Cre/hetero-
zygous for floxed Nav1.7 allele with each other. The result-
ing progeny are all Nav1.8 Knockout. However, 25% will
have wildtype Nav1.7 alleles, 50% will be heterozygous
for the floxed Nav1.7 allele and 25% will be homozygous
for the floxed Nav1.7 allele (i.e. DKO) figure 1. In the present study we investigated the role of the Nav1.7
channel in neuropathic pain using nociceptor-specific
deletion of Nav1.7 in mouse. In addition, we readdressed
the role of Nav1.8 in neuropathic pain by generating a
double knockout of Nav1.8 and Nav1.7. We reasoned that
the co-deletion of Nav1.7 in Nav1.8-expressing neurons
should reveal any potential role for Nav1.8 in neuropathic
pain. Eight weeks old C57BL6 inbred mice were used as
wildtype (WT) control (wildtype Nav1.8 and Nav1.7)
because it would be impractical and undesirable to gener-
ate them as littermates to the test groups (DKO and
Nav1.8 KO). The expected ratio of each of desired strains
would be only 6.25% if the parents were heterozygous for
both Nav1.8Cre and floxed Nav1.7 alleles compared with Page 2 of 9
(page number not for citation purposes) Background Nav1.7
is expressed principally in peripheral neurons with very
weak expression detected in CNS [10,11]. As there are no
subunit specific blockers, gene knockouts in mice have
provided insights into the role of individual α-subunits
genes in pain [5]. Deletion of the Nav1.8 gene [12] and
nociceptor-specific knockout of the Nav1.7 [13] gene have
identified a role for these two α-subunits in setting
mechanical and, to a lesser extent, thermal pain thresh-
olds. In addition, behavioral studies have revealed deficits
in inflammatory pain models, most dramatically in the
nociceptor-specific Nav1.7 knockout [12,13]. These find-
ings suggest that these α-subunits could be targets for new
anti-inflammatory drugs. Generation of Nav1.7 and Nav1.8 double knockouts in
nociceptors groups, figure 2b (WT 145.6 ± 13.12 sec, n = 4; Nav1.8 KO
146.4 ± 11.37, n = 7 and DKO 108.6 ± 11.5, n = 7). 25% as in our breeding strategy. The Nav1.8 KO/Nav1.7
heterozygous group was analysed in behavioural tests and
was found to be similar to the Nav1.8 KO group (data not
shown). Acute pain thresholds We measured thermal pain thresholds using the hotplate
and Hargreave's tests. In the hotplate test, which involves
supraspinal activity, the response latency was not different
between the three mouse groups (WT 36.1 ± 6.5 sec, n =
7; Nav1.8 KO 29.0 ± 5.6, n = 7 and DKO 32.7 ± 6.35, n =
7), figure 3a. However, the response latency in the Har-
greave's test was doubled in DKO group (15.30 ± 0.91 sec,
n = 7) compared to both WT (6.88 ± 0.28, n = 11) and
Nav1.8 KO (8.47 ± 0.78, n = 7) groups, figure 3b. The
latency in the Nav1.8 KO was higher that that of the WT
group which is in agreement with the finding of Akopian Generation of Nav1.7 and Nav1.8 double knockouts in
nociceptors Generation of Nav1.7 and Nav1.8 double knockouts in
nociceptors Nociceptor-specific Nav1.7 knockout mice and their litter-
mate controls were generated as described previously
[13]. It is not possible to generate global knockouts of
both Nav1.8 and Nav1.7 since global deletion of Nav1.7 is Page 2 of 9
(page number not for citation purposes) Page 2 of 9
(page number not for citation purposes) Molecular Pain 2005, 1:24 http://www.molecularpain.com/content/1/1/24 B A Development and motor coordination
Figure 2
Development and motor coordination. A) Males and females mice have similar weights among the three groups being
compared. In males the P value for WT vs. Nav1.8 KO is 0.09, for WT vs. DKO is 0.89 and for Nav1.8 KO vs. DKO is 0.08. In
females the P value for WT vs. Nav1.8 KO is 0.27, for WT vs. DKO is 0.71 and for Nav1.8 KO vs. DKO is 0.27. B) Perform-
ance on the rotarod apparatus is not significantly different between the three mice groups. The P value for WT vs. Nav1.8 KO
is 0.98, for WT vs. DKO is 0.38 and for Nav1.8 KO vs. DKO is 0.39. P values were calculated using two-tailed T-test. WT in
white, Nav1.8 KO in grey and DKO in black. 0
10
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40
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Females
Weight (grams)
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0
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Figure 2 Development and motor coordination
Figure 2
Development and motor coordination. A) Males and females mice have similar weights among the three groups being
compared. In males the P value for WT vs. Nav1.8 KO is 0.09, for WT vs. DKO is 0.89 and for Nav1.8 KO vs. DKO is 0.08. In
females the P value for WT vs. Nav1.8 KO is 0.27, for WT vs. DKO is 0.71 and for Nav1.8 KO vs. DKO is 0.27. B) Perform-
ance on the rotarod apparatus is not significantly different between the three mice groups. The P value for WT vs. Nav1.8 KO
is 0.98, for WT vs. DKO is 0.38 and for Nav1.8 KO vs. DKO is 0.39. P values were calculated using two-tailed T-test. WT in
white, Nav1.8 KO in grey and DKO in black. Development and motor coordination The weight of male (WT 26.72 ± 1.60 gram, n = 6; Nav1.8
KO 23.44 ± 0.76, n = 9 and DKO 27.05 ± 1.63, n = 9) and
female (WT 20.50 ± 1.77 gram, n = 3; Nav1.8 KO 21.43 ±
0.95, n= 3 and DKO 19.66 ± 1.03, n = 4) mice in each
group was very similar and was not significantly different,
figure 2a. Motor coordination as tested through perform-
ance on the rotarod apparatus was unchanged between
the mouse groups studied. The time spent on the rotarod
was not significantly different between the three mouse Page 3 of 9
(page number not for citation purposes) Molecular Pain 2005, 1:24 http://www.molecularpain.com/content/1/1/24 Acute pain thresholds are increased in the DKO mice
Figure 3
Acute pain thresholds are increased in the DKO mice. A) Latency to respond in the hotplate test was not different
between all groups. The P value for WT vs. Nav1.8 KO is 0.67, for WT vs. DKO is 0.72 and for Nav1.8 KO vs. DKO is 0.43
B) Latency to paw withdrawal in the Hargreave's test was doubled in the DKO mice. The P value for WT vs. Nav1.8 KO is
0.08, for WT vs. DKO is <0.0001 and for Nav1.8 KO vs. DKO is <0.0001. C) The 50% withdrawal threshold to stimulation
with von Frey hairs was not different between all groups. The P value for WT vs. Nav1.8 KO is 0.21, for WT vs. DKO is 0.2
and for Nav1.8 KO vs. DKO is 0.84. D) The Nav1.8 KO and DKO mice showed profound analgesia to noxious mechanical
pressure applied to the tail using the Randall-Selitto apparatus. The P value for WT vs. Nav1.8 KO is <0.0001, for WT vs. DK
is <0.0001 and for Nav1.8 KO vs. DKO is 0.67. P values were calculated using two-tailed T-test. WT in white, Nav1.8 KO in
grey and DKO in black. Page 4 of 9
(page number not for citation purposes) Development and motor coordination A
B
C
D
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(grams) D Acute pain thresholds are increased in the DKO mice
Figure 3
Acute pain thresholds are increased in the DKO mice. A) Latency to respond in the hotplate test was not different
between all groups. The P value for WT vs. Nav1.8 KO is 0.67, for WT vs. DKO is 0.72 and for Nav1.8 KO vs. DKO is 0.43. B) Latency to paw withdrawal in the Hargreave's test was doubled in the DKO mice. The P value for WT vs. Nav1.8 KO is
0.08, for WT vs. DKO is <0.0001 and for Nav1.8 KO vs. DKO is <0.0001. C) The 50% withdrawal threshold to stimulation
with von Frey hairs was not different between all groups. The P value for WT vs. Nav1.8 KO is 0.21, for WT vs. DKO is 0.25
and for Nav1.8 KO vs. DKO is 0.84. D) The Nav1.8 KO and DKO mice showed profound analgesia to noxious mechanical
pressure applied to the tail using the Randall-Selitto apparatus. The P value for WT vs. Nav1.8 KO is <0.0001, for WT vs. DKO
is <0.0001 and for Nav1.8 KO vs. DKO is 0.67. P values were calculated using two-tailed T-test. WT in white, Nav1.8 KO in
grey and DKO in black. Page 4 of 9
(page number not for citation purposes) Page 4 of 9
(page number not for citation purposes) Molecular Pain 2005, 1:24 http://www.molecularpain.com/content/1/1/24 Reduced pain behavior in formalin test in DKO
Figure 4
Reduced pain behavior in formalin test in DKO. A) The pain response after injection of 5% formalin in hindpaw showed
the typical biphasic course. B) The second phase of the formalin response was reduced to 30% in the DKO mice compared to
WT and Nav1.8 KO mice. The P value for WT vs. Nav1.8 KO is 0.94, for WT vs. DKO is 0.001 and for Nav1.8 KO vs. DKO
is 0.08. The first phase was not significantly different between all groups. Development and motor coordination The P value for WT vs. Nav1.8 KO is 0.60, for WT vs. DKO is 0.30 and for Nav1.8 KO vs. DKO is 0.96. P values were calculated using two-tailed T-test. WT in open circles, Nav1.8
KO in grey triangles and DKO in black squares. A
B
0
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Figure 4 Reduced pain behavior in formalin test in DKO
Figure 4
Reduced pain behavior in formalin test in DKO. A) The pain response after injection of 5% formalin in hindpaw showed
the typical biphasic course. B) The second phase of the formalin response was reduced to 30% in the DKO mice compared to
WT and Nav1.8 KO mice. The P value for WT vs. Nav1.8 KO is 0.94, for WT vs. DKO is 0.001 and for Nav1.8 KO vs. DKO
is 0.08. The first phase was not significantly different between all groups. The P value for WT vs. Nav1.8 KO is 0.60, for WT vs. DKO is 0.30 and for Nav1.8 KO vs. DKO is 0.96. P values were calculated using two-tailed T-test. WT in open circles, Nav1.8
KO in grey triangles and DKO in black squares. Reduced pain behavior in formalin test in DKO. A) The pain response after injection of 5% formalin in hindpaw showed
the typical biphasic course. B) The second phase of the formalin response was reduced to 30% in the DKO mice compared to
WT and Nav1.8 KO mice. The P value for WT vs. Nav1.8 KO is 0.94, for WT vs. DKO is 0.001 and for Nav1.8 KO vs. DKO
is 0.08. The first phase was not significantly different between all groups. The P value for WT vs. Nav1.8 KO is 0.60, for WT vs. DKO is 0.30 and for Nav1.8 KO vs. DKO is 0.96. P values were calculated using two-tailed T-test. WT in open circles, Nav1.8
KO in grey triangles and DKO in black squares. et al using the conventional Nav1.8 global knockout [12]. Development and motor coordination The nociceptor-specific Nav1.7 knockout alone shows a
40% increase in latency in the Hargreave's test [13] Nav1.8 and the DKO mice in this test, figure 3d, and sim-
ilar results have been obtained with the nociceptor-spe-
cific Nav1.7 knockout [13] Nav1.8 and the DKO mice in this test, figure 3d, and sim-
ilar results have been obtained with the nociceptor-spe-
cific Nav1.7 knockout [13] Pain thresholds to punctate mechanical stimulation was
measured using calibrated von Frey hairs according to the
up and down method [20]. The 50% withdrawal thresh-
old was not different between the three groups (WT 0.56
± 0.07 gram, n = 11; Nav1.8 KO 0.45 ± 0.05, n = 13 and
DKO 0.49 ± 0.06, n = 17), figure 3c. In contrast pain
threshold to noxious mechanical pressure applied to the
tail using the Randall-Selitto apparatus was much higher
in both the Nav1.8 KO (412.4 ± 33.97 gram, n = 7) and
DKO (395.7 ± 21.03, n = 7) groups compared to that of
the WT (131.4 ± 16.54, n = 6) group, figure 3d. Usually
the cut off point (500 gram) was reached without an
observed escape response in the Nav1.8 KO and DKO
groups, indicating a high resistance to static blunt
mechanical pressure. There was no difference between the Inflammatory pain behaviour (B) WT (open circles), Nav1.8 KO (grey triangles) and DKO (black squares) developed profound
h
i
l ll d
i
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Test/baseline
0
0.2
0.4
0.6
0.8
1
1.2
5
10
15
20
25
30 B 10 30 Analysis o
Figure 5 y
p
p
p
p
g
Analysis of neuropathic pain in nociceptor-specific Nav1.7 knockout mice and double knockout mice. (A) Both
control (white circles, mean 0.56 ± 0.9, n = 8) and nociceptor-specific Nav1.7 KO (black squares, 0.48 ± 0.8, n = 11) developed
robust mechanical allodynia after ligation of spinal nerve L5. There is no difference in the extent of pain behavior at any time
point (P = 0.49 ANOVA). (B) WT (open circles), Nav1.8 KO (grey triangles) and DKO (black squares) developed profound
mechanical allodynia after ligation of spinal nerve L5. commonly used inflammatory pain models [13]. It would
be impossible therefore to measure a further reduction in
inflammatory pain behaviour in the DKO. spinal nerve) in the Nav1.7 nociceptor-specific knockout
and homozygous floxed-Nav1.7 as controls. Both groups
developed a robust mechanical allodynia starting form
the third day post surgery, figure 5a. The extent and time
course of development of increased mechanosensitivity
was identical in both nociceptor-specific Nav1.7 knock-
out and their littermate controls. Inflammatory pain behaviour The pain response elicited by injection of 20µl of 5% for-
malin intradermally in the hindpaw showed the typical
biphasic response in the three groups, figure 4a. The first
phase (1–10 minutes) was not different between the three
groups (WT 107.8 ± 10.12 sec n = 8, Nav1.8 KO 155 ±
11.00 n = 4, DKO 120.4 ± 5.96 n = 8), figure 4b. In con-
trast, the second phase (10–60 minutes) was much
reduced in the DKO group (105.4 ± 28.43 sec) compared
to both the Nav1.8 KO (309 ± 80.85, P = 0.08) and the WT
(216 ± 43.67, P = 0.0016) groups, figure 4b. This effect
was even more dramatic than that observed in the nocice-
ptor specific Nav1.7 knockout [13] We did not study other
inflammatory pain models since the nociceptor-specific
Nav1.7 knockout mouse is completely deficient in Page 5 of 9
(page number not for citation purposes) http://www.molecularpain.com/content/1/1/24 Molecular Pain 2005, 1:24 Analysis of neuropathic pain in nociceptor-specific Nav1.7 knockout mice and double knockout mice
Figure 5
Analysis of neuropathic pain in nociceptor-specific Nav1.7 knockout mice and double knockout mice. (A) Both
control (white circles, mean 0.56 ± 0.9, n = 8) and nociceptor-specific Nav1.7 KO (black squares, 0.48 ± 0.8, n = 11) developed
robust mechanical allodynia after ligation of spinal nerve L5. There is no difference in the extent of pain behavior at any time
point (P = 0.49 ANOVA). (B) WT (open circles), Nav1.8 KO (grey triangles) and DKO (black squares) developed profound
mechanical allodynia after ligation of spinal nerve L5. A
B
0
0.2
0.4
0.6
0.8
1
1.2
0
5
10
15
20
25
Test/baseline
Test/baseline
Time post surgery (days)
0
0.2
0.4
0.6
0.8
1
1.2
5
10
15
20
25
30 A
0
0.2
0.4
0.6
0.8
1
1.2
0
5
10
15
20
25
Test/baseline A Analysis of neuropathic pain in nociceptor-specific Nav1.7 knockout mice and double knockout mice
Figure 5
Analysis of neuropathic pain in nociceptor-specific Nav1.7 knockout mice and double knockout mice. (A) Both
control (white circles, mean 0.56 ± 0.9, n = 8) and nociceptor-specific Nav1.7 KO (black squares, 0.48 ± 0.8, n = 11) developed
robust mechanical allodynia after ligation of spinal nerve L5. There is no difference in the extent of pain behavior at any time
point (P = 0.49 ANOVA). Neuropathic pain We investigated the role of Nav1.7 expressed in nocicep-
tors in neuropathic pain. Analysis of neuropathic pain in
the nociceptor-specific Nav1.7 knockout using the Chung
model showed that they developed mechanical allodynia
to the same extent as the littermate control mice
(homozygous floxed Nav1.7), figure 5. This clearly shows
that unlike its critical role in inflammatory pain [13],
Nav1.7 does not contribute to neuropathic pain. In addition, the Nav1.8 knockout and WT as well as the
DKO mice developed a profound mechanical allodynia
the level of which was indistinguishable between the three
groups. This provides further evidence that Nav1.8 does
not contribute to mechanical allodynia as reported by
Kerr et al [17]. In addition our results rule out Nav1.7 up-
regulation in the Nav1.8 knockout as the reason behind
the discrepancy between the finding of Kerr et al [17] and
Lai et al [18]. Surprisingly Lai et al [18] failed to detect the
changes in acute pain thresholds observed in the Nav1.8
KO both in this study and that of Akopian et al [12],
which would be expected to occur if Nav1.8 had been
down-regulated. Acute pain thresholds p
analysis of acute mechanical thresholds in DKO mice con-
firmed previous findings that the knockout of either
Nav1.8 or Nav1.7 renders the mice resistant in the Randel-
Selitto test of noxious mechanical pressure while the
responses to von Frey hairs remain unchanged compared
to controls [12,13]. Furthermore, it has been shown that
both Nav1.8 and nociceptor-specific Nav1.7 knockouts
have small increases of about 20–40% in their thermal
threshold [12,13]. Interestingly, we found that the ther-
mal threshold in the Hargreave's test was doubled in the
DKO compared to Nav1.8 knockout or WT controls, figure
3b. The increase is more than the sum of the phenotype in
the individual knockouts and may indicate that Nav1.8
and Nav1.7 are the major VGSC isoforms present in noci-
ceptive terminals and their deletion dramatically increases
pain threshold. This is supported by the fact that Nav1.7 is
transported to nerve terminals of sensory neurons [10]
and that Nav1.8 currents are localized in the terminals
[23]. http://www.molecularpain.com/content/1/1/24 Molecular Pain 2005, 1:24 only investigated the pain response in the formalin
model. While the nociceptive-specific Nav1.7 knockout
showed a reduction in the second phase to about 50% of
controls [13] the DKO showed a slightly bigger reduction
to 30% of controls, figure 4b. This is in contrast to Nav1.8
mice that showed no reduction in the second phase com-
pared to controls, figure 4b. Therefore, the reduction in
the second phase in the DKO is a new phenotype rather
than a mere summation of the phenotype in the individ-
ual knockouts. This highlights further the predominant
role for Nav1.7 in inflammatory pain. Expression of
Nav1.7 has been reported to increase in sensory neurons
subsequent to induction of inflammation [24]. Neuropathic pain in Nav1.7 Nav1.8 double knockout mice
To address the issue of compensatory up-regulation of
Nav1.7 in Nav1.8 knockout we induced peripheral nerve
injury in WT, Nav1.8 KO and DKO. All mice groups
studied developed a robust mechanical allodynia starting
form the third day post surgery, figure 5b. There were no
statistically meaningful differences in the behaviour of the
groups of mice. Discussion This study confirms that the two sodium channel α- sub-
units Nav1.7 and Nav1.8, expressed selectively in nocicep-
tive sensory neurons, have important roles in nociception
and in pain. In the absence of specific pharmacological
blockers, the use of genetic approaches, a combination of
global and nociceptor-specific knockouts, has enabled us
to carry out studies exploring the contribution of these
two isoforms of VGSCs in different pain conditions. Nav1.8 knockouts are already known to have deficits in
inflammatory and visceral but not neuropathic pain [12]. Nav1.7 nociceptor-specific knockouts are almost totally
refractory to changes in peripheral pain thresholds evoked
by inflammatory mediator [13]. This mirrors the inflam-
matory phenotype of Nav1.7 gain-of-function mutations
in erythermalgia [21]. This heritable disorder is caused by
point mutations that lead to altered thresholds of activa-
tion in Nav1.7 [22] This study and our previous [13] work have explored the
role of Nav1.7 in Nav1.8-expressing neurons, since our
strategy exploits the Nav1.8 promoter to drive Cre expres-
sion. We have shown previously using non-quantitative
RT-PCR that not all the Nav1.7 mRNA in DRGs from the
nociceptor-specific Nav1.7 knockout is deleted [13]. This
indicates that there is a population of sensory neurons,
the size of which has yet to be determined, that express
Nav1.7 but not Nav1.8. Therefore, the role of Nav1.7 in
sensory neurons where Nav1.8 is not expressed (and
which do not express Cre) remains to be studied. From all the above, our results indicate that Nav1.8 and
Nav1.7 subunits contribute to the excitability of periph-
eral nerve terminals and their modulation is critical for
peripheral sensitisation in inflammatory pain Neuropathic pain in nociceptor-specific Nav1.7 knockout
mice To study neuropathic pain behavior we induced periph-
eral nerve injury using to the Chung model (ligation of L5 Page 6 of 9
(page number not for citation purposes) Page 6 of 9
(page number not for citation purposes) http://www.molecularpain.com/content/1/1/24 Page 7 of 9
(page number not for citation purposes) Genotyping of mice strains g
5. Wood JN, Abrahamsen B, Baker MD, Boorman JD, Donier E, Drew
LJ, Nassar MA, Okuse K, Seereeram A, Stirling CL, Zhao J: Ion chan-
nel activities implicated in pathological pain. Novartis Found
Symp 2004, 261:32-40. discussion 40–54 Nav1.8Cre and floxed Nav1.7 lines were produced as
described [13]. Both strains were back-crossed at last 5
times onto a C57/BL6 background. y p
6. Lai J, Porreca F, Hunter JC, Gold MS: Voltage-gated sodium chan-
nels and hyperalgesia. Annu Rev Pharmacol Toxicol 2004,
44:371-397. Genomic DNA was prepared from tail biopsies and geno-
typed using PCR. Primers for Nav1.7 are (CAGAGATTTCT-
GCATTAGAATTTGTTC)
and
(AGTCTTTGTGGCACACGTTACCTC) which give a WT
band of 317 bp and a floxed band of 461 bp. Primers for
Nav1.8 are (TGTAGATGGACTGCAGAGGATGGA) and
(ttacccggtgtgtgctgtagaaag) which give a WT band of 460
bp. Nav1.8Cre was detected by primers (aaatttgcctgcattac-
cggtcga) and (aaatgttgctggatagtttttactgcc) located inside
Cre sequence which give a band of 249 bp. Genomic DNA was prepared from tail biopsies and geno-
typed using PCR. Primers for Nav1.7 are (CAGAGATTTCT-
GCATTAGAATTTGTTC)
and 7. Djouhri L, Newton R, Levinson SR, Berry CM, Carruthers B, Lawson
SN: Sensory and electrophysiological properties of guinea-
pig sensory neurones expressing Nav 1.7 (PN1) Na+ channel
alpha subunit protein. J Physiol 2003, 546:565-576. (AGTCTTTGTGGCACACGTTACCTC) which give a WT
band of 317 bp and a floxed band of 461 bp. Primers for
Nav1.8 are (TGTAGATGGACTGCAGAGGATGGA) and
(ttacccggtgtgtgctgtagaaag) which give a WT band of 460
bp. Nav1.8Cre was detected by primers (aaatttgcctgcattac-
cggtcga) and (aaatgttgctggatagtttttactgcc) located inside
Cre sequence which give a band of 249 bp. 8. Djouhri L, Fang X, Okuse K, Wood JN, Berry CM, Lawson SN: The
TTX-resistant sodium channel Nav1.8 (SNS/PN3): expres-
sion and correlation with membrane properties in rat nocic-
eptive primary afferent neurons. J Physiol 2003, 550:739-752. p
p
y
J
y
9. Akopian AN, Sivilotti L, Wood JN: A tetrodotoxin-resistant volt-
age-gated sodium channel expressed by sensory neurons. Nature 1996, 379:257-262. 10. Toledo-Aral JJ, Moss BL, He ZJ, Koszowski AG, Whisenand T, Levin-
son SR, Wolf JJ, Silos-Santiago I, Halegoua S, Mandel G: Identifica-
tion of PN1, a predominant voltage-dependent sodium
channel expressed principally in peripheral neurons. Proc Natl
Acad Sci U S A 1997, 94:1527-1532. Acknowledgements The authors would like to acknowledge Mark D Baker for critical reading
of the manuscript and Elizabeth A Matthew for great help in establishing the
Chung model. We also thank the Wellcome Trust, the MRC and the Lon-
don Pain Consortium for funding this work. The authors would like to acknowledge Mark D Baker for critical reading
of the manuscript and Elizabeth A Matthew for great help in establishing the
Chung model. We also thank the Wellcome Trust, the MRC and the Lon-
don Pain Consortium for funding this work. Competing interests p
g
The author(s) declare that they have no competing
interests. References 2. Baker MD, Wood JN: Involvement of Na+ channels in pain
pathways. Trends Pharmacol Sci 2001, 22:27-31. 3. Felts PA, Yokoyama S, Dib-Hajj S, Black JA, Waxman SG: Sodium
channel alpha-subunit mRNAs I, II, III, NaG, Na6 and hNE
(PN1): different expression patterns in developing rat nerv-
ous system. Brain Res Mol Brain Res 1997, 45:71-82. y
4. Meisler MH, Kearney JA: Sodium channel mutations in epilepsy
and other neurological disorders. The Journal of Clinical
Investigation 2005, 115:2010-2017. Inflammatory pain We have not studied pain behavior in inflammatory mod-
els where the nociceptive-specific Nav1.7 knockout has
shown complete deficits in inflammatory pain behavior
[13], because it would not be possible to observe further
changes brought about by the co-deletion of Nav1.8. We Page 7 of 9
(page number not for citation purposes) Page 7 of 9
(page number not for citation purposes) Molecular Pain 2005, 1:24 http://www.molecularpain.com/content/1/1/24 The lack of changes in mechanical allodynia in any indi-
vidual or even in double knockouts of Nav1.8 and Nav1.7
subunits is consistent with recent data by Flake et al. They
reported that changes in sodium currents shortly after
nerve injury do not correlate with an increase in neuronal
excitability and that the sum of changes to ionic currents,
and not a single class of voltage-gated ion channel, under-
lie increased neuronal excitability [25]. This does not,
however, exclude a potential role for both Nav1.7 and
Nav1.8 in spontaneous neuropathic pain as suggested by
the finding that neuromas in the Nav1.8 knockout mouse
display less ectopic discharges than wildtype littermates
[26]. These studies confirm the importance of Nav1.7 and
Nav1.8 in the physiological processes that underlie altered
peripheral thresholds in inflammatory pain. Neuropathic
pain that arises as a consequence of nerve damage and
neuronal dysfunction is, however, not dependent on the
presence of these two sodium channels. In terms of the
contribution of other α-subunits to neuropathic pain, a
strong correlation has been found between the expression
of Nav1.3 and the appearance of neuropathic pain. GDNF
that reverses neuropathic pain behaviour also normalises
Nav1.3 expression [27], and recent antisense studies have
supported the view that increased Nav1.3 expression con-
tributes to neuropathic pain development [28] formed in a Home Office designated room at 22 ± 2°C. Experiments were performed on animals of at least 8
weeks of age. Behavioral tests were done as before [19] LCS Analysed the Nav1.8Cre mouse. LCS Analysed the Nav1.8Cre mouse. JNW:
supervised
experiments
and
corrected
the
manuscript. References References
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pathways. Trends Pharmacol Sci 2001, 22:27-31. 3. Felts PA, Yokoyama S, Dib-Hajj S, Black JA, Waxman SG: Sodium
channel alpha-subunit mRNAs I, II, III, NaG, Na6 and hNE
(PN1): different expression patterns in developing rat nerv-
ous system. Brain Res Mol Brain Res 1997, 45:71-82. 4. Meisler MH, Kearney JA: Sodium channel mutations in epilepsy
and other neurological disorders. The Journal of Clinical
Investigation 2005, 115:2010-2017. 5. Wood JN, Abrahamsen B, Baker MD, Boorman JD, Donier E, Drew
LJ, Nassar MA, Okuse K, Seereeram A, Stirling CL, Zhao J: Ion chan-
nel activities implicated in pathological pain. Novartis Found
Symp 2004, 261:32-40. discussion 40–54
6. Lai J, Porreca F, Hunter JC, Gold MS: Voltage-gated sodium chan-
nels and hyperalgesia. Annu Rev Pharmacol Toxicol 2004,
44:371-397. 7. Djouhri L, Newton R, Levinson SR, Berry CM, Carruthers B, Lawson
SN: Sensory and electrophysiological properties of guinea-
pig sensory neurones expressing Nav 1.7 (PN1) Na+ channel
alpha subunit protein. J Physiol 2003, 546:565-576. 8. Djouhri L, Fang X, Okuse K, Wood JN, Berry CM, Lawson SN: The
TTX-resistant sodium channel Nav1.8 (SNS/PN3): expres-
sion and correlation with membrane properties in rat nocic-
eptive primary afferent neurons. J Physiol 2003, 550:739-752. 9. Akopian AN, Sivilotti L, Wood JN: A tetrodotoxin-resistant volt-
age-gated sodium channel expressed by sensory neurons. Nature 1996, 379:257-262. 10. Toledo-Aral JJ, Moss BL, He ZJ, Koszowski AG, Whisenand T, Levin-
son SR, Wolf JJ, Silos-Santiago I, Halegoua S, Mandel G: Identifica-
tion of PN1, a predominant voltage-dependent sodium
channel expressed principally in peripheral neurons. Proc Natl
Acad Sci U S A 1997, 94:1527-1532. 11. Sangameswaran L, Fish LM, Koch BD, Rabert DK, Delgado SG, Ilnicka
M, Jakeman LB, Novakovic S, Wong K, Sze P, Tzoumaka E, Stewart
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auxiliary. Neuroscientist 2001, 7:42-54. 2. Baker MD, Wood JN: Involvement of Na+ channels in pain
pathways. Trends Pharmacol Sci 2001, 22:27-31. Conclusion In summary, our results indicate a critical role for Nav1.7
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terminals and in their sensitisation following tissue dam-
age and inflammation. However, the presence of these
channels, or changes to their expression and function are
not required for the establishment of mechanical allody-
nia arising from nerve injury. Authors' contributions MAN generated the mice, performed behavioral tests and
wrote the manuscript. MAN generated the mice, performed behavioral tests and
wrote the manuscript. AL carried out the neuropathic pain study of the DKO and
Nav1.8 KO. Behavioural analysis All tests were approved by the United Kingdom Home
Office Animals (Scientific Procedures) Act 1986 and per- 11. Sangameswaran L, Fish LM, Koch BD, Rabert DK, Delgado SG, Ilnicka
M, Jakeman LB, Novakovic S, Wong K, Sze P, Tzoumaka E, Stewart
GR, Herman RC, Chan H, Eglen RM, Hunter JC: A novel tetrodo- Page 8 of 9
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states. Science 2000, 290:124-127. Publish with BioMed Central and every
scientist can read your work free of charge
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Your research papers will be:
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BioMedcentral
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(page number not for citation purposes) Publish with BioMed Central and every
scientist can read your work free of charge
"BioMed Central will be the most significant development for
disseminating the results of biomedical research in our lifetime."
Sir Paul Nurse, Cancer Research UK
Your research papers will be:
available free of charge to the entire biomedical community
peer reviewed and published immediately upon acceptance
cited in PubMed and archived on PubMed Central
yours — you keep the copyright
Submit your manuscript here:
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BioMedcentral Publish with BioMed Central and every
scientist can read your work free of charge 28. Hains BC, Klein JP, Saab CY, Craner MJ, Black JA, Waxman SG:
Upregulation of sodium channel Nav1.3 and functional
involvement in neuronal hyperexcitability associated with
central neuropathic pain after spinal cord injury. J Neurosci
2003, 23:8881-8892.
|
https://openalex.org/W2116766958
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https://juser.fz-juelich.de/record/276177/files/bg-12-6443-2015.pdf
|
English
| null |
Speciation and distribution of P associated with Fe and Al oxides in aggregate-sized fraction of an arable soil
|
Biogeosciences
| 2,015
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cc-by
| 9,123
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Speciation and distribution of P associated with Fe and Al oxides
in aggregate-sized fraction of an arable soil
X. Jiang1, R. Bol1, S. Willbold2, H. Vereecken1, and E. Klumpp1
1Institute of Bio- and Geosciences, Agrosphere Institute (IBG-3), Jülich, Germany
2Central Institute for Engineering, Electronics and Analytics, Analytics (ZEA-3), Forschungszentrum
Jülich GmbH, Wilhelm Johnen Straße, 52425 Jülich, Germany Correspondence to: X. Jiang (x.jiang@fz-juelich.de) Received: 24 February 2015 – Published in Biogeosciences Discuss.: 2 July 2015
Revised: 21 October 2015 – Accepted: 23 October 2015 – Published: 11 November 2015 these oxides. Intriguingly, however, Fe/Al oxides were not
the main bonding sites for pyrophosphate. Residual P con-
tained similar amounts of total P associated with both a- (11–
15 % of total P) and c-Fe oxides (7–13 % of total P) in var-
ious aggregate-sized fractions, suggesting that it was likely
occluded within the a- and c-Fe oxides in soil. This implies
that, with the dissolution of Fe oxides, this P may be released
and thus available for plants and microbial communities. these oxides. Intriguingly, however, Fe/Al oxides were not
the main bonding sites for pyrophosphate. Residual P con-
tained similar amounts of total P associated with both a- (11–
15 % of total P) and c-Fe oxides (7–13 % of total P) in var-
ious aggregate-sized fractions, suggesting that it was likely
occluded within the a- and c-Fe oxides in soil. This implies
that, with the dissolution of Fe oxides, this P may be released
and thus available for plants and microbial communities. Abstract. To maximize crop productivity fertilizer P is gen-
erally applied to arable soils, a significant proportion of
which becomes stabilized by mineral components and in part
subsequently becomes unavailable to plants. However, little
is known about the relative contributions of the different or-
ganic and inorganic P bound to Fe/Al oxides in the smaller
soil particles. Alkaline (NaOH–Na2EDTA) extraction with
solution 31P-nuclear magnetic resonance (31P-NMR) spec-
troscopy is considered a reliable method for extracting and
quantifying organic P and (some) inorganic P. However, any
so-called residual P after the alkaline extraction has remained
unidentified. Therefore, in the present study, the amorphous
(a) and crystalline (c) Fe/Al oxide minerals and related P
in soil aggregate-sized fractions (> 20, 2–20, 0.45–2 and
< 0.45 µm) were specifically extracted by oxalate (a-Fe/Al
oxides) and dithionite–citrate–bicarbonate (DCB, both a- and
c-Fe/Al oxides). These soil aggregate-sized fractions with
and without the oxalate and DCB pre-treatments were then
sequentially extracted by alkaline extraction prior to solu-
tion 31P-NMR spectroscopy. This was done to quantify the
P associated with a- and c-Fe/Al oxides in both alkaline ex-
traction and the residual P of different soil aggregate-sized
fractions. 1
Introduction Phosphorus is, after nitrogen, the most limiting soil nutri-
ent for agricultural crops and plays an important role in the
overall functioning of the agro-ecosystems (Bowman and
Moir, 1993; Wei et al., 2014). To increase or maintain crop
productivity, P in the form of synthetic and animal-based
fertilizer is applied to arable soils. However, the long-term
(over)application of P in excess of plant crop requirements
has increased P in surface and subsurface soils, generally
over time, transforming it into various unavailable forms
(Arai et al., 2005; He et al., 2009; Sims et al., 2000). Soil organic and inorganic P are, in the majority of cases,
stabilized by association with mineral components. Nega-
tively charged phosphorus compounds attach themselves to
mineral components such as aluminosilicate minerals and
hydrous Fe or Al oxides (Solomon and Lehmann, 2000;
Turner et al., 2005). The P reactivity in Fe/Al oxyhydroxides
turns large proportions of soil P into unavailable forms (Arai
and Livi, 2013; Solomon and Lehmann, 2000; Solomon et
al., 2002). Different Fe/Al oxides, however, have varying P The results showed that overall P contents increased with
decreasing size of the soil aggregate-sized fractions. How-
ever, the relative distribution and speciation of varying P
forms were found to be independent of soil aggregate-size. The majority of alkaline-extractable P was in the a-Fe/Al
oxide fraction (42–47 % of total P), most of which was or-
thophosphate (36–41 % of total P). Furthermore, still sig-
nificant amounts of particularly monoester P were bound to Biogeosciences, 12, 6443–6452, 2015
www.biogeosciences.net/12/6443/2015/
doi:10.5194/bg-12-6443-2015
© Author(s) 2015. CC Attribution 3.0 License. Biogeosciences, 12, 6443–6452, 2015
www.biogeosciences.net/12/6443/2015/
doi:10.5194/bg-12-6443-2015
© Author(s) 2015. CC Attribution 3.0 License. 2.1
Site description and soil fractionation Composite topsoil sample (0–15 cm, pH: 7.64) was col-
lected from a cultivated field in the German TERENO (Ter-
restrial Environmental Observatories) experimental platform
site Selhausen (50◦52′08′′ N, 6◦26′59′′ E; arable soil, Luvi-
sol) in March 2013. The field was cultivated with winter
wheat in October 2011–2012 and winter barley in Septem-
ber 2013. No P fertilization was applied in the years 2011–
2013. More details about soil physicochemical characteris-
tics and N fertilization of the test field are provided by Ta-
ble S1 in the Supplement and Kupisch et al. (2015). The
soil sample was fractionated using the soil particle-size frac-
tionation method (Séquaris and Lewandowski, 2003), which
explicitly avoids the destruction of small aggregates in soil
(Berns et al., 2008). In brief, 100 g of soil sample was sus-
pended in 200 mL of ultrapure water (Milli-Q) in a 1 L Du-
ran bottle (Schott, Mainz, Germany) and horizontally shaken
at 150 rpm for 6 h. After that, 600 mL of ultrapure water was
added and mixed allowed to settle. Soil phases containing
varying soil-aggregated sizes were collected by the pipette
method based on Stoke’s law. The soil fraction > 20 µm
(sand-sized aggregates, with mass ratio of 82.3 % of total
soil) was obtained by removing supernatant after settling for
6 min. The supernatant was subsequently settled for 12 h and
then the new supernatant was removed to obtain a soil frac-
tion from 2 to 20 µm (silt-sized aggregates, with mass ra-
tio of 12.9 % of total soil). The newly obtained supernatant
was subsequently separated into particle size of 0.45–2 µm Alkaline extraction and solution 31P-NMR spectroscopy
are widely thought as reliable methods for quantifying or-
ganic phosphorus (Turner, 2008). Inorganic phosphate (Pi)
in alkaline soil extracts is assumed to originate from com-
plexes with both Fe and Al (Turner, 2008). However, the dis-
tribution of organic P (Po) related to Fe/Al oxides in the al-
kaline extracts is still unclear. Our study aims to provide a
novel method to understand the distribution of Pi and Po on
Fe/Al oxides in the alkaline extract of soil. X. Jiang et al.: Speciation and distribution of P associated with Fe and Al oxides Few studies have dis-
tinguished organic and inorganic P associated with different
Fe/Al oxides (i.e., amorphous and crystalline Fe/Al oxides)
(Mahieu et al., 2000), despite the fact that P speciation will
ultimately determine the different P bioavailability, mobil-
ity and mineralization in soil (Dick and Tabatabai, 1978; Liu
et al., 2014). The study by Mahieu et al. (2000) found that
amorphous Fe oxides had strong correlations with some or-
ganic P forms in the humic acid and calcium humate extracts
of lowland rice soils. Long-term soil management and devel-
opment (e.g., cultivation, fertilized management, and forest
growth and development) affect soil chemistry, such as the
transformation of Fe oxides (Li et al., 2008). Inevitably, the
ecosystem-driven transformation of Fe oxides alters the re-
lated P retention and bioavailability in soil. Therefore, the
study of various P/Fe oxide relationships is helpful for un-
derstanding soil P dynamics in long timescales. However,
little is known about speciation and distribution of P asso-
ciated with varying Fe/Al oxides. Additionally, finer-sized
soil fractions were found to be enriched in highly active and
easily mineralized organic P than larger particles (Rubæk et
al., 1999). Similarly, organic P forms in general increased
with decreasing particle size in forest and cropland soils
(Cade-Menun, 2005). It seems that smaller soil particles are
more reactive and have higher P-retaining capacity due to in-
creased surface area than larger soil particles. In this context,
it is important that different soil aggregate-sized fractions be
also considered when studying P associated with Fe/Al ox-
ides in soil. Our current study aimed (i) to provide a comprehensive
insight into the distribution of P bound to amorphous and
crystalline Fe and Al (hydr)oxides in different soil aggregate-
sized fractions, (ii) to identify the speciation and distribution
of Pi and Po associated with amorphous and crystalline Fe/Al
(hydr)oxides in NaOH–Na2EDTA soil extracts, and (iii) to
provide detailed information on residual P distribution after
alkaline extraction. This was done in order to provide fur-
ther information on the partition and quantification of these
(unavailable) P forms, especially for the P/Fe oxide frac-
tion of the arable soil, which may help to improve current
soil P sustainable development management strategies. In
this study, we extracted amorphous and crystalline Fe/Al ox-
ides from a bulk soil and different soil aggregate-sized frac-
tions of an arable field. X. Jiang et al.: Speciation and distribution of P associated with Fe and Al oxides Additionally, the isolated amorphous
Fe oxide-free and all Fe oxide-free aggregate-sized fractions
were then treated by alkaline extraction. Simultaneously, the
original bulk soil and various aggregate-sized fractions were
also treated with alkaline extraction and studied by solution
31P-NMR spectroscopy. X. Jiang et al.: Speciation and distribution of P associated with Fe and Al oxides X. Jiang et al.: Speciation and distribution of P associated with Fe and Al oxides X. Jiang et al.: Speciation and distribution of P associated with Fe and Al oxides 6444 adsorption capacities and some studies showed amorphous
Fe/Al oxides carried the majority of P in soils, despite higher
abundance of crystalline Fe oxides (Arai and Livi, 2013; Arai
et al., 2005; Rick and Arai, 2011). Few studies have dis-
tinguished organic and inorganic P associated with different
Fe/Al oxides (i.e., amorphous and crystalline Fe/Al oxides)
(Mahieu et al., 2000), despite the fact that P speciation will
ultimately determine the different P bioavailability, mobil-
ity and mineralization in soil (Dick and Tabatabai, 1978; Liu
et al., 2014). The study by Mahieu et al. (2000) found that
amorphous Fe oxides had strong correlations with some or-
ganic P forms in the humic acid and calcium humate extracts
of lowland rice soils. Long-term soil management and devel-
opment (e.g., cultivation, fertilized management, and forest
growth and development) affect soil chemistry, such as the
transformation of Fe oxides (Li et al., 2008). Inevitably, the
ecosystem-driven transformation of Fe oxides alters the re-
lated P retention and bioavailability in soil. Therefore, the
study of various P/Fe oxide relationships is helpful for un-
derstanding soil P dynamics in long timescales. However,
little is known about speciation and distribution of P asso-
ciated with varying Fe/Al oxides. Additionally, finer-sized
soil fractions were found to be enriched in highly active and
easily mineralized organic P than larger particles (Rubæk et
al., 1999). Similarly, organic P forms in general increased
with decreasing particle size in forest and cropland soils
(Cade-Menun, 2005). It seems that smaller soil particles are
more reactive and have higher P-retaining capacity due to in-
creased surface area than larger soil particles. In this context,
it is important that different soil aggregate-sized fractions be
also considered when studying P associated with Fe/Al ox-
ides in soil. observation by He et al. (2008), which suggested that most
of this residual P could be orthophosphate. adsorption capacities and some studies showed amorphous
Fe/Al oxides carried the majority of P in soils, despite higher
abundance of crystalline Fe oxides (Arai and Livi, 2013; Arai
et al., 2005; Rick and Arai, 2011). Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. X. Jiang et al.: Speciation and distribution of P associated with Fe and Al oxides X. Jiang et al.: Speciation and distribution of P associated with Fe and Al oxides 6445 Figure 1. A schematic diagram of the soil sample treatments. (with mass ratio of 4.0 % of total soil) by spinning the sam-
ples at 2525 × g for 4 min (Biofuge, Heraeus), according to
Stoke’s law calculations with an assumed particle density
of 2.65 g cm−3. Lastly, a < 0.45 µm fraction (with mass ratio
of 0.57 % of total soil) was obtained by spinning the super-
natant at 2525 × g for 68 min (Biofuge, Heraeus). The final
supernatant only contained the electrolyte phase as well as
a small quantity of nanoparticles; this has been investigated
in our other concurrent work (Jiang et al., 2015). The soil
aggregate-sized fractions were then freeze-dried for the ele-
ments analysis (i.e., Fe, Al, P and Ca) by inductively coupled
plasma mass spectrometry (ICP-MS). 2.2
Oxalate and dithionite–citrate–bicarbonate (DCB)
treatments Figure 1. A schematic diagram of the soil sample treatments. Acid ammonium oxalate was used to dissolve amorphous,
poorly crystalline and organo-Fe and Al (hydr)oxides (Kle-
ber et al., 2005; Masiello et al., 2004; Neubauer et al., 2013). The bulk soil and different aggregate-sized fractions were
treated with acid ammonium oxalate according to the extrac-
tion method of Kiem and Kögel-Knabner (2002) and Schw-
ertmann (1964). Briefly, 200 mg of soil sample was dispersed
in 40 mL of 0.2 M acidic oxalate solution (0.113 M ammo-
nium oxalate and 0.087 M oxalic acid) and shaken for 2 h in
the dark, followed by centrifugation. The supernatants were
subsequently analyzed for Fe (Feox), Al (Alox), P (Pox) and
Ca (Caox) by ICP-MS. Na2S dissolved in D2O was added to the lyophilized ma-
terials to maintain reducing conditions for the removal of
paramagnetic ions (i.e., Fe and Mn) during precipitation
(Vestergren et al., 2012). The solution was centrifuged at
13 200 × g for 20 min (Centrifuge 5415R, Eppendorf). Solu-
tion 31P-NMR spectra were obtained using a Bruker Avance
600 MHz spectrometer equipped with a prodigy probe, op-
erating at 242.95 MHz for 31P (Izarova et al., 2014). Ex-
tracts were measured with a D2O-field lock at room temper-
ature. Chemical shifts were referenced to 85 % orthophos-
phoric acid (0 ppm). The NMR parameters generally used
were 32 000 data points, 0.5 s repetition delay (we tested the
bulk soil with 0.5 and 2 s repetition delay and no differences
for the solution 31P-NMR spectra were observed), 30◦pulse
width, 24 000 scans and 0.7 s acquisition time. The DCB mainly extracts organically bound, amorphous
and crystalline Fe oxides and only parts of dissolved oxides
for Al and Si. For DCB extraction the protocol of Kiem and
Kögel-Knabner (2002) was used. Briefly, 200 mg of sample
was extracted using 30 mL of 0.3 M sodium citrate solution,
10 mL of 1 M sodium hydrogen carbonate solution and 1 g
of sodium dithionite. After shaking for 16 h at room tem-
perature, the soil suspension was centrifuged. The residue
was then dispersed in 32 mL of magnesium sulfate (0.05 M)
to exchange the adsorbed Fe with magnesium and thereafter
centrifuged a second time. The two supernatant phases were
combined and ICP-MS was used to measure Fe (FeDCB), Al
(AlDCB), P (PDCB) and Ca (CaDCB). 2.4
Calculations The schematic diagram of whole soil sample preparation is
shown in Fig. 1. The P contents in the various extracted frac-
tions were calculated as follows: (1)
(2)
(3) (Pa-Fe)N = PN −PN-I,
(1)
(Pc-Fe)N = PN-I −PN-II,
(2)
(Pother)N = PN-II,
(3) (2)
(3) 2.3
Solution 31P-NMR spectroscopy (3) where (Pa-Fe)N is NaOH–Na2EDTA-extractable P associ-
ated with amorphous Fe/Al oxides, (Pc-Fe)N is NaOH–
Na2EDTA-extractable P associated with crystalline Fe ox-
ides, (Pother)N is NaOH–Na2EDTA-extractable P associ-
ated with other soil compounds, PN is NaOH–Na2EDTA-
extractable P in soil samples, PN-I is NaOH–Na2EDTA-
extractable P in “amorphous FeO-free” soil samples, and
PN-II is NaOH–Na2EDTA-extractable P in “FeO-free” soil
samples. To highlight the chemical composition of inorganic and or-
ganic P species in the bulk soil and different aggregate-
sized fractions (> 20, 2–20, 0.45–2 and < 0.45 µm), these
aggregate-sized fractions without and with oxalate and DCB
pre-treatments were shaken for 16 h with a mixture of 0.25 M
NaOH and 50 mM Na2EDTA (soil and solution mass ratio
is 1 : 20–25, pH ≥13) (Cade-Menun and Preston, 1996) and
centrifuged at 2525 × g for 68 min (Biofuge, Heraeus). The
P (P-NaOH), Fe (Fe-NaOH), Al (Al-NaOH) and Ca (Ca-NaOH)
were analyzed by ICP-MS for the supernatants. These su-
pernatants were then frozen and subsequently lyophilized. Relative distributions of Pi and Po in NaOH–Na2EDTA
extracts of soil samples, a-Fe/Al oxide-free and Fe/Al oxide- 2.1
Site description and soil fractionation Furthermore, the
residual P after alkaline extraction still accounts for signifi-
cant proportion of P for some soils, most apparent in high-
pH soil or for unamended soils with low P concentration
(Cade-Menun and Liu, 2014; Cheesman et al., 2012; Mc-
Dowell et al., 2007; Turner et al., 2007; Turner et al., 2003). The residual P is by definition unidentified and is simply pre-
sumed to be recalcitrant in the environment (Cheesman et al.,
2010). Richter et al. (2006) indicated that recalcitrant P as
slowly cycling P did still contribute to P bioavailability on
decadal timescales. Therefore, an improved understanding
of the residual P distribution, its bonding and “true” recal-
citrance in soil is important for developing strategies to ex-
ploit this P as an “in part additional” source for plant uptake
or microbial usage. This is probably feasible in line with the www.biogeosciences.net/12/6443/2015/ Biogeosciences, 12, 6443–6452, 2015 3.1
Total and oxalate- and DCB-extractable P, Fe, Al
and Ca in soil aggregate-sized fractions In contrast, c-Al content was low (only
0–0.34 g kg−1, 0–0.6 % of total Al) in the various aggregate-
sized fractions (Table 2). Similarly, a high amount of c-Fe
and a low amount of c-Al were also found in sandy soils
(Borggaard et al., 1990). This is probably because organic
matter in soil inhibits Al oxides crystallization (Borggaard et
al., 1990). There is currently ongoing discussion in the liter-
ature (Acebal et al., 2000; Borggaard et al., 1990; Ryan and
Gschwend, 1991) with respect to the extent to which c-Al
is extracted by DCB and what reasons for such an incom-
plete extraction of this Al are. Only 12–20 % of total P (c-P,
the difference between PDCB and Pox, Table 2) was released
with crystalline Fe and Al oxides. This shows that amorphous
Fe/Al oxides are more important for P bonding compared
to crystalline Fe/Al oxides. Several researchers have already
reported that P was dominantly associated with amorphous
Fe and Al oxides fractions in various soil types in the USA
(Arai et al., 2005; Rick and Arai, 2011). It was presumed
in those studies that specific anion adsorption (e.g., P) sup-
pressed the transformation from amorphous to crystalline Fe
oxides (Arai et al., 2005; Biber et al., 1994), which could free soil samples were characterized by solution 31P-NMR
spectroscopy (Figs. 2 and S1 in the Supplement). Here only
orthophosphate, phosphate monoesters and pyrophosphate
were scaled to 100 %. Therefore, the distribution of Pi and Po
associated with a- and c-Fe/Al oxides in NaOH–Na2EDTA
extracts (Table 1) was calculated with the combination of
NaOH–Na2EDTA extraction (Table S2) and corresponding
solution 31P-NMR spectra (Figs. 2 and S1). 3.1
Total and oxalate- and DCB-extractable P, Fe, Al
and Ca in soil aggregate-sized fractions Table 2 represents ICP-MS results of elemental contents (i.e.,
P, Fe, Al and Ca) in the bulk soil and different aggregate-
sized fractions, as well as their oxalate and DCB extracts. To-
tal P was 0.73 g kg−1 in bulk soil with a significant increase
from large particles to small particles (range between 0.54
and 2.47 g kg−1). Total Fe and Al in different soil aggregate-
sized fractions showed the same gradient with total P. To-
tal P had a strong positive relationship with total Fe and
Al (R2 > 0.99; P < 0.006) in these aggregate-sized fractions,
which indicated that P was, to a large extent, associated with
Fe and Al in all aggregate-sized fractions. The high correla-
tion also indicated the reason why smaller particles contained
more P. There was more Fe and Al in the smaller size frac-
tions, and the minerals containing these elements (i.e., Fe and
Al) acted as important P carriers. Orthophosphate
(%)
P-monoesters
(%)
Pyrophosphate*
(%)
Bulk soil
79.4
19.1
1.4
Bulk soil after oxalate
60.8
35.0
4.2
Bulk soil after DCB
51.5
43.0
5.5
*All other P forms are not included. Figure 2. Solution phosphorus-31 nuclear magnetic resonance
spectra of NaOH–Na2EDTA extracts of bulk soil without and with
oxalate and dithionite (DCB) pre-treatments (all subfigures have
been reduced by the same factors). The associated table only shows
the relative mass distribution (%) of orthophosphate, P-monoesters
and pyrophosphate in NaOH–Na2EDTA extracts of bulk soil with-
out and with oxalate and DCB pre-treatments. Oxalate-extractable P, Fe and Al (Pox, Feox and Alox) also
had an increasing trend with smaller particle sizes. Oxalate-
extractable P corresponded to 53–62 % of the total P in vary-
ing soil aggregate-sized fractions, although the amounts of
amorphous Fe and Al oxides fractions were low with re-
spect to total Fe (15–17 %) and total Al (2–4 %; Table 2). DCB-extractable P (PDCB) was 70–75 % of the total P in
these aggregate-sized fractions, which implied that Fe/Al
oxides were the major carriers of P in the soil (Table 2). The differences between DCB-extractable Fe and Al ox-
ides and oxalate-extractable Fe and Al oxides represent the
amounts of extracted crystalline Fe (c-Fe) and Al (c-Al) ox-
ides. There was a large amount of c-Fe (6.31–28.4 g kg−1,
31–37 % of total Fe). X. Jiang et al.: Speciation and distribution of P associated with Fe and Al oxides X. Jiang et al.: Speciation and distribution of P associated with Fe and Al oxides 8
6
4
2
0
-2
-4
-6
ppm
P-monoesters
Bulk soil after DCB
Bulk soil after oxalate
Bulk soil
Pyrophosphate
P-diesters
Orthophosphate
Orthophosphate
(%)
P-monoesters
(%)
Pyrophosphate*
(%)
Bulk soil
79.4
19.1
1.4
Bulk soil after oxalate
60.8
35.0
4.2
Bulk soil after DCB
51.5
43.0
5.5
*All other P forms are not included. Figure 2. Solution phosphorus-31 nuclear magnetic resonance
spectra of NaOH–Na2EDTA extracts of bulk soil without and with
oxalate and dithionite (DCB) pre-treatments (all subfigures have
been reduced by the same factors). The associated table only shows
the relative mass distribution (%) of orthophosphate, P-monoesters
and pyrophosphate in NaOH–Na2EDTA extracts of bulk soil with-
out and with oxalate and DCB pre-treatments. 8
6
4
2
0
-2
-4
-6
ppm
P-monoesters
Bulk soil after DCB
Bulk soil after oxalate
Bulk soil
Pyrophosphate
P-diesters
Orthophosphate www.biogeosciences.net/12/6443/2015/ Biogeosciences, 12, 6443–6452, 2015 6446 X. Jiang et al.: Speciation and distribution of P associated with Fe and Al oxides Although there was a smaller amount of Ca in dif-
ferent aggregate-sized fractions compared to Fe and Al (Ta-
ble 2), DCB also dissolved between 48 and 100 % of total Ca
in varying aggregate-sized fractions (Table S3). Therefore, it
could be assumed that a small amount of the measured c-P
(Table 2) was related to Ca minerals (Ca–P precipitates) in
the soil. The orthophosphate and phosphate monoesters were
major detected inorganic and organic P forms in all
soil aggregate-sized fractions (Fig. 3). NaOH–Na2EDTA-
extractable orthophosphate (31P-NMR chemical shift of 5–
7 ppm; Cade-Menun, 2005) ranged from 0.25 to 1.27 g kg−1,
accounting for the majority of P (47–51 % of total P) in all the
aggregate-sized fractions (Fig. 3). Phosphate monoesters (3–
6 ppm; Bol et al., 2006; Cade-Menun, 2005; Fuentes et al.,
2012) were always the dominant organic P, with contents of
53–332 mg kg−1 (10–14 % of total P; Fig. 3). Phosphate mo-
noesters are generally the dominant group of organic phos-
phorus compounds in most soils: they occur mainly as in-
ositol phosphates, a family of phosphoric esters of hexahy-
droxy cyclohexane (inositol; Turner et al., 2005). The con-
tent of pyrophosphate (−4 to −5 ppm; Cade-Menun, 2005)
was only up to 15 mg kg−1 (less than 1 % of total P). Phos-
phonates (12 ppm) and phosphate diesters (2.5 to −1 ppm;
Cade-Menun, 2005) only contributed 0.6–1.0 % of total P
(Fig. 3). We found that there were only few, if any, P diesters
in fine particles (Fig. 3). Many microbial products such as
teichoic acid P or DNA-P exhibit these structures (Makarov
et al., 2002). Although there were higher amounts of total Fe, Al and
P in smaller particles compared with larger particles, there
were no clearly discernible proportional differences in ox-
alate and DCB-extractable Fe, Al and P fractions with re-
spect to total Fe, Al and P between various aggregate-sized
fractions. Therefore, there was a clear indication that the
relative proportional distributions of amorphous, crystalline
Fe/Al oxides and related P in this soil were independent of
the aggregate size of soil particles, a result not found previ-
ously. Kiem and Kögel-Knabner (2002) suggested that there
was a similar Feox / FeDCB ratio across three size separates
of arable soil fractions < 6 µm. Fe/Al (hydr)oxides and or-
ganic matter bind clay particles together into soil aggregates
(Tisdall and Oades, 1982). X. Jiang et al.: Speciation and distribution of P associated with Fe and Al oxides 6447 Table 1. NaOH–Na2EDTA-extractable P and residual P distribution (%, with respect to total P of each soil aggregate-sized fraction) associ-
ated with amorphous and crystalline oxides, and other soil compounds as shown in Eqs. (1–6). Table 1. NaOH–Na2EDTA-extractable P and residual P distribution (%, with respect to total P of each soil aggregate-sized fraction) associ-
ated with amorphous and crystalline oxides, and other soil compounds as shown in Eqs. (1–6). NaOH–Na2EDTA-extractable P
Residual P
(Pa-Fe)N
(Pc-Fe)N
(Pother)N
(Pa-Fe)resi
(Pc-Fe)resi
(Pother)resi
Ortho. Mono. Ortho. Mono. Ortho. Mono. Bulk soil
39
6
5
2
5
4
12
9
18
Sand-sized > 20 µm
36
6
6
1
4
4
11
13
18
Silt-sized 2–20 µm
39
7
3
1
6
5
13
7
18
Clay 0.45–2 µm
37
8
4
1
6
5
13
9
16
Clay < 0.45 µm
41
6
6
1
6
5
15
8
12
Ortho. stands for orthophosphate and Mono. for P monoesters. (Pa-Fe)N: NaOH–Na2EDTA-extractable P associated with amorphous Fe/Al oxides. (Pc-Fe)N:
NaOH–Na2EDTA-extractable P associated with crystalline Fe oxides. (Pother)N: NaOH–Na2EDTA-extractable P associated with other soil compounds. (Pa-Fe)resi: residual P associated with amorphous Fe oxides. (Pc-Fe)resi: residual P associated with crystalline Fe oxides. (Pother)resi: residual P associated
with other soil compounds. Ortho. stands for orthophosphate and Mono. for P monoesters. (Pa-Fe)N: NaOH–Na2EDTA-extractable P associated with amorphous Fe/Al oxides. (Pc-Fe)N:
NaOH–Na2EDTA-extractable P associated with crystalline Fe oxides. (Pother)N: NaOH–Na2EDTA-extractable P associated with other soil compounds. (Pa-Fe)resi: residual P associated with amorphous Fe oxides. (Pc-Fe)resi: residual P associated with crystalline Fe oxides. (Pother)resi: residual P associated
with other soil compounds. explain the low amount of P associated with crystalline Fe
oxides. was found in a recent study of dried wetland soil samples
(Cheesman et al., 2012). Inorganic and organic P contents
in NaOH–Na2EDTA extracts were characterized by solution
31P-NMR spectroscopy. Inorganic (orthophosphate and py-
rophosphate) and organic (phosphate monoesters, phosphate
diesters and phosphonates) P forms were present in our soil
aggregate-sized fractions. Oxalate extracted P also had a strong positive linear
correlation with oxalate extracted Fe (R2 = 0.997) and Al
(R2 = 0.976). Similarly high correlation between oxalate-
extractable P and Fe oxides has already been shown in acidic
and neutral soils (Forsmann and Kjaergaard, 2014). Never-
theless, c-Fe and c-Al contents were not strongly correlated
with c-P. X. Jiang et al.: Speciation and distribution of P associated with Fe and Al oxides Therefore, these Fe/Al oxides as
inorganic binding agents and associated P were distributed
on soil aggregates with all varying sizes proportionally. All the identified P forms were present in higher concen-
trations in < 2 µn soil fraction when compared to larger-sized
particles. Small-sized soil fractions, such as soil colloids iso-
lated in this study, are well established as being efficient
carriers of nutrients (especially phosphorus; Haygarth et al.,
2006). Therefore, soil managements, which control soil col-
loids loss from productive land (e.g., tillage and wind/water
erosion), are crucial to restrain any unwanted strong declines
in soil phosphorus levels and thus help to retain the poten- 3.1
Total and oxalate- and DCB-extractable P, Fe, Al
and Ca in soil aggregate-sized fractions The residual P after alkaline extraction was separated into
P bound to a-Fe oxides, c-Fe oxides and other soil com-
pounds as follows: (Pa-Fe)resi = Pa-Fe −(Pa-Fe)N,
(4)
(Pc-Fe)resi = “Pc-Fe′′ −(Pc-Fe)N =
(PFe −Pa-Fe) −(Pc-Fe)N,
(5)
(Pother)resi = PT −PFe −PN-II,
(6) (Pa-Fe)resi = Pa-Fe −(Pa-Fe)N,
(4)
(Pc-Fe)resi = “Pc-Fe′′ −(Pc-Fe)N =
(PFe −Pa-Fe) −(Pc-Fe)N,
(5)
(Pother)resi = PT −PFe −PN-II,
(6) (4) (5)
(6) (6) where (Pa-Fe)resi is residual P associated with amorphous
Fe oxides, (Pc-Fe)resi is residual P associated with crys-
talline Fe oxides, (Pother)resi is residual P associated with
other soil compounds, Pa-Fe is P associated with amorphous
Fe/Al oxides (i.e., oxalate-extractable P), PFe is P associ-
ated with amorphous and crystalline Fe/Al oxides (i.e., DCB-
extractable P), Pc-Fe is P associated with crystalline Fe/Al
oxides, and PT is total P. www.biogeosciences.net/12/6443/2015/ Biogeosciences, 12, 6443–6452, 2015 3.2
Speciation and distribution of P forms in soil
aggregate-sized fractions We found that 58–65 % of total P was extracted by NaOH–
Na2EDTA in different soil size fractions (Table S2). A
similar extraction efficiency (average of 63 % of total P) X. Jiang et al.: Speciation and distribution of P associated with Fe and Al oxides 6448 Table 2. The amounts of P, Fe, Al and Ca in bulk soil and soil aggregate-sized fractions, and the amounts of these elements extracted
by oxalate and dithionite–citrate–bicarbonate (DCB) (g kg−1). Fractions with respect to total P, Fe, and Al values of each aggregate-sized
fraction shown in Table 2 are given in parentheses. Data are the mean values of three replicates with standard deviations. Table 2. The amounts of P, Fe, Al and Ca in bulk soil and soil aggregate-sized fractions, and the amounts of these elements extracted
by oxalate and dithionite–citrate–bicarbonate (DCB) (g kg−1). Fractions with respect to total P, Fe, and Al values of each aggregate-sized
fraction shown in Table 2 are given in parentheses. Data are the mean values of three replicates with standard deviations. Aggregate-sized
Pc
T
Fec
T
Alc
T
Cac
T
Pdox
Fedox
Aldox
Pe
DCB
Fee
DCB
Ale
DCB
c-Pf
c-Fef
c-Alf
fractions
Bulk soila, b
0.73 ± 0.03
24.0 ± 0.7
44.6 ± 2.1
4.03 ± 0.12
0.41 ± 0.01
3.75 ± 0.10
0.88 ± 0.03
0.53 ± 0.02
11.6 ± 0.5
1.03 ± 0.08
0.12
7.80
0.15
(56)
(16)
(2)
(72)
(48)
(2)
(16)
(33)
(0.3)
Sand-sized
0.54 ± 0.05
19.5 ± 2.9
37.4 ± 2.3
3.63 ± 0.32
0.29 ± 0.01
2.92 ± 0.24
0.59 ± 0.04
0.40 ± 0.04
9.24 ± 0.53
0.81 ± 0.07
0.11
6.31
0.22
> 20 µma, b
(53)
(15)
(2)
(73)
(47)
(2)
(20)
(32)
(0.6)
Silt-sized
1.40 ± 1.00
44.7 ± 3.9
73.8 ± 0.9
5.63 ± 0.40
0.82 ± 0.04
7.00 ± 0.23
1.83 ± 0.07
0.98 ± 0.04
20.7 ± 0.5
1.93 ± 0.04
0.17
13.7
0.10
2–20 µma, b
(58)
(16)
(2)
(70)
(46)
(3)
(12)
(31)
(0.1)
Clay
2.37 ± 0.06
69.3 ± 3.5
108 ± 2
7.10 ± 0.30
1.38 ± 0.01
11.6 ± 0.1
3.28 ± 0.05
1.72 ± 0.06
36.8 ± 0.7
3.62 ± 0.31
0.33
25.2
0.34
0.45–2 µma ,b
(58)
(17)
(3)
(73)
(53)
(3)
(14)
(36)
(0.3)
Clay
2.47 ± 0.06
76.4 ± 3.3
114 ± 2
8.23 ± 0.35
1.54 ± 0.07
12.4 ± 0.5
4.36 ± 0.28
1.85 ± 0.09
40.8 ± 1.1
4.11 ± 0.35
0.31
28.4
ND
< 0.45 µma, b
(62)
(16)
(4)
(75)
(53)
(4)
(13)
(37)
a Amounts increase with decreasing particle size. b Relative proportions of isolated fractions are independent of decreasing particle size. 3.3
NaOH–Na2 EDTA-extractable P associated with Fe
and Al oxides in soil aggregate-sized fractions It is worth noting that only small amounts of Fe (0–1 %
of total Fe) from “untreated” soil aggregate-sized fractions
were dissolved by NaOH–Na2EDTA (Table S3). Although
the presence of Na2EDTA caused some dissolved Fe ions to
precipitate in humic–iron compounds, the precipitation was
negligible at hydroxide ion concentrations ≥100 mM (≥pH
13; Turner, 2008). The NaOH–Na2EDTA extracted P was not
occluded in the Fe-containing minerals (e.g., Fe oxides) but
probably associated with the surface of Fe-containing min-
erals and bound to other minerals. Therefore, a- and c-Fe
oxide-associated P present in NaOH–Na2EDTA extracts was
probably attached to the surface of Fe oxides. Alkaline ex-
traction could remove P held to Fe and Al components of soil
surface by chemisorption (Hedley et al., 1982; McLaughlin
et al., 1977; Ryden et al., 1977). It is worth noting that there
was unavoidable P release from P–goethite complexes after
oxalate treatment by ligand exchange; the amount of released
P was negligible at low initial P level (Johnson and Loeppert,
2006), as was the case in our soil samples. The bulk soil and aggregate-sized fractions after oxalate
and DCB pre-treatments were also extracted by NaOH–
Na2EDTA for 31P-NMR analysis. We found that the bulk
soil (Fig. 2) and other soil aggregate-sized fractions (Fig. S1)
after oxalate pre-treatment revealed a higher phosphate mo-
noester / orthophosphate ratio in NaOH–Na2EDTA extracts
compared to that without oxalate treatment. The bulk soil
(Fig. 2) and other soil aggregate-sized fractions (Fig. S1)
after DCB pre-treatment had the highest phosphate mo-
noester / orthophosphate ratio among the three types of sam-
ples. This finding implies that relatively high amounts of or-
thophosphate were removed by oxalate and particularly DCB
– i.e., it was mainly orthophosphate that was bound to a-
and c-Fe oxides in aggregate-sized fractions rather than or-
ganic P. Furthermore, most pyrophosphate still remained in
Fe/Al oxide-free aggregate-sized fractions (Figs. 2 and S1). Pyrophosphate is thought to be contained within live fungal
tissue (Koukol et al., 2008) or sorbed to the mineral soil ma-
trix (Gunary, 1966). However, the present study suggests that
Fe/Al oxides (part of mineral matrix) were not the main sites
for pyrophosphate adsorption. High contents of dissolved Al were still found in NaOH–
Na2EDTA extracts; these were similar to those in oxalate
and DCB extracts of soil aggregate-sized fractions (Tables 2
and S3). X. Jiang et al.: Speciation and distribution of P associated with Fe and Al oxides c Total P, Fe, Al and Ca. d Oxalate-extractable P, Fe and Al. e DCB-extractable P, Fe and Al. f c-(P, Fe, Al)
means the difference between (P, Fe, Al)DCB and (P, Fe, Al)ox. Na2EDTA extracts accounted for 49–54 % of total P in vari-
ous aggregate-sized fractions, including orthophosphate (41–
47 % of total P) and phosphate monoesters (7–9 % of total
P; Table 1). The amorphous oxides carried more orthophos-
phate and phosphate monoesters (36–41 and 6–8 % of total P,
respectively) than did the crystalline oxides (3–6 and 1–2 %
of total P, respectively) in the NaOH–Na2EDTA extracts (Ta-
ble 1). The P adsorption experiment from Borggaard (1983)
showed that a-Fe oxides absorbed more phosphate than c-Fe
oxides in soils. Generally, phosphate monoesters are com-
monly assumed to be stabilized in soils by association with
amorphous metal oxides, which have greater sorption affin-
ity compared to crystalline metal oxides (Celi and Barberis,
2007; Turner and Engelbrecht, 2011). tial bioavailability of phosphorus over longer time periods. However, the distributions of different P forms in our study
seemed to be independent of soil aggregate size (Fig. 3). In contrast, some studies have found that soil size fractions
drive the differential P distributions in forest and arable soils
(Liu et al., 2014; Solomon et al., 2002). Our novel finding
about the similar P distribution in various aggregate-sized
fractions of this soil was probably due to the comparable Fe
and Al oxide distribution in aggregate-sized fractions when
considering that Fe and Al oxides were domain P carriers in
the soil. www.biogeosciences.net/12/6443/2015/ Biogeosciences, 12, 6443–6452, 2015 www.biogeosciences.net/12/6443/2015/ 3.3
NaOH–Na2 EDTA-extractable P associated with Fe
and Al oxides in soil aggregate-sized fractions Similarly, high Al and low Fe contents were found in
NaOH–Na2EDTA extracts from a tropical forest soil (Turner,
2008). Considering that little Si was dissolved in NaOH– Because orthophosphate and phosphate monoesters were
the dominant P forms, only these are discussed below (Ta-
ble 1). The amount of P bound to Fe/Al oxides in NaOH– www.biogeosciences.net/12/6443/2015/ Biogeosciences, 12, 6443–6452, 2015 X. Jiang et al.: Speciation and distribution of P associated with Fe and Al oxides
* NaOH-Na2EDTA extractable P. ᵟ NaOH-Na2EDTA non-extractable P. 0
300
600
900
1200
1500
1800
Bulk soil
Sand-sized >20 µm Silt-sized 2-20 µm
Clay 0.45-2 µm
Clay <0.45 µm
NaOH-P * (n=3)
Residual P ᵟ (n=3)
Orthophosphate (n=2)
P-monoesters (n=2)
P-diesters (n=2)
Pyrophosphate (n=2)
Phosphonate (n=2)
mg kg-1
Relative proportion of various chemical P forms
is independent of soil particle aggregate-size. X. Jiang et al.: Speciation and distribution of P associated with Fe and Al oxides 6449 * NaOH-Na2EDTA extractable P. ᵟ NaOH-Na2EDTA non-extractable P. Figure 3. NaOH–Na2EDTA-extractable P, residual P content, and phosphorus forms/contents (mg kg−1) from the NaOH–Na2EDTA extracts
in different aggregate-sized fractions as identified by solution phosphorus-31 nuclear magnetic resonance spectroscopy. The varying chemical
P contents were determined by multiplying the proportion of spectral area by the total concentration of the NaOH–Na2EDTA-extractable P. Figure 3. NaOH–Na2EDTA-extractable P, residual P content, and phosphorus forms/contents (mg kg−1) from the NaOH–Na2EDTA extracts
in different aggregate-sized fractions as identified by solution phosphorus-31 nuclear magnetic resonance spectroscopy. The varying chemical
P contents were determined by multiplying the proportion of spectral area by the total concentration of the NaOH–Na2EDTA-extractable P. Na2EDTA in all aggregate-sized fractions (data not shown),
it was indicative that NaOH–Na2EDTA extracted Al mainly
stemmed from amorphous Al oxides (rare c-Al oxides in the
soil, Table 2) rather than aluminosilicate minerals in the soil. Therefore, the Al–P in NaOH–Na2EDTA extracts probably
contained P on the surface of Al oxides as well as occluded
in Al oxides. aggregate-sized fractions was bound to a-Fe oxides and 7–
13 % of total P was associated with c-Fe oxides (Table 1). The 20–24 % of total P associated with Fe oxides (Table 1)
was equivalent to 53–66 % of the P present in the residual
fraction. Therefore, the majority of P in residual fraction was
associated with Fe oxides (Table 1). 3.3
NaOH–Na2 EDTA-extractable P associated with Fe
and Al oxides in soil aggregate-sized fractions The remaining 12–18 %
of total P (i.e., 34–47 % of the P present in residual fractions)
was bound to other minerals or organic matter which was not
dissolved or released in either DCB or NaOH–Na2EDTA of
different aggregate-sized fractions (Table 1). Between 8 and 11 % of total P (including 4–6 % or-
thophosphate and 4–5 % phosphate monoesters) was still
present in NaOH–Na2EDTA extracts which were not associ-
ated with Fe/Al oxides of different aggregate-sized fractions
(Table 1). The NaOH–Na2EDTA extraction included P con-
tained within microbial cells (Turner and Blackwell, 2013). Furthermore, inositol phosphate is stabilized mainly through
strong adsorption on the surface of amorphous metal oxides
and clay minerals (Celi and Barberis, 2007), so we assumed
that a part of these P compounds was adsorbed on the sur-
faces of clay minerals. Additionally, some P was probably
associated with Ca minerals in this NaOH–Na2EDTA ex-
tracts considering that 43–98 % of total Ca was dissolved in
NaOH–Na2EDTA extracts of different aggregate-sized frac-
tions (Table S3). Our novel results suggest that the a- and c-Fe oxide-
associated P in the residual P fraction was most likely oc-
cluded inside of a- and c-Fe oxides. This is in contrast
to the a- and c-Fe oxide-associated P present in NaOH–
Na2EDTA extracts which were absorbed on the surface of
Fe oxides. The P associated with Fe oxides in residual frac-
tions was probably held at the internal surfaces of Fe ox-
ides by chemisorption at protonated surface sites or by the
replacement of surface hydroxyls in soil aggregates (Hedley
et al., 1982; Ryden et al., 1977) or diffused into the bulk of
Fe oxides or into pores and defects (McLaughlin et al., 1977). This means that, with the changes in soil conditions (e.g., soil
anoxic condition, acidic condition, high concentrations of or-
ganic acid or siderophores; Forsmann and Kjaergaard, 2014;
Kraemer, 2004) when Fe oxides become dissolved, P associ-
ated with the Fe oxides may become available to plants or the
microbial population. The a-Fe/Al oxides adsorbed more or-
thophosphate and monoesters than c-Fe/Al oxides (Table 1:
NaOH–Na2EDTA-extractable P). However, there were simi- 3.4
Residual P in soil aggregate-sized fractions Using our specific separation method, the residual P was sep-
arated into P bound to a-Fe oxides, c-Fe oxides and other
soil compounds (Eqs. 4–6 and Table 1). The P in resid-
ual fractions accounted to 35–42 % of total P in aggregate-
sized fractions. We found that 11–15 % of total P in different X. Jiang et al.: Speciation and distribution of P associated with Fe and Al oxides 6450 lar amounts of occluded P inside of both a- and c- Fe oxides
(Table 1: Residual P). diffraction, and mössbauer spectroscopy, Clay. Clay Miner., 48,
322–330, 2000. The Ca–P complexes present in residual fractions of soil
aggregate-sized fractions should contribute to a proportion of
the remaining residual P, ca. 12–18 % of total P. Cheesman et
al. (2010) suggested that there were inorganic Ca phosphates
in the residual P from detritus and surface soils. In the present
study, 12 % of total P was not associated with Fe oxides in
residual P of < 0.45 µm soil fractions (Table 1), despite the
fact that almost all of the Ca was already extracted by NaOH–
Na2EDTA and DCB (Table S3). Therefore, it seemed that
the majority of the 12–18 % of total P was bound to other
materials. In our other concurrent work (Jiang et al., 2015),
we found that some P in the soil nanoparticles after DCB
treatment was still bound to Fe-bearing phyllosilicate miner-
als (i.e., Fe structurally embedded into the crystalline struc-
ture of phyllosilicate minerals via isomorphic substitution;
Regelink et al., 2014). It is possible that these Fe-bearing
phyllosilicate minerals may also be responsible for the bond-
ing of the remaining P in the NaOH–Na2EDTA non-extracts. It was worth noting that soil aggregate-sizes do not have a
significant effect on the relative distribution of P forms bond
to a- and c-Fe/Al oxides for NaOH–Na2EDTA extracts and
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Molecular speciation of phosphorus present in readily dispersible
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1 A prominent example is the special tseg issue 2014:4 on the book of Oscar Gelderblom, Cities of com
merce. The institutional foundations of international trade in the Low Countries, 1250-1650 (Princeton
2013). Conflict Management and Interdisciplinary History
Presentation of a New Project and an Analytical Model Justyna Wubs-Mrozewicz tseg 15 (1): 89-107
doi: 10.18352/tseg.1017 tseg 15 (1): 89-107
doi: 10.18352/tseg.1017 Abstract This article advances the idea that the concept of conflict resolution, which is usu-
al in social, economic, political and legal history of premodern Europe and in the
social sciences in general, is too goal-oriented and therefore unsatisfactory for the
analyses of conflicts. Instead, a problem-oriented concept of conflict management
is proposed. It consists of prevention, provocation, maintenance of the status quo,
escalation and de-escalation, as well as resolution. Such a concept allows to analyse
multi-level conflicts (individual, group, large-scale) pertaining diverse yet interrelat-
ed issues. This article shows how it will be used to analyse contentious issues which
are related to mercantile cities in premodern cities in northern Europe (1350-1570),
in a recently granted nwo vidi project. Conflicts are fascinating. When crises occur, relations between people
are revealed and discussed in far greater detail than when all is plain
sailing. It is therefore not surprising that historians of very diverse spe-
cialisations have delved into conflicts. In particular, the attempts to solve
conflicts, and the institutions created to this end, have been in the spot-
light in economic, social, political and legal history. The topic of pre-
modern conflict resolution, especially from a mercantile perspective,
has also been frequently addressed in tseg.1 Yet when analysing con-
flicts in the past, it is not enough to focus on conflict resolution only. This becomes especially apparent once we move beyond the traditional
socio-economic framing of premodern conflicts and take into account 89 WUBS-MROZEWICZ TSEG the dynamics of political, diplomatic and more broadly defined social
conflicts. One of the main conclusions of my recent research project on
conflict resolution in premodern Danzig (Gdańsk), the Baltic and North
Sea areas, was that many conflicts were never truly resolved.2 Some of
them lingered on, others were revived even after a formal resolution or
morphed into a new conflict. This concerned both small-scale and large-
scale conflicts, as the project analysed not only mercantile disputes be-
tween individuals, but also more broadly defined social conflicts be-
tween groups and political clashes at the level of cities and states. A full
resolution where contentious issues did not return, and where relations
were at least in part restored, was certainly an ideal to be pursued. How-
ever, the reality was often different. Attempts at resolution in courts and
outside of them, as well as formal and informal diplomacy, were often
ineffective. 2 nwo (The Netherlands Organization for Scientific Research) veni, 2011-2015.
3 nwo vidi 2018-2023, granted in May 2017. Abstract This would be a pessimistic conclusion after several months of in-
tensive perusals in archives in the region. However, another conclusion
emerged which was far more exciting: individuals, groups and rulers
made deliberate use of a plethora of strategies and tactics to channel,
control and skilfully employ conflicts, both on land and sea. It was by
far not only conflict resolution which was on their mind, as for instance
prevention or escalation proved to be effective tools. Also, conflicts per-
taining to social, economic and political matters were often intertwined
because large-scale could result in individual disputes and vice versa. For example, war could lead to private commercial losses or inheritance
quarrels could escalate to a diplomatic crisis. The examination of the
process of dealing with various conflicts gives far more clues about how
society worked at this time, than focussing on one specific type of con-
flicts or a narrow outcome. As it will be outlined in more detail below,
this is what has been standard in the various historiographies, including
social and economic history. This was the starting point for the vidi project on the management
of multi-level conflicts in commercial cities in northern Europe circa
1350-1570, spanning from London to Reval (Tallinn).3 Multi-level con-
flicts refer here to individual, group and large-scale conflicts, on matters
related to economy, politics and social relations. This is thus, emphati-
cally, a broader scope of interest than what has been usual in socio-eco-
nomic analyses of premodern commercial cities. Due to their far-reach- VOL. 15, NO. 1, 2018 90 CONFLICT MANAGEMENT AND INTERDISCIPLINARY HISTORY ing autonomy and economic clout, the governments and inhabitants of
these cities could deal with these conflicts in their own way, in particular
in the cities belonging to the Hanse. This included diplomacy, legal pro-
ceedings, arbitration, mediation, social pressure, as well as the inven-
tion of institutions and instruments which regulated relations across
a considerable geographical distance.4 Commercial cities were chosen
because there was a high level of mobility to and from them, both in the
form of (semi)permanent migration, and seasonal influx of traders and
craftsmen. Such mobility heightened the complexity of conflicts which
appeared in the cities, whether it concerned shipwreck, inheritances or
breaches of contracts. Abstract It also required a considerable level of knowledge
of handling conflicts, both within the city itself and how specific cases
could be approached elsewhere, as Europe was characterised by legal
and political fragmentation.5 Interestingly, at a time when there was no division of power into leg-
islative, executive and judicial branches in the urban governments, the
same people could be involved in handling individual and large-scale
conflicts. They could be involved in conflict prevention, escalation or
resolution. At the same time, they were part of this large network of
urban mobility, travelling in various capacities, for instance as traders
and as magistrates, and thus acquiring knowledge on various parts of
Europe. The fact that next to magistrates and representatives of the rul-
ers, also ‘good men’ from the wider urban society were regularly called in 4 The bibliography in the footnotes concerning the project can only be selective. Here concerning the
interplay between states and cities, and the particular cities which will be subject to analysis: W. Block-
mans, ‘Voracious states and obstructing cities. An aspect of state formation in preindustrial Europe’,
Theory and society 18 (1989) 733-755; P. Stabel, ‘Economic development, urbanisation and political or-
ganisation in the late medieval southern Low Countries’, in: P. Bemholz, M.E. Streit and R. Vaubel (eds.),
Political competition, innovation and growth. A historical analysis (Berlin 1998) 183-204; J. Puttevils, Mer
chants and trading in the sixteenth century. The golden age of Antwerp (London 2015); M. Bogucka, Baltic
commerce and urban society, 1500-1700. Gdańsk/Danzig and its Polish context (Aldershot 2003); E. Isen-
mann, Die deutsche Stadt im Mittelalter 1150-1550 (Cologne 2014 2nd ed.); S. Gustafsson, Svenska städer
i medeltidens Europa. En komparativ studie av stadsorganisation och politisk kultur (Stockholm 2006);
R.I. Frost, The Oxford history of Poland-Lithuania. The making of the Polish-Lithuanian union, 1385-1569
(Oxford 2015). The four articles in W. Blockmans, M. Krom and J. Wubs-Mrozewicz (eds.), The Routledge
handbook of maritime trade around Europe 1300-1600. Commercial Networks and Urban Autonomy (Lon-
don 2017) give an overview of the state of the art of research, the relevant bibliography and primary
sources on Lübeck, Reval, London and Danzig; C. Jahnke, ‘Lübeck and the Hanse. A queen without its
body’, 231-247; I. Leimus and A. Mänd, ‘Reval (Tallinn). A city emerging from maritime trade’, 273-291;
M. Kowaleski, ‘The maritime trade networks of late medieval London’, 383-410 and J. Wubs-Mrozewicz,
‘Danzig (Gdańsk). Abstract Seeking stability and autonomy’, 248-272. 5 Cities, at all times, offer interesting cases of studying social conflicts, see: P. Le Galès, European cities. Social conflicts and governance (Oxford 2002). 91 4 The bibliography in the footnotes concerning the project can only be selective. Here concerning the
interplay between states and cities, and the particular cities which will be subject to analysis: W. Block-
mans, ‘Voracious states and obstructing cities. An aspect of state formation in preindustrial Europe’,
Theory and society 18 (1989) 733-755; P. Stabel, ‘Economic development, urbanisation and political or-
ganisation in the late medieval southern Low Countries’, in: P. Bemholz, M.E. Streit and R. Vaubel (eds.),
Political competition, innovation and growth. A historical analysis (Berlin 1998) 183-204; J. Puttevils, Mer
chants and trading in the sixteenth century. The golden age of Antwerp (London 2015); M. Bogucka, Baltic
commerce and urban society, 1500-1700. Gdańsk/Danzig and its Polish context (Aldershot 2003); E. Isen-
mann, Die deutsche Stadt im Mittelalter 1150-1550 (Cologne 2014 2nd ed.); S. Gustafsson, Svenska städer
i medeltidens Europa. En komparativ studie av stadsorganisation och politisk kultur (Stockholm 2006);
R.I. Frost, The Oxford history of Poland-Lithuania. The making of the Polish-Lithuanian union, 1385-1569
(Oxford 2015). The four articles in W. Blockmans, M. Krom and J. Wubs-Mrozewicz (eds.), The Routledge
handbook of maritime trade around Europe 1300-1600. Commercial Networks and Urban Autonomy (Lon-
don 2017) give an overview of the state of the art of research, the relevant bibliography and primary
sources on Lübeck, Reval, London and Danzig; C. Jahnke, ‘Lübeck and the Hanse. A queen without its
body’, 231-247; I. Leimus and A. Mänd, ‘Reval (Tallinn). A city emerging from maritime trade’, 273-291;
M. Kowaleski, ‘The maritime trade networks of late medieval London’, 383-410 and J. Wubs-Mrozewicz,
‘Danzig (Gdańsk). Seeking stability and autonomy’, 248-272. 6 I have done preliminary research in the archives in Lübeck, Stockholm, Antwerp, London, Tallinn,
Gdańsk (also for Elbing), Berlin, Copenhagen. Also the availability of appropriate sources in Bruges and
Toruń has been established. WUBS-MROZEWICZ WUBS-MROZEWICZ TSEG to partake in the handling of conflicts, makes the pool of ‘conflict man-
agers’ of various backgrounds and capacities even more flexible and di-
verse. Looking beyond institutions which were geared towards the nar-
rower concept of conflict resolution, like courts or peace treaties, turned
my attention to such social actors. Conflicts are created and handled by
specific people, and these people can open a new door to our under-
standing of the past. Since conflicts and people create paperwork, they left a fantastic im-
print in the form of very rich sources in urban archives, most of which
have not been analysed from the point of view of dealing with discord.6
And on a more general level, the preparatory work with primary sources
became the basis for the drafting of a process-oriented model of histor-
ical conflict management. The model comprises conflict resolution, but
insistently goes beyond it: by including mechanisms such as prevention,
provocation, maintenance of status quo, escalation and de-escalation
(discussed in more detail below). The ambition is that the project will
provide ample test cases for this model of interdisciplinary, multi-level
conflict analysis from a historical perspective, which then can be de-
veloped further and applied to other areas and periods. In this research
note, the main premises of the project will be presented. I will sketch
why there is a need for historically-informed, systematic research on
conflict management, briefly present the state of the art of historical re-
search on conflict resolution and conflict management, and outline the
aspects of my approach which set the agenda for this project and histor-
ical conflict studies in general. From the point of view of social and eco-
nomic history readership of this journal, this approach changes the use
of the very popular concept of ‘conflict resolution’. It shows why social
and economic historians, along with legal and diplomatic historians,
can benefit from moving beyond this narrow concept. 7 N. Elias, The civilising process (Oxford 1969 [1939]); S. Pinker, The better angels of our nature. Why vio
lence has declined (New York 2012).
8 G. Simmel, Conflict and the web of group affiliation (New York 1964); M. Deutsch, The resolution of
conflict. Constructive and destructive processes (New Haven 1977).
9 https://www.ted.com/talks/scilla_elworthy_fighting_with_non_violence, last accessed 1.09.2017.
10 http://www.wetenschapsagenda.nl/publicatie/portfolio/.
11 For instance, J. Barrett and J.T. Barrett, A history of alternative dispute resolution. The story of a polit
ical, social, and cultural movement (San Francisco 2004); A. Fiadjoe, Alternative dispute resolution. A de
veloping world perspective (London 2015). Why bother about conflict management in the past? In present-day society, there is an urgent need for historically-informed
analyses. For a long time, there has been an idea of a progress of civili-
zation, either in a positivist way or in the framing of Norbert Elias. Such 92 VOL. 15, NO. 1, 2018 CONFLICT MANAGEMENT AND INTERDISCIPLINARY HISTORY progress would result in steadily declining rates of violence and increas-
ingly peaceful ways of handling conflicts now than in the past.7 But too
much evidence runs counter to this view: we have not eliminated con-
flicts or figured out how to deal with them very quickly and effectively. Conflict on various levels is present everywhere. The view that conflict
is an integral part of human interaction, not an exception, as argued
by sociologist Georg Simmel and social psychologist Morton Deutsch,
therefore captures the human experience, now as in the past, more ac-
curately.8 This means that we have to rethink how we can become better
at dealing with it. If improvement in the future is not self-evident, the
past is the only place to look for inspiration. It comes with the benefit
of hindsight on the whole process of handling a conflict. In her stirring
ted talk on fighting with non-violence, the peace builder Scilla Elwor-
thy has advocated the use of powerful, telling examples of handling con-
flicts in the past. Those (hi)stories can be transformative in our under-
standing of conflicts, and provide concrete ideas for dealing with them.9
The urgent need to reconsider classical paradigms for dealing with con-
flicts at various levels and anchor the change in historical reflection has
been underscored by the Dutch ‘Portfolio for research and innovation’.10
Apparently, we need more examples than those which have been un-
earthed until now and we need to reconsider how they are to be framed Apparently, we need more examples than those which have been un-
earthed until now, and we need to reconsider how they are to be framed. From the point of view of the project, there are two relevant areas
of conflict studies which are currently reframing their approach. Alter-
native Dispute Resolution (adr) is a field in the study and practice of
law which focuses on finding efficient alternatives to legal proceedings,
namely arbitration and mediation.11 Due to the popularity of adr in
the usa, the profession of mediation has had a growing impact on legal
practice in Europe. 12 N. Bayne and S. Woolcock, The new economic diplomacy. Decision-making and negotiation in interna
tional economic relations (New York 2011 3rd updated ed.); M. Okano-Heijmans, ‘Conceptualizing eco-
nomic diplomacy. The crossroads of international relations, economics ipe and diplomatic studies’, The
Hague Journal of Diplomacy 6 (2011) 7-36; P. Bergeijk, M. Okano-Heijmans and J. Melissen (eds.), Eco
nomic diplomacy. Economic and political perspectives (Leiden 2011). Illustration 1 Workshop of
Rogier van der Weyden. A
Man Reading (Saint Ivo?)
(© The National Gallery,
London). 13 J. Bercovitch, Conflict resolution in the twenty-first century. Principles, methods, and approaches (Ann
Arbor 2009); P.T. Coleman, M. Deutsch and E.C. Marcus (eds.), The handbook of conflict resolution. The
ory and practice (s.l. 2014, 3rd updated ed.); O. Ramsbotham, T. Woodhouse and H. Miall, Contemporary
conflict resolution (Malden 2016, 4th updated ed.).
14 Ramsbotham, Contemporary conflict resolution, 11, 34.
15 For instance J.P. Lederach, The little book of conflict transformation (Intercourse 2003); for conflict
engagement, see: Ramsbotham, Conflict resolution.
16 See for instance the content versus the ambitions of the Journal of Conflict Management and the In
ternational Journal of Conflict Management.
17 See for instance the journal Conflict Management and Peace Studies; E. Muller, G. Frerks and B. de
Graaf (eds.), Conflict. Over conflict en conflictbeheersing (Deventer 2016).
18 Ramsbotham, Conflict resolution, 11. Why bother about conflict management in the past? This is presented as a novel, progressive approach to
handling conflicts. The project which we are starting casts light on a pe-
riod when such strategies were commonly used, and were practiced by
many actors. It can thus provide a historical basis for the current discus- 93 WUBS-MROZEWICZ TSEG Illustration 1 Workshop of
Rogier van der Weyden. A
Man Reading (Saint Ivo?)
(© The National Gallery,
London). Illustration 1 Workshop of
Rogier van der Weyden. A
Man Reading (Saint Ivo?)
(© The National Gallery,
London). sion regarding the pros and cons of involving new specialists in handling
conflicts between individuals and groups. The project’s findings can
also be important for modern economic diplomacy, another field which
combines theory and practice.12 Until very recently, scholars in this field
concentrated solely on state actors in large-scale conflicts, i.e. diplomats
representing a government. They have now begun to devote more at-
tention to the influence of business firms and ngos. The variety of con-
flict managers in commercial cities who fulfilled diplomatic roles, both
front- and back-channel, can bring new insights to this field, especially
because many of the conflicts concerned economic relations. Both Alternative Dispute Resolution and economic diplomacy are
embedded in the large field of conflict studies, where conflict resolu-
tion is the predominant concept used by scientists, as exemplified by 94 VOL. 15, NO. 1, 2018 CONFLICT MANAGEMENT AND INTERDISCIPLINARY HISTORY the influential Journal of Conflict Resolution.13 This focus is not surpris-
ing: there is a broad consensus that discord entails high costs in terms
of economic, political and social capital, rendering swift conflict resolu-
tion all the more desirable. Even in those analyses which take other dy-
namics into account under the umbrella of resolution, the implicit goal
is indeed solving conflicts. Yet as the past shows us, it may be more effec-
tive to give equal attention to other stages of conflict and other ways of
handling it. This could further broaden the impact of adr (by expanding
it to actual alternative conflict management) and economic diplomacy
(by evaluating the flexible role of diplomats in all conflict stages). Nowadays in social sciences, the concept of conflict management is
quite restricted: it is mostly employed in analyses of problems in orga-
nizations and teams. It is thus often connected to organizational psy-
chology and leadership questions. 19 For instance A. Bonoldi, ‘Mercanti a processo. La risoluzione delle controversie tra operatori alle
fiere di Bolzano (secc. xvii-xviii)’, in: A. Bonoldi, A. Leonardi and K.O. Occhi (eds.), Interessi e regole.
Operatori e istituzioni nel commercio transalpino in età moderna (secoli xvi-xix) (Bologna 2012); O. Gel-
derblom, ‘The resolution of commercial conflicts in Bruges, Antwerp and Amsterdam (1250-1650)’, in:
D. Ma and J.L. van Zanden (eds.), Law and long-term economic change (Stanford 2011) 244-276; D. Heir-
baut, ‘Rules for solving conflicts of laws in the Middle Ages. Part of the solution, part of the problem’, in:
A. Musson (ed.) Boundaries of the law. Geography, gender, and jurisdiction in medieval and early modern
Europe (Aldershot 2005) 118-129; S.E. Sachs, ‘Conflict resolution at a medieval English fair’, in: A. Cordes
and S. Dauchy (eds.), Eine Grenze in Bewegung. Öffentliche und private Justiz im Handels-und Seerecht
(Munich 2013) 19-38. Why bother about conflict management in the past? It is also, paradoxically, seen by some
as more narrow than conflict resolution or conflict settlement.14 Alter-
native, emerging concepts are conflict transformation and conflict en-
gagement, the latter being particularly applied to intractable (seemingly
unsolvable) conflicts.15 However, there are clear signs that the concept
of conflict management is one of the candidates for a broader applica-
tion, and at this stage the terms conflict management, conflict resolu-
tion and dispute resolution are sometimes used interchangeably.16 It has
also been used more and more in the field of international relations and
peace studies, i.e. in reference to large-scale conflicts.17 If there are such strong reasons to put the concept of ‘conflict reso-
lution’ under scrutiny, why is it still so dominant in the social sciences? The main criticism of the alternatives has been that they are too indeter-
minate. For the time being, the researchers voicing this reservation pre-
fer therefore to stick to resolution.18 A clear, historically informed model
of conflict management can thus be a contribution to the academic field 95 WUBS-MROZEWICZ TSEG TSEG of conflict studies by delineating the concepts; to the practice-orient-
ed fields of adr and economic diplomacy by giving actual and probably
surprising content to the concept of conflict management. This can pro-
vide a concrete answer to the call for showing historical examples which
not only make us understand mechanisms of conflicts better, but which
also inspire us. The state of the art in economic, political, legal and
social history The predominant use of the concept of ‘conflict resolution’ in social
sciences has had a major impact on historical research: this term has
been clearly favoured as it is reflected in numerous publications. For his-
torians, this concept conveys the notion of obstacles removed, thresh-
olds crossed and change over time. Accordingly, this term and variants
such as dispute settlement have become central concepts in historical
debates. Specifically, they are standard in economic, legal and political
history of commercial cities, which are relevant to this project.19 In the
following section, I will outline how these concepts are used in these
fields of history, and then discuss how the emerging concept of ‘conflict
management’ has been used in some social and political historical stud-
ies of medieval history. This historiographical overview shows why there
is a gap in social and economic, as well as other historical, analyses. Economic historians have concentrated on commercial disputes, for
example contract breaches, which affected individual traders or privi-
leged foreign groups. There is a heated debate in economic history on
whether guilds, rulers or city councils were most effective in dealing
with such conflicts. Handling them adequately meant facilitating for-
eign trade, so this question has been seen as a litmus test for who was
responsible for the economic growth of premodern Europe. Making use 96 VOL. 15, NO. 1, 2018 CONFLICT MANAGEMENT AND INTERDISCIPLINARY HISTORY of the tenets of the New Institutional Economics, researchers have de-
fined the ‘who’ as institutions, while the people behind them and their
networks have not yet been a subject of study.20 This is apparent in the
aforementioned 2014 debate issue of tseg on the book by Oscar Gelder-
blom, Cities of Commerce. The Institutional Foundations of Internation
al Trade in the Low Countries, 1250-1650 (Princeton 2013), which ar-
gues that city magistrates (as institutions) and inter-urban competition
should be seen as forces behind the growth. The book adds a new and
very interesting element to the older ‘polity/ruler vs. guild/merchants’
debate. The tseg issue, in turn, is an exemplary academic discussion on
the topic of urban and economic growth. However, both the book and
the journal issue stay within the conceptual framework of discussing
conflicts which is usual in economic history. The focus is on institutions,
and the role of social actors in handling conflicts is not put into ques-
tion. 20 D. Acemoglu, S. Johnson and J. Robinson, ‘The Rise of Europe. Atlantic trade, institutional change,
and economic growth’, American Economic Review 95:3 (2005) 546-579; A. Greif, Institutions and the
path to the modern economy. Lessons from medieval trade (New York 2006); O. Gelderblom, Cities of com
merce; S. Epstein, Freedom and growth. The rise of states and markets in Europe, 1300-1750 (London
2000); O. Gelderblom and R. Grafe. ‘The rise and fall of the merchant guilds. Re-thinking the comparative
study of commercial institutions in premodern Europe’, Journal of Interdisciplinary History 40:4 (2010)
477-511; F. Apellániz, ‘Venetian trading networks in the medieval Mediterranean’, The Journal of Inter
disciplinary History 44:2 (2013) 157-179; T. Ertman, Birth of the Leviathan. Building states and regimes
in medieval and early modern Europe (Cambridge 1997); E. Lindberg, ‘Club goods and inefficient institu-
tions. Why Danzig and Lübeck failed in the early modern period’, The Economic History Review 62 (2009)
604-628; S. Ogilvie, Institutions and European trade. merchant guilds 1000-1800 (Cambridge 2011);
D. Stasavage, States of credit. Size, power, and the development of European polities (Princeton 2011).
21 A. Cordes, ‘The search for a medieval Lex Mercatoria’, in: V. Piergiovanni (ed.), From lex mercatoria
to commercial law (Berlin 2005) 53-68; A. Cordes, ‘Litigating abroad. Merchants’ expectations regarding
procedure before foreign courts according to the Hanseatic privileges (12th-16th century)’, working pa- 21 A. Cordes, ‘The search for a medieval Lex Mercatoria’, in: V. Piergiovanni (ed.), From lex mercatoria
to commercial law (Berlin 2005) 53-68; A. Cordes, ‘Litigating abroad. Merchants’ expectations regarding
procedure before foreign courts according to the Hanseatic privileges (12th-16th century)’, working pa- The state of the art in economic, political, legal and
social history Even more importantly, the concept of conflict resolution itself
is not questioned, but rather treated as a given. Conflict resolution re-
mains the implicit and explicit concept, and the debate revolves around
judicial versus extra-judicial means of resolving conflicts, the efficien-
cy and use of courts and institutions of conflict resolution provided by
guilds, cities and rulers. This institutional and resolution focus is also visible in legal history,
including the most innovative approaches. In the context of merchants
and mercantile cities, the recently established absence of one universal
Lex Mercatoria in Europe has led to the question of which judicial and
extra-judicial institutions and tools (litigation in various types of courts,
arbitration and mediation) were best-suited to resolve mercantile con-
flicts.21 Taking one step further, the question of ‘who’ was best-suited as a 97 per (Frankfurt a.M., 2013); D. De ruysscher, ‘Law merchant in the mould. The transfer and transforma-
tion of commercial practices into Antwerp customary law (16th-17th centuries)’, in: V. Duss et al. (eds.),
Rechtstransfer in der Geschichte. Legal transfer in history (Munich 2006) 433-445; D. De Ruysscher, ‘Naer
het Romeinsch recht alsmede den stiel mercantiel’. Handel en recht in de Antwerpse rechtbank (16de-17de
eeuw) (Kortrijk-Heule 2009). 22 W. Decock, Theologians and contract law. The moral transformation of the Ius commune (ca. 1500-
1650) (Leiden 2013). Compare for the High Middle Ages the role of monasteries in the society as a
whole, S. Vanderputten and A.-J. Bijsterveld. ‘Penitential discourse and conflict management in the
late-eleventh- and early-twelfth-century southern low countries’, Revue belge de philologie et d’histoire 90
(2012) 471-492, here 480. 23 For instance: A. Wijffels, ‘Krieg, Diplomatie und Recht. Die englisch-hansischen Konflikte 1468-
1603’, in: A. Cordes (ed.), Mit Freundschaft oder mit Recht? Inner- und außergerichtliche Alternativen
zur kontroversen Streitentscheidung im 15.-19. Jahrhundert (Cologne 2015) 85-100; A. Wijffels, ‘Intro-
duction. Commercial quarrels-and how (not) to handle them’, Continuity and Change 32:1 (2017) 1-9,
which is the introduction to a special issue co-edited by F. Miranda and myself, see note 30. See also:
J. Wubs-Mrozewicz and A. Wijffels. ‘Diplomacy and advocacy. The case of the king of Denmark v. Dutch
skippers before the Danzig city council (1564-1567), The Legal History Review 84:1-2 (2016) 1-53. This
approach has recently also been employed in the project of Albrecht Cordes, see note 31. per (Frankfurt a.M., 2013); D. De ruysscher, ‘Law merchant in the mould. The transfer and transforma-
tion of commercial practices into Antwerp customary law (16th-17th centuries)’, in: V. Duss et al. (eds.),
Rechtstransfer in der Geschichte. Legal transfer in history (Munich 2006) 433-445; D. De Ruysscher, ‘Naer
het Romeinsch recht alsmede den stiel mercantiel’. Handel en recht in de Antwerpse rechtbank (16de-17de
eeuw) (Kortrijk-Heule 2009).
22 W. Decock, Theologians and contract law. The moral transformation of the Ius commune (ca. 1500-
1650) (Leiden 2013). Compare for the High Middle Ages the role of monasteries in the society as a
whole, S. Vanderputten and A.-J. Bijsterveld. ‘Penitential discourse and conflict management in the
late-eleventh- and early-twelfth-century southern low countries’, Revue belge de philologie et d’histoire 90
(2012) 471-492, here 480.
23 For instance: A. Wijffels, ‘Krieg, Diplomatie und Recht. Die englisch-hansischen Konflikte 1468-
1603’, in: A. Cordes (ed.), Mit Freundschaft oder mit Recht? Inner- und außergerichtliche Alternativen
zur kontroversen Streitentscheidung im 15.-19. Jahrhundert (Cologne 2015) 85-100; A. Wijffels, ‘Intro-
duction. Commercial quarrels-and how (not) to handle them’, Continuity and Change 32:1 (2017) 1-9,
which is the introduction to a special issue co-edited by F. Miranda and myself, see note 30. See also:
J. Wubs-Mrozewicz and A. Wijffels. ‘Diplomacy and advocacy. The case of the king of Denmark v. Dutch
skippers before the Danzig city council (1564-1567), The Legal History Review 84:1-2 (2016) 1-53. This
approach has recently also been employed in the project of Albrecht Cordes, see note 31. 25 Here more extensively, as the high middle ages are not part of the analysis in the project itself: G. Alt
hoff, Spielregeln der Politik im Mittelalter. Kommunikation in Frieden und Fehden (Darmstadt 1997);
S. Patzold, ‘Konflikte als Thema in der modernen Mediävistik’, in: H.-W. Goetz, Moderne Mediävistik.
Stand und Perspektiven der Mittelalterforschung (Darmstadt 1999) 198-205; the introduction and contri-
butions in W.C. Brown and P. Górecki (eds.), Conflict in medieval Europe. Changing perspectives on society
and culture (Aldershot 2003); F. Cheyette, ‘Suum cuique tribuere’, French Historical Studies 6 (1970) 287- WUBS-MROZEWICZ WUBS-MROZEWICZ TSEG social actor to deal with mercantile matters has been recently taken up,
showing that in premodern times, for instance theologians could be of
more use to the mercantile community than lawyers.22 The actor-centred
approach is thus gaining prominence in this field and providing very in-
teresting insights on unlikely actors. The tradition of legal history, how-
ever, directs most of these analyses towards the framework of conflict
resolution. The recurring question is who could end a conflict, whether
in a court or outside of it, as this has been seen as the task of lawyers
and mediators. A noteworthy exception is the work of Alain Wijffels,
who has been advocating the use of the concept of ‘conflict manage-
ment’. His insights stem from bridging legal and political history, and
his studies have been one of the sources of inspiration for this project.23 p
p
j
The connection with political history is thus certainly fruitful for
those who want to go beyond conflict resolution. Yet paradoxically, these
branches of political history which are relevant to commercial cities
are still operating within the resolution framework, albeit at a different
level. Specifically, political historians have discussed conflicts regard-
ing commercial towns in terms of ongoing state formation. The power
struggles concerning autonomy, the control of hinterland, the income
from trade and military contributions which could be expected from
both sides when the relationship grew closer, have therefore received
most attention. These wars and diplomatic crises, kings and rebellious
cities, have constituted the core of writing national historiographies of
northern Europe since the nineteenth century. It is therefore one of the 98 VOL. 15, NO. 1, 2018 CONFLICT MANAGEMENT AND INTERDISCIPLINARY HISTORY most traditional branches of historical conflict studies, with a bibliogra-
phy which cannot be adequately dealt with in any single journal article. In these analyses, the focus has been on the causes and effects of large-
scale conflicts and on the most prominent actors in these conflicts. This
had been apparent in the more narrowly defined subdiscipline of diplo
matic history, where diplomats as representatives of rulers have been
seen as the prime conflict fixers. Only very recently have New Diplomat-
ic Historians begun to expand this category to urban councillors. 24 J. Watkins, ‘Toward a new diplomatic history of medieval and early modern Europe’, Journal of Me
dieval and Early Modern Studies 38:1 (2008) 1-14; M. Ebben and L. Sicking, ‘New diplomatic history in
the premodern age. An Introduction’, Tijdschrift voor Geschiedenis 127:4 (2014) 541-552; H. Kugeler,
C. Sepp and G. Wolf (eds.), Internationale Beziehungen in der Frühen Neuzeit. Ansätze und Perspektiven
(Hamburg 2006). WUBS-MROZEWICZ A her-
alded next step in the development of the field is the connection with
other branches of history, taking interest in a broader range of sources
and topics.24 p
The state of the art in these fields reveals strikingly little dialogue be-
tween premodern economic, legal and political history on the conceptu
al level, and specifically on the level of the concept of conflict and ways
of handling it. This concerns not only research on commercial cities, but
premodern historical research in general. Conflict as a topic has obvi-
ously been present in historical studies since the discipline has emerged. Conflict as a concept, on the other hand, has been most problematized
in studies from the 1970s on, but with a rather restricted chronological
and thematic range. The focus has been on the High Middle Ages and on
the complex relations between kings, lords, clergy and peasants in the
absence of strong states as a thematic topic. Consequently, the role of
rituals, negotiation, the formulation of law, the Peace of God movement,
the impact of kinship and various types of communities, the use of vi-
olence, spatial divisions, the question of feudal relations (and the no-
tion of feudalism as such) have been discussed extensively for the tenth
to thirteenth centuries, often framed as dispute settlement. The excit-
ing aspects of this research have been the influence of legal anthropol-
ogy, and more generally the interdisciplinary take which makes histor-
ical conflict studies part of cultural history.25 Scholars have also delved The state of the art in these fields reveals strikingly little dialogue be-
tween premodern economic, legal and political history on the conceptu
al level, and specifically on the level of the concept of conflict and ways
of handling it. This concerns not only research on commercial cities, but
premodern historical research in general. Conflict as a topic has obvi-
ously been present in historical studies since the discipline has emerged. 99 WUBS-MROZEWICZ WUBS-MROZEWICZ TSEG into other aspects of handling conflicts, most notably the prevention of
conflicts.26 This has resulted in a more frequent use of the term conflict
management/Konfliktbewältigung/conflictbeheersing in these studies,
but these historians have often used conflict or dispute settlement and
conflict management interchangeably.27 From the point of view of our
project, it is important to note that studies of the high Middle Ages have
not led to a more defined notion of conflict management which could
work at various levels. This is not surprising, given the fact that social
structures like guilds, cities and states which became arenas of conflict,
became far more developed in the later periods. It would seem logical that studies of the Late Middle Ages and Early
Modern period would take up this thread and spin it further, but it has
not happened so far in a systematic way. Only some elements of the in-
terdisciplinarity and conceptual approach developed for this earlier pe-
riod, have been taken up. For instance, the roles and definitions of rit-
uals, feuds or faction strife in (urban) societies, have also been applied 26 As pointed out by W.C. Brown and P. Górecki, ‘What conflict means. The making of medieval con-
flict studies in the United States, 1970-2000’, in: Brown and Górecki (eds.), Medieval Conflict, 19, 23,
25; S.D. White, Custom, kinship, and gifts to saints. The laudatio parentum in western France, 1050-1150
(Chapel Hill 1988); E. Tabuteau, Transfers of property in eleventh-century Norman law (Chapel Hill 1988);
P. Górecki, ‘Ad Controversiam Reprimendam. Family groups and dispute prevention in medieval Poland,
c. 1200’, Law and History Review 14:2 (1996) 213-243; Davies and Fouracre (eds.), The settlement of dis
putes. 27 For instance Patrick Geary, see: P.J. Geary and J. Falquevert, ‘Vivre en conflit dans une France sans
État. Typologie des mécanismes de règlement des conflits (1050-1200)’, Annales (1986) 1107-1133, lat-
er translated as conflict management; ‘Moral obligations and peer pressure. Conflict resolution in the
medieval aristocracy’, in: P.J. Geary, F. Curta, C. Spinei (eds.), Writing history. Identity, conflict, and memo
ry in the Middle Ages (Bucarest 2012) 89-94; A.-J. Bijsterveld, Do ut des. Gift giving, memoria, and conflict
management in the medieval Low Countries (Hilversum 2007); Vanderputten and Bijsterveld, ‘Peniten-
tial discourse’, 471-492; D. Barthélemy, ‘La vengeance, le jugement et le compromis’, Actes des congrès
de la Société des historiens médiévistes de l’enseignement supérieur public 31:1 (2000) 11-20; O. 299; S.D. White, ‘Pactum ... Legem Vincit et Amor Judicium. The settlement of disputes by compromise in
eleventh-century Western France’, The American Journal of Legal History 22:4 (1978) 281-308; S. Reyn-
olds, Kingdoms and communities in Western Europe, 900-1300 (Oxford 1997); E.R. Brown, ‘The tyranny of
a construct. Feudalism and historians of medieval Europe’, The American Historical Review (1974) 1063-
1088; W. Davies and P. Fouracre (eds.), The settlement of disputes in early medieval Europe (Cambridge
1992); W.I. Miller, Bloodtaking and peacemaking. Feud, law, and society in Saga Iceland (Chicago 2009).
The main anthropological influence has been by Simon Roberts, see for instance: S. Roberts, ‘The study
of dispute. Anthropological perspectives’, in: J. Bossy (ed.) Disputes and settlements. Law and human rela
tions in the West (Cambridge 1983) 1-24. WUBS-MROZEWICZ Auge et
al. (eds.), Bereit zum Konflikt. Strategien und Medien der Konflikterzeugung und Konfliktbewältigung im
europäischen Mittelalter (Ostfildern 2008). An interesting work to use as comparison is conflict manage-
ment in te sixth century, P.N. Bell, Social conflict in the age of Justinian. Its nature, management, and medi
ation (Oxford 2013). VOL. 15, NO. 1, 2018 100 CONFLICT MANAGEMENT AND INTERDISCIPLINARY HISTORY to the later medieval period.28 Moreover, peace-making and arbitration
have received some interdisciplinary attention.29 Again, no comprehen-
sive model of conflict management, which reflects upon various strate-
gies of approaching conflict, has been developed for the period up to the
end of the sixteenth century. The special issue of Continuity and Change
(2017) on merchants and commercial conflicts in Europe 1200-1600,
edited by Flávio Miranda and myself, with an introduction by Alain
Wijffels, has put forward some ideas in this direction, on the basis of
conferences held in 2013-2015.30 There is, however, a surge in interest in
the topic and the approach, as exemplified by recent collaborative pro
jects on maritime conflict management in this period.31 This means it is
time to tackle the thematic, conceptual and chronological lacuna in the
analysis of historical conflicts in a systematic way. A systematic analysis is the very goal of the project. We aim to con-
nect the threads from political, economic, legal and cultural research
traditions where the handling of conflicts appears in various guises. Also, we aim to build further on the conceptual considerations to cre-
ate a working model of historical conflict management. And because a
model only comes to life when it serves a content, we will test it through
cases coming from extensive primary source analysis. 28 For northern Europe, especially research on the Low Countries is of interest: J. Braekevelt, F. Buylaert
and J. Dumolyn, ‘The politics of factional conflict in late medieval Flanders’, Historical Research 85:227
(2012) 13-31; S. ter Braake, ‘Parties and factions in the late middle ages. The case of the Hoeken and
Kabeljauwen in The Hague (1483-1515)’, Journal of Medieval History 35:1 (2009) 97-111; F. Buylaert,
‘Familiekwesties. De beheersing van vetes en private conflicten in de elite van laatmiddeleeuws Gent’,
Stadsgeschiedenis 2 (2007) 1-19; P. Hoppenbrouwers, ‘Bloedwraak en vete in de late middeleeuwen’,
Tijdschrift voor Geschiedenis 123:2 (2010) 158-177; J. Firnhaber-Baker and Dirk Schoenaers (eds.), The
Routledge history handbook of medieval revolt (London 2016). WUBS-MROZEWICZ As Petzold, ‘Konflikte’, 199 pointed out, it
is remarkable that German research on feuding and cultural elements of it, in the vein of Otto Brunner,
has not been integrated into Anglo-Saxond scholarship, see: O. Brunner, Land und Herrschaft. Grundfra
gen der territorialen Verfassungsgeschichte Österreichs im Mittelalter (Vienna 1939, revised in 1959). 29 E. Powell, ‘Arbitration and the law in England in the late Middle Ages’, Transactions of the Royal His
torical Society 33 (1983) 49-67; G. Naegle (ed.), Frieden schaffen und sich verteidigen im Spätmittelalter
(Munich 2012). 29 E. Powell, ‘Arbitration and the law in England in the late Middle Ages’, Transactions of the Royal His
torical Society 33 (1983) 49-67; G. Naegle (ed.), Frieden schaffen und sich verteidigen im Spätmittelalter
(Munich 2012). 30 F. Miranda and J. Wubs-Mrozewicz (eds.), Merchants and commercial conflicts in Europe, 1250-1600,
special issue of Continuity and Change 32:1 (2017). 31 L. Sicking, ‘Introduction. Maritime conflict management, diplomacy and international law, 1100-
1800’, Comparative Legal History 5:1 (2017) 2-15, outlines an nwo internationalisation project started
in 2016; sfb 1095 ‘Die Hanse und ihr Recht: Ressourcenschwäche und Funktionalität’, led by Albrecht
Cordes at Goethe University Frankfurt am Main. 101 A project on conflict management: an outline of the
approach The project consists of three subprojects (a PhD and two postdoc),
namely an analysis of conflicts, conflict management and conflict man-
agers in the commercial cities of (1) Lübeck, Stockholm, Reval (Tallinn),
(2) Bruges, Antwerp, London, (3) Danzig (Gdańsk), Thorn (Toruń), El-
bing (Elbląg). Each of the subprojects has an own emphasis due to the
specific contexts in these cities, but the subprojects have a shared ap-
proach which will also result in a synthesising monograph. p
y
g
g p
First, we approach the concept of conflict with a very open mind. Conflict is a very broad and internally connected category in this pro
ject: we investigate commercial disputes (debts, cheating, bankruptcies)
and conflicts related to the mobility of traders and skippers (shipwreck,
clashes about inheritances and religion), which could lead to large-scale
conflicts. At the same time, big politics (war, privateering, withdrawal
of privileges, bans and embargoes) caused serious problems for indi-
viduals. The conflict management cases will be drawn from a variety of
representative northern European towns, ranging from highly autono-
mous ones (Lübeck) to towns where the king had a clear influence (Lon-
don). The core of the analysis is constituted by multi-level conflicts, be-
cause they affected many types of inhabitants in the commercial towns
of northern Europe. Not only traders, but also skippers, craftsmen, their
wives and children, who were travelling and settling in another town. In
particular, inheritance conflict cases offer a unique opportunity to hear
the premodern voices of women in testimonials and petitions. By zoom-
ing in on how these conflicts were handled, we can uncover the inter-
play between the individual, group and large-scale conflicts and show
through a new lens how urban Europe responded to state formation and
complex political, institutional, demographic and economic changes
between 1350 and 1570. Second, the shared point of departure is the defined concept of con-
flict management, based on preliminary archival research. It is a pro-
cess-oriented model, consisting of 1) prevention as part of the task of
urban governments to accommodate citizens, strangers and the city’s
overlord, 2) provocation to force change, 3) maintenance of the status
quo when no solution could be found, 4) escalation and de-escalation
to steer the conflict and 5) resolution with a marked end such as a hand-
shake, court sentence or peace treaty. Consequently, it was conducted
using a host of tactics, e.g. WUBS-MROZEWICZ TSEG 32 Ramsbotham, Conflict resolution, 15-16, e.g. the escalation/de-escalation model or the hourglass
model.
33 M.L. Pelus-Kaplan, ‘Merchants and immigrants in Hanseatic cities, c. 1500-1700’, in: D. Calabi, and
S.T. Christensen (eds.), Cultural exchange in early modern Europe. ii Cities and cultural exchange in Europe
(Cambridge 2007) 132-53; D.W. Poeck, Die Herren der Hanse. Delegierte und Netzwerke (Frankfurt 2010);
J. Puttevils, Merchants and trading; P. Spufford, Power and profit. The merchant in medieval Europe (New
York 2002). 32 Ramsbotham, Conflict resolution, 15-16, e.g. the escalation/de-escalation model or the hourglass
model. A project on conflict management: an outline of the
approach specific urban policy or litigation. We consid- 102 VOL. 15, NO. 1, 2018 CONFLICT MANAGEMENT AND INTERDISCIPLINARY HISTORY er these strategies as one package and we pose the question who em-
ployed them, how and when. The elements of the model are strategies,
i.e. deliberate choices made in handling conflicts. They should indeed
be seen as elements in a conflict, not stages of it, which is often the case
in models of conflict resolution.32 This means that they could occur sev-
eral times during a conflict in various sequences (e.g. alternating cycles
of escalation and de-escalation), that some of the elements could be
entirely absent during a conflict (e.g. maintenance of the status quo)
and, as mentioned in the introduction to this paper, there might have
not been any clear resolution at all. Conflicts, when seen from a his-
torical perspective, often appear to be highly complex or even messy. Approaching the model as a composite of elements allows to evaluate
the process of each historical conflict in its own right, and not accord-
ing to a sequential matrix. I take into account that the model might be
adapted as a result of the findings from the empirical sources, and that
some of its elements will turn out to be more important than others. There will thus be continuous reflection on the use and usefulness of
the model. Third, the category of ‘conflict managers’ is also intentionally broad. The main common denominator is that in these premodern commer-
cial cities, they were connected either directly or through kith and kin
to local and overseas trade.33 However, the category was not fixed as one
profession. It was a given or accepted role. Mercantile city councillors
could be judges, arbitrators or urban diplomats; neighbours ‘of good rep-
utation’ and experience in complex mercantile relations acted as me-
diators; merchant sons became bishops and reverends involved in pol-
itics, or academically trained lawyers and diplomats for kings, exerting
his influence on even micro-conflicts. They accompanied cases to courts
of appeal, or brought them to the attention of their ruler. Also, taking a
cue from economic sociology, we take into account that these managers
were embedded in social relations with conflict participants. This could
influence the choices they made. 34 Wubs-Mrozewicz and Wijffels, ‘Diplomacy and advocacy’.
35 J. Wubs-Mrozewicz, ‘Kopieergedrag. De vormen en functies van afschriften in het laatmiddeleeuwse
politieke briefverkeer van de Hanzesteden’, Tijdschrift voor Geschiedenis 127:4 (2014) 603-624.
36 J. Wubs-Mrozewicz, ‘Neutrality before Grotius. A city, a state and seven salt ships in the Baltic (1564-
1567)’, Journal of Early Modern History 22 (2018) 1-28. A project on conflict management: an outline of the
approach Even though they were almost exclu-
sively male (with the exception of queens and female regents), they 103 WUBS-MROZEWICZ TSEG dealt with conflicts which affected women, men and children. Finally,
as they were part of a highly mobile group in northern Europe, they con-
nected cities and conflicts, and could spread the knowhow on handling
them in various contexts. To date, premodern conflicts have not been re-
searched systematically from the perspective of those who directly dealt
with them. From the point of view of social and economic history, this
is a clear shift from the institutional approach originating in New Insti-
tutional Economics, to an ‘agents making the institutions’ approach. In
other words, it brings the social and economic history of premodern
cities much closer together. Fourth, the integral and multi-level approach means that various
types of tactics will be considered – to a much broader extent than
what has been usual for the premodern mercantile conflict resolution
research tradition. Mediation, arbitration, litigation in urban and high-
er courts, diplomacy, violence on land and sea, embargoes and black-
mail are typical examples. They could be used by various types of con-
flict managers, at various stages of a conflict, and as part of various
strategies. For instance, a city magistrate could suggest mediation as
conflict resolution, but it could also be a way to stall a conflict. A rep-
resentative of a ruler could act as a diplomat, but he could also start le-
gal proceedings. These legal proceedings could then turn out to be not
an attempt to resolve a conflict, as we would traditionally expect, but
a provocation.34 We also seek to uncover tactics that have escaped at-
tention until now or seem to be unusual, surprising or even illogical. Taking examples from my recent research, it could be sophisticated in-
direct communication, like the purposeful circulation of copies of let-
ters and documents, which could show the intention of escalating or
de-escalating a conflict, depending on the context.35 Also, the formu-
lation of new legal concepts, like neutrality, can also be seen as a con-
flict management strategy to prevent further escalation of the conflict. A project on conflict management: an outline of the
approach And more in general, when analysing a historical conflict, one could see
how legal means, for instance court proceedings, were not meant to re-
solve a group conflict, but to provoke a large-scale one.36 Conversely,
tactics like not writing down a loan contract between business partners
could be seen as a serious negligence in conflict prevention, but it was 104 VOL. 15, NO. 1, 2018 CONFLICT MANAGEMENT AND INTERDISCIPLINARY HISTORY in fact a common way to maintain trust and good relations in northern
Europe.37 This brings us to the fifth aspect of the approach. In order to estab-
lish why and how one strategy was chosen instead of another, and who
made this decision, the concepts of subsidiarity and game theory will be
applied. Subsidiarity is usually applied to analysing on which level of a
legal forum a matter was handled or to studying the organisational divi-
sion of power. It has been effectively applied to and further developed
for historical studies, as shown in the work of Peter Stabel.38 Here, it will
be broadened to various social actors who managed conflicts. Game the-
ory, a favoured economic approach, has been used with some caution, if
not reluctance, by scholars of the arts. The mathematical discourse has
often been seen as a difficult threshold. However, its tenets are highly in-
spiring also for historians. Specifically, they draw the attention not only
to the choices which were eventually made, but they help to consider
all options which were available in a conflict context. This means that
roads not taken also become part of the analysis, thus avoiding a path
dependency.39 There are interesting insights on the application of game
theory to conflict studies, exactly where individual, group and large col-
lective interests are at stake.40 Choices for one strategy or tactic above the other are not made in
a void. Complex circumstances influence decision-making processes. This means that the context of the conflict also determined what was
desired or expected, and in turn what was effective. In all three subpro
jects, the use of strategies will therefore be viewed from three sides. First, the policy or policies or the urban governments will be charted,
in other words the content and background of the theory on how to
deal with conflicts. In this broad take, this means law, diplomatic guide-
lines and a narrative of the public good. 37 J. Wubs-Mrozewicz, ‘Mercantile conflict resolution and the role of the language of trust. A Danzig
case in the middle of the sixteenth century’, Historical Research 88:241 (2015) 417-440; O. Gelderblom,
‘The governance of early modern trade. The case of Hans Thijs, 1556-1611’, Enterprise & Society iv (2003)
606-39.
38 P. Stabel, ‘Economic development’, 183-204: I would like to thank Peter Stabel for drawing my atten-
tion to this concept.
39 A. Colman, Game theory and its applications in the social and biological sciences (London 2013); Greif,
Institutions; P.C. Ordeshook, Game theory and political theory (Cambridge 1986); J. Wubs-Mrozewicz,
‘Game theory and the Hanse. An epilogue’, in: J. Wubs-Mrozewicz and S. Jenks (eds.), The Hanse in late
medieval and early modern Europe (Leiden 2013) 283-288.
40 G. Bornstein, ‘Intergroup conflict. Individual, group, and collective interests’, Personality and Social
Psychology Review 7:2 (2003) 129-145; Ramsbotham, Conflict resolution, 20-21. A project on conflict management: an outline of the
approach Second, the practice of conflict 105 41 nwo veni ‘Dealing with foreign traders, dealing with conflict. Strategies of conflict resolution and
their role in trade relations in the Baltic c. 1450-1580’.
42 For instance, the main series: H.A. Poelman (ed.), Bronnen tot de geschiedenis van den Oostzeehandel
1122-1499; F.G. von Bunge (ed.), Hanserecesse/Hanserezesse (Leipzig 1870-1970), 4 series and a total of
26 volumes. Hansisches Urkundenbuch (Halle 1876-1939), 11 vols.; Liv-, Esth- und Curländisches Urkun
denbuch nebst Regesten, vol. 1, (Reval 1853); R. Häpke (ed.), Niederländische Akten und Urkunden zur
Geschichte der Hanse und zur deutschen Seegeschichte (Munich 1913-1923), 2 vols.; Stockholms stads
böcker från äldre tid (incl. ämbetsbok) (1917-1944), 9 vols.; Urkundenbuch der Stadt Lübeck: 1139-1470
(1843-1905) 11 vols.
43 As demonstrated for instance in Wubs-Mrozewicz, ‘Mercantile conflict resolution’ and ‘Neutrality’. 43 As demonstrated for instance in Wubs-Mrozewicz, ‘Mercantile conflict resolution’ and ‘Neutrality’. WUBS-MROZEWICZ WUBS-MROZEWICZ TSEG management, i.e. how it was actually done will be examined and finally
the perception of it will be taken into account. By perception we mean
the contemporary evaluation of the effectiveness and expectations ex-
pressed of various conflict participants and onlookers. Again, this will
apply to macro-, meso- and micro-conflicts. Here, the interdisciplinary
approach of the project comes particularly to the fore. This means that the envisaged empirical basis is broad. The manu-
script sources include diplomatic (letters and instructions), normative
(laws and ordinances), administrative (entries in town books, accounts
on travels) and narrative (contemporary historiographies on conflicts)
sources, legal proceedings in urban courts and courts of higher instances,
and petitions to town councils and rulers. Preliminary research has been
already conducted in various archives in the years 2011-2015.41 Also,
published sources will be used.42 One of the strengths of the project is
that so much ‘new’ material can be used for the multi-level approach,
i.e. sources which have not been used for conflict analyses or which have
never been used for research at all.43 These sources are unique not only
in the context of northern Europe, but in premodern Europe as a whole. Of course, the availability of the various categories of sources differs de-
pending on e.g. the degree of preservation, so the gaps will have to be ac-
counted for in the analyses. The overarching method, containing all the described elements of
the approach, is contextualised comparison of conflict management
connected to specific cities. The comparison concerns case studies in
three subprojects centred around these commercial cities. The cities,
from Reval to London, are chosen on the basis of the availability of suit-
able sources, the varying degree of their autonomy and a representative
geographical spread. Such comparison of premodern cities, informed
by extensive source analysis, has not been undertaken yet for north-
ern Europe. For specific research questions, historical network analysis
and the analysis of the language of conflict management will be applied 106 VOL. 15, NO. 1, 2018 CONFLICT MANAGEMENT AND INTERDISCIPLINARY HISTORY comparatively. Yet the key connectors between the micro-, meso- and
macro-levels, the various strategies and cities are the conflict managers. This way, we can view institutions of conflict management, as well as
their forms and functions, from a different angle. WUBS-MROZEWICZ The questions which
will recur in all three subprojects will thus concern not only conflicts
and conflict management, but also the background and embeddedness
of these conflict managers, their strategies and motivations, and the per-
ception of their effectiveness. Conclusions All in all, the project aims to bridge the gaps between the historical sub-
disciplines, and between the ways conflicts in the past and the present
are being analysed. From the perspective of view of social and econom-
ic historical readership, the major points will be the broadening of the
concept of conflict resolution by making it part of a concept of con-
flict management; the emphasis on agency in the study of institutions
of conflict management; considering individual and group mercantile
conflicts together with large-scale political conflicts, all as embedded
in social relations; and connecting the practice of dealing with conflicts
(most prominent in economic history) with the policy of it (more usual
in legal and political history) as well as perception (usually the domain
of cultural and social history). We take into account that the project set-
up will be adapted while the empirical research will be conducted, and
welcome comments on the project idea. This nwo vidi project will be conducted at the University of Amster-
dam, Faculty of Humanities, January 2018-March 2023. About the author Justyna Wubs-Mrozewicz (1976) is a vidi pi and senior lecturer in Medi
eval History at the University of Amsterdam. Her recent publications include
‘The late medieval and early modern Hanse as an institution of conflict man-
agement’, Continuity and Change 32:1, May 2017, 59-84 and W. Blockmans,
M. Krom, J. Wubs-Mrozewicz (eds.), The Routledge Handbook of Maritime Trade
Around Europe (Routledge 2017). E-mail: j.j.wubs-mrozewicz@uva.nl 107 WUBS-MROZEWICZ WUBS-MROZEWICZ
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Clinical and therapeutic implications of psychiatric comorbidity in high functioning autism/Asperger syndrome: An Italian study
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Open journal of psychiatry
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cc-by
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ABSTRACT netic mechanisms and an improvement of the diagnostic
criteria, clinical practitioners have a really poor under-
standing that Autism Spectrum Disorders (ASDs) occur
most frequently than thought in general population
(prevalence rate for all ASD is estimated at 1%) [1]. ASDs are neurobiological disorders, caused by complex
gene-environment interactions, not yet fully identified
[2]. The present study describes the occurrence of psychi-
atric comorbid disorders in a cohort of 86 high func-
tioning autism (HFA)/Asperger syndrome (AS) pa-
tients, examined at Child Neurology and Psychiatry
Unit of Tor Vergata University. 38 patients out of 86
(44.2%) presented one or more psychiatric comorbid-
ities, such as mood disorders, Attention Deficit/Hy-
peractivity Disorder (ADHD), Tourette syndrome
(TS), anxiety disorders, including obsessive-compul-
sive disorder (OCD), and psychotic symptoms. We
compared our sample with the evidences from the
scientific literature on psychiatric comorbidity in
ASD patient, in particular in HFA/AS. In this paper
we focus on the high frequency of comorbid psychiat-
ric disorders in HFA/AS patients, such as mood dis-
orders,
Attention
Deficit/Hyperactivity
Disorder
(ADHD), Tourette syndrome (TS), anxiety disorders,
including obsessive-compulsive disorder (OCD), and
psychosis, including schizophrenia. We analyzed rates
of all psichiatric comorbidities diagnosed in a sample
of HFA/AS subjects and we compared findings from
our study with the evidences from the scientific lit-
erature on psychiatric comorbidity in ASD patients,
in particular HFA/AS. We point out that comorbid
psychiatric symptoms can be hardly diagnosed, be-
cause they could present atipically in ASDs then in
general population. Furthermore, they could be mask-
ed by ASD core symptoms. Several studies on patients with ASDs indicate that up
to 70.8% of them are affected by one or more psychiatric
condition in comorbidity [3]. Psychiatric comorbidity
occurs highly in patients with High Functioning Autism
(HFA) and Asperger syndrome (AS) [4-6], which are
milder forms of autism, according to some authors. HFA
individuals have a good cognitive level, expressed by an
IQ > 70. AS is characterized by high verbal IQ, impaired
social interaction, restricted interests and ritualistic be-
haviours, in absence of language delay. It is still debated if a comorbid disorder could occur in
ASD patients with a range of different symptoms from
those ones that are pathognomonic in general population. The identification of psychiatric comorbidity is difficult
in ASD patients: any psychiatric symptoms could overlap
with or be masked by ASD symptoms themselves. OJPsych OJPsych Open Journal of Psychiatry, 2013, 3, 329-334
http://dx.doi.org/10.4236/ojpsych.2013.33034 Published Online July 2013 (http://www.scirp.org/journal/ojpsych/) Open Journal of Psychiatry, 2013, 3, 329-334
h
//d d i
/10 4236/ j
h 2013 33034 P b ABSTRACT In this regard, HFA/AS patients could provide clinical
information about their emotional states, due to their
high cognitive level and great verbal communication. Moreover, those characteristics allow them to attend
mainstream school and so they often experience more
bullying and less social support, which seem to be re-
lated to increasing psychiatric comorbidities, like con-
duct problems and disruptive behaviors [1]. Keywords: Autistic Spectrum Disorders; Psychiatric
Comorbidity On the other hand, it was found that ASDs are much
less recognized and diagnosed in those with milder core
symptoms. HFA/AS patients may therefore present to
health services for other psychiatric symptoms and these Clinical and therapeutic implications of psychiatric
comorbidity in high functioning autism/Asperger
syndrome: An Italian study 1Department of Child Neurology and Psychiatry, Tor Vergata University, Rome, Italy
2Department of Neuroscience, Child and Adolescence Psychiatry Unit, Children Hospital Bambino Gesù, Rome, Italy
Email: silviagiovinazzo@yahoo.it Received 10 March 2013; revised 12 April 2013; accepted 20 April 2013 Received 10 March 2013; revised 12 April 2013; accepted 20 April 2013 Copyright © 2013 Silvia Giovinazzo 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. Keywords: Autistic Spectrum Disorders; Psychiatric
Comorbidity 1. BACKGROUNDS Despite of the increased knowledge about the pathoge- OPEN ACCESS S. Giovinazzo et al. / Open Journal of Psychiatry 3 (2013) 329-334 330 at Child Neurology and Psychiatry Unit of Tor Vergata
University, in Rome, from 2009 to 2012. surface features may not clearly signal the presence of an
ASD [1]. The patient’s mean age was 14.9 years (median 13.1,
SD 6.7), with a mean age at evaluation of 11.9 years (SD
7.1). According to DSM-IV-TR [7], ADHD, OCD, schizo-
phrenia are excluded from ASD diagnostic criteria and
diagnosis of anxiety disorders is to be avoided. But many
people with ASD also have a range of psychiatric symp-
toms quite corresponding to diagnostic criteria for those
psychiatric disorders. Male patients were 77 out of 86 (89%), with a male/
female ratio of 8.5. Autistic symptoms were assessed by comparing the
core clinical features with the DSM-IV diagnostic crite-
ria for ASDs; furthermore, the diagnosis of ASD was
supported by the Autism Diagnostic Observation Sched-
ule (ADOS; Lord et al., 2000) and the Autism Diagnostic
Interview-Revised (ADI-R; Lord et al., 1995). Furthermore, according new DSM-V diagnostic crite-
ria, AS will not be provided for in ASDs. Nevertheless,
we led our study while DSM-IV-TR criteria were still
valid and we could recruit AS patients. However, both
our and the other findings about psychiatric comorbidity
in AS, hereafter should alert clinicians about the high risk
of psychopathology in an “AS-like” people, although it
can no longer be considered as “autistic” people. Cognitive assessment was performed by the Wechsler
Intelligence Scales (WISC-III; Wechsler, 1991) and non
verbal Leiter International Performance Scale-Revised
(Leiter-R-Visualization and Reasoning, Roid & Miller,
1997). Have not yet been identified specific risk factors for
the development of co-morbidity in ASDs. Risk factors
that are well recognized in general population to be asso-
ciated with child psychiatric disorders show similar rela-
tionship in children with ASDs [1]. It has been demo-
strated that individuals with less severe ASDs, as HFA
and AS, are equally likely to show additional psychiat-
ric disorders [1]. 2.1. Objectives Swanson, Nolan, and Pelham-IV (SNAP-IV), a ques-
tionnaire designed to assist in diagnosing a child’s
behavioral problems, like Attention Deficit Disorder
with Hyperactivity (ADHD) and Oppositional Defi-
ant Disorder; Our clinical study aimed to: Describe the psychiatric comorbid disorders in a co-
hort of HFA/AS patients; Define the prevalence of every psychiatric conditions
in the sample, comparing our data with the literature; Children’s Depression Inventory (CDI), a brief self-
report test that assesses signs of depression in chil-
dren and adolescents 7 to 17 years old; p
p
g
Identify how many comorbid psychiatric conditions
have arisen in each patient; Multidimensional Anxiety Scale for Children (MASC),
a self-report test that assesses the presence of symp-
toms related to anxiety disorders in youth aged 8 to 19
years. Identify a possible relationship between age of pa-
tients and the development of a specific comorbidity; Assess the drugs used for the treatment of psychiatric
symptoms, defining how many patients needed to set
up a polytherapy. 1. BACKGROUNDS The presence of psychiatric disorders in comorbidity
was evaluated by: Kiddie-Schedule for Affective Disorders and Schizo-
phrenia (K-SADS; Kaufman et al., 1997), a semi-
structured interview designed to assess present and
past episodes of psychiatric disorders in children and
adolescents between the ages of 6 - 18, according to
DSM-IV criteria; Frequently are diagnosed anxiety and depression, be-
havioral disorders like ADHD or oppositional defiant
disorder (ODD) and obsessive disorders [2,8,9]. Some
studies report rare cases of comorbidity with schizophre-
nia and other disorders, including Tourette’s syndrome,
tics, trichotillomania, enuresis or encopresis. Many pa-
tients present multiple comorbid disorders: respectively
80% of ASDs plus ADHD, 60% of ASDs plus behavioral
disorder, 40% of ASDs plus affective disorder have an-
other or more comorbidities [3]. Child Behavior Checklist (CBCL) 4/18 years, a ques-
tionnaire completed by parents, about children/ado-
lescent behavioral (e.g. internalizing problems-with-
drawal, somatic complaints, anxiety/depression-, de-
linquent behavior and externalizing behavior aggres-
sive and also attention difficulties, social problems
and thought problems); Conners’ Parent and Conners’ Teacher Rating Scale,
which are questionnaires filled by parents or teachers,
which provide to assess and quantify oppositional be-
havior, inattention, hyperactivity and ADHD Index; 2.2. Methods and Materials In our sample, all 86 subjects were high functioning,
with full scale IQ > 70. Mean IQ was 92.4 (SD = 20.9). The sample consisted of 86 HFA/AS patients, examined OPEN ACCESS Copyright © 2013 SciRes. S. Giovinazzo et al. / Open Journal of Psychiatry 3 (2013) 329-334 331 Table 3. Distribution of comorbidities within our sample,
according to age ranges. 38 patients out of 86 (44.2%) had one or more psy-
chiatric comorbidities. Attention deficit/Hyperactivity Disorder (ADHD) was
the most common comorbidity, diagnosed in 25 patients
(66%). Age ranges
Comorbidity [6,9] ys
18.4%
[9,14] ys
23.7%
[14,18] ys
28.9%
>18 ys
28.9%
Attention deficit/Hyperactivity
disorder
X
X
X
X
Bipolar disorder
X
X
X
Psychosis
X
X
Depression
X
Tics and Tourette’s syndrome
X
X
Generalized anxiety disorder
X
X
X
Oppositional defiant disorder
X
X
Eating disorder
X
Obsessive/Compulsive disorder
X
Learning disorder
X Were also diagnosed bipolar disorder (n = 7; 18.4%),
depression (n = 2; 5.3%), generalized anxiety disorder
(GAD) (n = 2; 5.3%), positive psychotic symptoms (n =
6; 15.8%), learning disorder (n = 1; 2.6%), obsessive-
compulsive disorder (n = 1; 2.6%), eating disorders (n =
1; 2.6%), oppositional defiant disorder (n = 2; 5.3%), tics
and Tourette’s syndrome (n = 2; 5.3%) (Table 1). These 38 patients was divided in 3 groups: 1) only
with one comorbidity (71%), 2) with 2 comorbidity
(24%), 39 with 3 or more (5%) (Table 2). The association between ADHD and Bipolar Disorder
occurred most frequently in patients with multiple
comorbidities (36.4%). We studied the distribution of comorbidities within the
sample (Table 3), according to four age ranges: creased in puberty and in adolescence. creased in puberty and in adolescence. creased in puberty and in adolescence. Childhood, including 6- to 8-year and 11 months-old:
n = 7 (18.4%); Every age range was characterized by suffering from
specific types of comorbidity. p
yp
y
Of the 38 patients with comorbidities, 19 (50%) re-
ceived pharmacological treatment. Regarding disrup-
tive behaviours, most of our ASD plus ADHD patients
responded to MPH, one patient did not tolerate that drug
and had to change with atomoxetine, and another patient
was treated only with atomoxetine. The patients with
ODD also were treated with methylphenidate. Table 1. Comorbid disorders diagnosed in our sample. Table 1. Comorbid disorders diagnosed in our sample. Comorbid Disorder
Rate of patients (%)
Attention deficit/Hyperactivity disorder
66
Bipolar disorder
18.4
Psychosis
15.8
Depression
5.3
Tics and Tourette’s syndrome
5.3
Generalized anxiety disorder
5.3
Oppositional defiant disorder
5.3
Eating disorder
2.6
Obsessive/Compulsive disorder
2.6
Learning disorder
2.6 The patient with GAD received treatment with par-
oxetine (SSRI) and OCD was treated with sertraline
(SSRI). Comorbid Disorder
Rate of patients (%) The patients with depressive disorder received benzo-
diazepines or venlafaxine (NSRI) and that with BD re-
ceived Valproic Acid, which is the first line treatment for
BD in youth. The case of eating disorders was treated
with mirtazapine (NARI). 74% of the cases received a
polypharmacotherapy. 2.2. Methods and Materials Pre-puberty: including 9- to 13-year and 11 months-
old subjects: n = 9 (23.7%); Adolescence, including 14- to 17-year and 11 months-
old subjects: n = 11 (28.9%); responded to MPH, one patient did not tolerate that drug
and had to change with atomoxetine, and another patient
was treated only with atomoxetine. The patients with
ODD also were treated with methylphenidate. Adulthood, including 18 years-old subjects and over:
n = 11 (28.9%). There was a higher incidence of comorbidity in ado-
lescence. The prevalence of multiple comorbidities in- Psychotic symptoms were treated with atyical antipy-
chotics, as well as olanzapine or risperidone in most
cases, or aripiprazole in a single case. Copyright © 2013 SciRes. 3. DISCUSSION We compared findings from our study with the evidences
from the scientific literature on psychiatric comorbidity
in individuals with ASD. We consider at first the only
population-study dealing with several psychiatric comor-
bidities in ASD subject, involving a population-derived
sample. Table 2. Rate of patients presenting one or more psychiatric
comorbidities. Table 2. Rate of patients presenting one or more psychiatric
comorbidities. Comorbid psychiatric disorder
Rate of patients (%)
Any psychiatric disorder
44.2%
One psychiatric disorder
71%
Two psychiatric disorder
24%
Three or more psychiatric disorder
5% Comorbid psychiatric disorder
Rate of patients (%) We found that 44.2% of our HFA/AS patients had co-
morbid psychiatric disorders. Our results differ from the
study of Simonoff et al. [3]: they found that 70.8% of
112 ASD patients had at least one psychiatric comorbid-
ity. However, that sample also included patients with OPEN ACCESS OPEN ACCESS Copyright © 2013 SciRes. S. Giovinazzo et al. / Open Journal of Psychiatry 3 (2013) 329-334 332 low-functioning autism, which are more frequently and
severely affected by psychiatric symptoms, as well as
disruptive or repetitive behaviours, aggression, irritabil-
ity [5]. Moreover, they employed more effective diag-
nostic tools for screening and diagnosis of psychiatric
disorders in ASD, like Child and Adolescent Psychiatric
Assessment (CAPA; Angold and Costello, 2000). in adolescence, as well as other mood disorders. These
data are in accord to the literature [13-16] (prevalence of
2% - 9%, usually in adolescence). Depression occurred in 5.2%, with onset in adulthood,
compared with 0.9% evidenced by Simonoff [3]. De-
pression can often arise with an increase of withdrawal
and aggressive behaviours: these symptoms may present
more clearly in patients with HFA/AS, rather than in
low-functioning autism (LFA), which could primarily
presenting withdrawal and aggression. From other stud-
ies has been reported a very broad range of prevalence of
mood disorders, from 6% up to 70% [4,5,15-19]. This
variability could depend to the fact that some authors
examined only AS patients, other ones considered de-
pressive symptoms rather then a diagnosis of major de-
pression. In 2009, Mukkades et al. [4] pointed out a >90%
prevalence of comorbidity in AS and HFA samples: the
high rate was probably due to referral bias in diagnostic
process. In 2009, Mukkades et al. [4] pointed out a >90%
prevalence of comorbidity in AS and HFA samples: the
high rate was probably due to referral bias in diagnostic
process. Copyright © 2013 SciRes. 3. DISCUSSION In 2010, Mattila et al. [5] conducted a study on com-
bined community- and clinic-sample of HFA/AS pa-
tients, aged from 9.8 to 16.3 years. They highlighted a
comorbidity prevalence of 74%, in a cohort aged more
restrictely than ours. We considered also the age corre-
lated with the comorbidity. This correlation is low and
this aspect influenced the global prevalence in our popu-
lation. Only one adolescent patient presented OCD and two
individuals had a GAD (5.26%), one at puberty, the other
at about 18 years. Simonoff et al. [3] found that 2% of
ASD patients developed a OCD and up to 44% an anxi-
ety disorders. According to the literature, OCD is the
anxiety disorder that appears most frequently in ASD
[3,20,21]. In our sample, 31% of patients had multiple comorbid-
ities, as compared to 33% reported by Simonoff et al. [3],
and to 37% by Mattila et al. [5]. The same comorbidities
have been identified in these studies, as well as in ours:
ADHD, GAD, OCD, ODD, eating behavior disorders,
BD and depression, tics and TS, learning disorders and
psychosis. This recurrent finding of the same associa-
tions between disorders emphasizes that they have a
common genetic and neurobiological substrate. In our
sample, ADHD is the most frequent comorbid disorder
(66%), specially presenting in childhood and puberty. In
literature, the prevalence of ADHD among individuals
with ASD is estimated to be 28.2% [3], particularly
among those with HFA/AS it’s 44% - 65% [6]. These
findings stress the clinical necessity to assess inattention
and hyperactivity/impulsivity in ASD [10], and point out
also the fact that often is difficult to establish early diag-
nosis of HFA/AS, because autistic symptoms could be
masked by ADHD symptoms. It is possible to recognize obsessions/compulsions
from ASD repetitive behaviours, [6], but standardized
diagnostic tools are currently not yet available. In our sample, 15.8% of patients presented psychotic
symptoms, with onset in adulthood. In literature, the
prevalence of schizophrenia in ASD is of 0.6% [11], in
HFA/AS up to 3.3% [4]. In adults with schizophrenia,
there is often a history of autistic symptoms in childhood;
autism and schizophrenia share some neurobiological
abnormalities and genetic backgrounds [17,18]. According to all literature examined [3], tics and
Tourette’s syndrome had an overall prevalence of 22% -
30% [4,5,6,22]. Indeed, TS is considered a risk factor for development
of further neuropsychiatric disorder, in ASD subjects [22,
23]. 4. CONCLUSIONS AND FUTURE
PROSPECTIVES [4]
Mukaddes, N.M., Hergüner S. and Tanidir, C. (2010)
Psychiatric disorders in individuals with high-functioning
autism and Asperger’s disorder: Similarities and differ-
ences. World Journal of Biological Psychiatry, 11, 964-
971. doi:10.3109/15622975.2010.507785 There is still no consensus about the belief that symp-
toms of new onset in ASD should be classified as a
manifestation of further psychiatric disorder. [5]
Mattila, M.L., Hurtig, T., Haapsamo, H., et al. (2010) Co-
morbid psychiatric disorders associated with Asperger
syndrome/high-functioning autism: A community- and
clinic-based study. Journal of Autism and Developmental
Disorders, 40, 1080-1093. doi:10.1007/s10803-010-0958-2 It could be useful to assess the possible common
pathogenetic mechanisms and risk factors between ASDs
and other psychiatric disorders, and to obtain additional
information on the underlying disfunction [25]. In literature there are only few data about comorbid
psychiatric disorders in ASDs. It could be necessary to
increase the sample size and to collect the largest number
of clinical-anamnestic information, for studying etiopa-
thogenesis and risk factors for the occurrence of comor-
bidity [26,27]; meta-analysis studies would be useful, to
compare individual researches. It would be interesting to
highlight a possible correspondence between age and the
development of a specific comorbid disease. This report
may be useful for lifetime monitoring patients, to predict
the onset of a specific disease, and for early recognition
of symptoms, to plan a focused and effective treatment. [6]
Mazzone, L., Ruta, L. and Reale, L. (2012) Psychiatric
comorbidities in Asperger syndrome and high functioning
autism: Diagnostic challenges. Annals of General Psychi-
atry, 11, 16. [7]
American Psychiatric Association (2000) Diagnostic and
statical manual of mental disorders. 4th Edition, APA,
Washington, DC. [8]
Kim, J.A., Szatmari, P., Bryson, S.E., Streiner, D.L. and
Wilson, F. (2000) The prevalence of Anxiety and mood
problems among children with autism and Asperger syn-
drome. Autism, 4, 117-132. doi:10.1177/1362361300004002002 [9]
Gadow, K.D., DeVincent, C.J. and Schneider, J. (2009)
Comparative study of children with ADHD only, autism
spectrum disorder + ADHD, and chronic multiple tic dis-
order + ADHD. Journal of Attention Disorders, 12,
474-485. doi:10.1177/1087054708320404 According to some authors, there is need for caution in
interpreting results that use generic measures in higlhy
specific and distinct populations, as well as ASDs, with-
out first characterizing the instrument properties, e.g. scoring and cut-offs, pertaining to that population [1]. [10] Yoshida, Y. and Uchiyama, T. 3. DISCUSSION In our sample, two individuals presented tics only as
comorbidity. Indeed, TS is considered a risk factor for development
of further neuropsychiatric disorder, in ASD subjects [22,
23]. In our sample, two individuals presented tics only as
comorbidity. The neurobiological point of view shows that ASD
and ADHD are deficits in executive functioning and re-
flect a common dysfunction of fronto-temporal circuits
[11]. ASD and ADHD also share genetic background:
some chromosomal regions such as 2q24, 16p13, 16p1,
17p11, 5p13, 15q have been identified as susceptibility
loci to both ADHD and ASD [12]. About pharmacoterapy in ASDs [24], atypical antipsy-
chotics or neuroleptics can be effective against aggres-
sion and core symptoms; but it is often necessary to ad-
minister multiple drugs to control comorbid psychiatric
symptoms. Methylphenidate is the first choice for the
treatment of ADHD, benzodiazepines for anxiety symp-
toms, SSRI’s for the treatment of OCD and mood disor-
ders. Furthermore, two patients had oppositional defiant
disorder (5.6%), compared to 1% in the study of Simon-
off, to 16% according to Mattila et al. [5], up to 30%
according Mukkades et al. [4]. Must be considered that
aggression, often found in ODD, is common in ASD pa-
tients, most of all in LFA; moreover, could be the
epiphenomenon of other types of comorbidity, e.g. mood
disorders or anxiety [5]. This study has some limitations, at first by having a
moderate sample size, a too wide age range and no com-
parison group. The age-ranged groups were not well-
matched in gender and number of patients. Second, the
assessment by the parents and children/adolescents may
have resulted in some recall and/or referral bias. Fur-
thermore, we have not distinctly considered current psy- Bipolar disorder occurred in 5.2% of cases, specially OPEN ACCESS OPEN ACCESS S. Giovinazzo et al. / Open Journal of Psychiatry 3 (2013) 329-334 333 of psychiatric problems in adolescents with autism spec-
trum disorders. Journal of Child Psychology and
Psychiatry, 54, 186-194. doi:10.1111/j.1469-7610.2012.02606.x chiatric diagnosis from past comorbidity. Third, the present study only provides the rate and
type of psychiatric disorders, and there was no informa-
tion provided regarding associated risk factors such as
family history and life events. [2] [2]
Abrahams, B.S. and Geschwind, D.H. (2008) Advances
in autism genetics: On the threshold of a new neurobiol-
ogy. Nature Genetics, 9, 341-353. 3. DISCUSSION In addition, concerning the diagnostic instruments
used in this study, there are no algorithms precisely for
ASD. [3] [3]
Simonoff, E., Pickles, A., Charman, T., Chandler, S.,
Loucas, T. and Baird, G. (2008) Psychiatric disorders in
children with autism spectrum disorders: Prevalence,
comorbidity, and associated factors in a population-de-
rived sample. Journal of the American Academy of Child
and Adolescent Psychiatry, 47, 921-929. doi:10.1097/CHI.0b013e318179964f Regarding to the pharmacological treatment, it would
be necessary to involve and monitorize a larger sample
of treated patients, to verify its short and long term effi-
cacy. 4. CONCLUSIONS AND FUTURE
PROSPECTIVES (2004) The clinical neces-
sity for assessing Attention Deficit/Hyperactivity Disor-
der (ADHD) symptoms in children with high-functioning
Pervasive Developmental Disorder (PDD). European
Child & Adolescent Psychiatry, 13, 307-314. doi:10.1007/s00787-004-0391-1 Clinical practicioners should be able to use diagnostic
tools which are specific for the diagnosis of comorbidity
in ASD patients, to recognize atypical or masked symp-
toms (e.g. Autism Comorbidity Interview Present and
Lifetime version; ACI-PL; Lefeyer et al., 2006; Devel-
opmental Disability Child Global Assessment Scale; DD-
CGAS; Wagner et al., 2007). It would also be necessary
to update the diagnostic criteria for psychiatric disorders
most commonly associated with ASD, adapting them to
the characteristics of these patients. [11] Sinzig, J., Morsch, D., Bruning, N., Schmidt, M.H. and
Lehmkuhl, G. (2008) Inhibition, flexibility, working
memory and planning in autism spectrum disorders with
and without comorbid ADHD-symptoms. Child and
Adolescent Psychiatry and Mental Health, 2, 4-15. doi:10.1186/1753-2000-2-4 [12] Rommelse N.N., Geurts H.M., Franke B., Buitelaar J.K.,
Hartman C.A. (2011) A review on cognitive and brain
endophenotypes that may be common in autism spectrum
disorder and attention-deficit/hyperactivity disorder and
facilitate the search for pleiotropic genes. Neuroscience & Biobehavioral Reviews, 35, 1363-1396.
doi:10.1016/j.neubiorev.2011.02.015 [20] Russel, A.J., Mataix-Cols, D., Anson, M. and Murphy,
D.G.M. (2005) Obsessions and compulsions in Asperger
syndrome and high-functioning autism. British Journal of
Psychiatry, 186, 525-528. doi:10.1192/bjp.186.6.525 [13] Raja, M. and Azzoni, A. (2008) Comorbidity of Asper-
ger’s syndrome and Bipolar disorder. Clinical Practice
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H. and Kikuchi, M. (2008) High prevalence of bipolar
disorder comorbidity in adolescents and young adults
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Disorders, 111, 170-175. doi:10.1016/j.jad.2008.02.015 [22] Burd, L., Li, Q., Kerbeshian, J., Klug, M. G. and Freeman,
R.D. (2009) Tourette syndrome and comorbid pervasive
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Wentz, E., Ståhlberg, O., et al. (2009) Psychiatric and
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E. (2008) Association of ADHD, tics, and anxiety with
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C. (2004) Bipolar disorder, schizophrenia, and other
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and/or autism spectrum disorders. REFERENCES [1]
Simonoff, E., Jones, C.R., Baird, G., Pickles, A., Happé,
F. and Charman, T. (2013) The persistence and stability OPEN ACCESS OPEN ACCESS Copyright © 2013 SciRes. 334 S. Giovinazzo et al. / Open Journal of Psychiatry 3 (2013) 329-334 Copyright © 2013 SciRes. & Biobehavioral Reviews, 35, 1363-1396.
doi:10.1016/j.neubiorev.2011.02.015 Journal of Neural
Transmission, 111, 891-902. doi:10.1007/s00702-004-0115-1 [27] Hurtig, T., Kuusikko, S., Mattila, M.L., et al. (2009) Mul-
ti-informant reports of psychiatric symptoms among
high-functioning adolescents with Asperger syndrome or
autism. Autism, 13, 583-598. doi:10.1177/1362361309335719 [19] Ghaziuddin, M., Ghaziuddin, N. and Greden, J. (2002)
Depression in persons with autism: Implications for re-
search and clinical care. Journal of Autism and Deve-
lopmental Disorders, 32, 299-306. doi:10.1023/A:1016330802348 OPEN ACCESS OPEN ACCESS Copyright © 2013 SciRes.
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Supplementary Figure from Cytokeratins 5 and 17 Maintain an Aggressive Epithelial State in Basal-Like Breast Cancer
| null | 2,023
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17
5
PI
ure S1. Immunocytochemistry was performed for CK5 (red), CK17 (green), and DAPI (blue) in the
cated cell lines and visualized by fluorescent microscopy. Pie charts indicate the proportion of cells that
CK17+ (green), CK5/17+ (yellow), CK5+ (red), or CK5/17−(grey). No CK5 or CK17 was detected in
D cells. Scale bars, 100 µm.
CK5−CK17−
CK5+CK17−
CK5−CK17+
CK5+CK17+
T47D
BT20
EWD8
MDA-MB-468
UCD46 17
5
PI
ure S1. Immunocytochemistry was performed for CK5 (red), CK17 (green), and DAPI (blue) in the
cated cell lines and visualized by fluorescent microscopy. Pie charts indicate the proportion of cells that
CK17+ (green), CK5/17+ (yellow), CK5+ (red), or CK5/17−(grey). No CK5 or CK17 was detected in
D cells. Scale bars, 100 µm. CK5−CK17−
CK5+CK17−
CK5−CK17+
CK5+CK17+
T47D
BT20
EWD8
MDA-MB-468
UCD46 CK5+CK17+ Figure S1. Immunocytochemistry was performed for CK5 (red), CK17 (green), and DAPI (blue) in the
indicated cell lines and visualized by fluorescent microscopy. Pie charts indicate the proportion of cells that
are CK17+ (green), CK5/17+ (yellow), CK5+ (red), or CK5/17−(grey). No CK5 or CK17 was detected in
T47D cells. Scale bars, 100 µm. CK8/18
VIM
DAPI
T47D
EWD8
MDA-MB-468
BT20
UCD46
Figure S2. Immunocytochemistry was performed for CK8/18 (green), Vimentin (red), and DAPI (blue)
indicated cell lines and visualized by fluorescent microscopy. Scale bars, 100 µm. EWD8 CK8/18
VIM
DAPI
T47D EWD8 BT20 MDA-MB-468 UCD46 Figure S2. Immunocytochemistry was performed for CK8/18 (green), Vimentin (red), and DAPI (blue) in the
indicated cell lines and visualized by fluorescent microscopy. Scale bars, 100 µm. Figure S3. Common differentially expressed genes and pathways in MDA-MB-468 and BT2
with CK5 knockdown. A. Venn diagram shows the number of common genes altered in MDA-MB-
BT20 shCK5-22 vs shCont cells by RNA-seq analysis (q<0.05). B. GSEA analysis identified sign
regulated pathways in in MDA-MB-468 and BT20 shCK5-22 vs shCont. Common altered pathw
indicated with P values for each cell line. C. MDA-MB-468 shCK5-22 cells were transfected with s
siZEB1 then assayed after 48 h by immunoblot for ZEB1, CK5, Ecad, VIM, and β-actin. Fold ch
MDA-MB-468
BT-20
1243
971
4446
Hallmark pathway
MDA-MB-468
P value
BT-20
P value
ESTROGEN_RESPONSE_LATE
0.001527
0.000677
KRAS_SIGNALING_DN
0.00157
0.000147
INTERFERON_ALPHA_RESPONSE
0.002463
0.000149
IL2_STAT5_SIGNALING
0.002725
0.003883
EPITHELIAL_MESENCHYMAL_TRANSITION
0.00277
0.000342
HYPOXIA
0.002793
0.00037
UV_RESPONSE_DN
0.005362
0.002051
INTERFERON_GAMMA_RESPONSE
0.00551
0.000139
A
B A MDA-MB-468
BT-20
1243
971
4446
A MDA-MB-468
BT-20
1243
971
4446 Hallmark pathway
MDA-MB-468
P value
BT-20
P value
ESTROGEN_RESPONSE_LATE
0.001527
0.000677
KRAS_SIGNALING_DN
0.00157
0.000147
INTERFERON_ALPHA_RESPONSE
0.002463
0.000149
IL2_STAT5_SIGNALING
0.002725
0.003883
EPITHELIAL_MESENCHYMAL_TRANSITION
0.00277
0.000342
HYPOXIA
0.002793
0.00037
UV_RESPONSE_DN
0.005362
0.002051
INTERFERON_GAMMA_RESPONSE
0.00551
0.000139
B Figure S3. Common differentially expressed genes and pathways in MDA-MB-468 and BT20 cells
with CK5 knockdown. A. Venn diagram shows the number of common genes altered in MDA-MB-468 and
BT20 shCK5-22 vs shCont cells by RNA-seq analysis (q<0.05). B. 17
5
PI
ure S1. Immunocytochemistry was performed for CK5 (red), CK17 (green), and DAPI (blue) in the
cated cell lines and visualized by fluorescent microscopy. Pie charts indicate the proportion of cells that
CK17+ (green), CK5/17+ (yellow), CK5+ (red), or CK5/17−(grey). No CK5 or CK17 was detected in
D cells. Scale bars, 100 µm.
CK5−CK17−
CK5+CK17−
CK5−CK17+
CK5+CK17+
T47D
BT20
EWD8
MDA-MB-468
UCD46 Six mice were used per group. Tumors were measured weekly
volumes estimated by the formula l(w2)/2, plotted as mean ± s.e.m. Two-way ANOVA with mult
comparisons was used to determine statistical significance, indicated for the last timepoint. *P<0
**P<0.01. B. Final tumor mass (mg) is plotted with mean ± s.e.m. One-way ANOVA/Tukey tests
indicated. *P<0.05, **P<0.01, ****P<0.0001. C. Representative images of animals from each group
necropsy under an Illumatool. D. The number of GFP+ metastases visible at necropsy is plotted per gro
ANOVA/Tukey significance is indicated, *P<0.05, ns=not significant. C
B
D A
B A B
D B A shCont
shCK5-22
shCK17-73
Figure S5. Knockdown of CK5 or CK17 in MDA-MB-468 cells decreases gross metastases. A. 1 x 106
GFP+ shCont, shCK5-22, or shCK17-73 cells were injected bilaterally into opposing fourth mammary fat
pads of 8-week-old female NSG mice. Six mice were used per group. Tumors were measured weekly and
volumes estimated by the formula l(w2)/2, plotted as mean ± s.e.m. Two-way ANOVA with multiple
comparisons was used to determine statistical significance, indicated for the last timepoint. *P<0.05,
**P<0.01. B. Final tumor mass (mg) is plotted with mean ± s.e.m. One-way ANOVA/Tukey tests are
indicated. *P<0.05, **P<0.01, ****P<0.0001. C. Representative images of animals from each group at
necropsy under an Illumatool. D. The number of GFP+ metastases visible at necropsy is plotted per group. ANOVA/Tukey significance is indicated, *P<0.05, ns=not significant. C
D shCont
shCK5-22
shCK17-73
Figure S5. Knockdown of CK5 or CK17 in MDA-MB-468 cells decreases gross metastases. A. 1 x 106
GFP+ shCont, shCK5-22, or shCK17-73 cells were injected bilaterally into opposing fourth mammary fat
pads of 8-week-old female NSG mice. Six mice were used per group. Tumors were measured weekly and
volumes estimated by the formula l(w2)/2, plotted as mean ± s.e.m. Two-way ANOVA with multiple
comparisons was used to determine statistical significance, indicated for the last timepoint. *P<0.05,
**P<0.01. B. Final tumor mass (mg) is plotted with mean ± s.e.m. One-way ANOVA/Tukey tests are
indicated. *P<0.05, **P<0.01, ****P<0.0001. C. Representative images of animals from each group at
necropsy under an Illumatool. D. The number of GFP+ metastases visible at necropsy is plotted per group. ANOVA/Tukey significance is indicated, *P<0.05, ns=not significant. C
D D shCK17-73 D shCont
shCK5-22
shCK17-73
C C Figure S5. Knockdown of CK5 or CK17 in MDA-MB-468 cells decreases gross metastases. A. 17
5
PI
ure S1. Immunocytochemistry was performed for CK5 (red), CK17 (green), and DAPI (blue) in the
cated cell lines and visualized by fluorescent microscopy. Pie charts indicate the proportion of cells that
CK17+ (green), CK5/17+ (yellow), CK5+ (red), or CK5/17−(grey). No CK5 or CK17 was detected in
D cells. Scale bars, 100 µm.
CK5−CK17−
CK5+CK17−
CK5−CK17+
CK5+CK17+
T47D
BT20
EWD8
MDA-MB-468
UCD46 GSEA analysis identified significantly
regulated pathways in in MDA-MB-468 and BT20 shCK5-22 vs shCont. Common altered pathways are
indicated with P values for each cell line. C. MDA-MB-468 shCK5-22 cells were transfected with siCont or
siZEB1 then assayed after 48 h by immunoblot for ZEB1, CK5, Ecad, VIM, and β-actin. Fold change to
siCont is indicated below the blots. Hallmark pathway
MDA-MB-468
P value
BT-20
P value
ESTROGEN_RESPONSE_LATE
0.001527
0.000677
KRAS_SIGNALING_DN
0.00157
0.000147
INTERFERON_ALPHA_RESPONSE
0.002463
0.000149
IL2_STAT5_SIGNALING
0.002725
0.003883
EPITHELIAL_MESENCHYMAL_TRANSITION
0.00277
0.000342
HYPOXIA
0.002793
0.00037
UV_RESPONSE_DN
0.005362
0.002051
INTERFERON_GAMMA_RESPONSE
0.00551
0.000139
B B Figure S3. Common differentially expressed genes and pathways in MDA-MB-468 and BT20 cells
with CK5 knockdown. A. Venn diagram shows the number of common genes altered in MDA-MB-468 and
BT20 shCK5-22 vs shCont cells by RNA-seq analysis (q<0.05). B. GSEA analysis identified significantly
regulated pathways in in MDA-MB-468 and BT20 shCK5-22 vs shCont. Common altered pathways are
indicated with P values for each cell line. C. MDA-MB-468 shCK5-22 cells were transfected with siCont or
siZEB1 then assayed after 48 h by immunoblot for ZEB1, CK5, Ecad, VIM, and β-actin. Fold change to
siCont is indicated below the blots. MDA-MB-468
BT20
Figure S4. Aspect ratios of BLBC cells with knockdown of CK5 or CK17. Brightfield images were used
to measure the length and width of each cell in five separate fields. Aspect ratios were calculated by dividing
the longer length by the shorter length and displayed as violin plots. Number of cells measured in eac
group is indicated below the graph. ANOVA/Kruskal-Wallis tests were used to determine statistica
significance. *P<0.05, ****P<0.0001. 558
184
251
162
247
363
217
133
124
185 BT20 MDA-MB-468
BT20
558
184
251
162
247
363
217
133
124
185 Figure S4. Aspect ratios of BLBC cells with knockdown of CK5 or CK17. Brightfield images were used
to measure the length and width of each cell in five separate fields. Aspect ratios were calculated by dividing
the longer length by the shorter length and displayed as violin plots. Number of cells measured in each
group is indicated below the graph. ANOVA/Kruskal-Wallis tests were used to determine statistical
significance. *P<0.05, ****P<0.0001. shCont
shCK5-22
shCK17-73
A
Figure S5. Knockdown of CK5 or CK17 in MDA-MB-468 cells decreases gross metastases. A. 1 x
GFP+ shCont, shCK5-22, or shCK17-73 cells were injected bilaterally into opposing fourth mammary
pads of 8-week-old female NSG mice. 17
5
PI
ure S1. Immunocytochemistry was performed for CK5 (red), CK17 (green), and DAPI (blue) in the
cated cell lines and visualized by fluorescent microscopy. Pie charts indicate the proportion of cells that
CK17+ (green), CK5/17+ (yellow), CK5+ (red), or CK5/17−(grey). No CK5 or CK17 was detected in
D cells. Scale bars, 100 µm.
CK5−CK17−
CK5+CK17−
CK5−CK17+
CK5+CK17+
T47D
BT20
EWD8
MDA-MB-468
UCD46 1 x 106
GFP+ shCont, shCK5-22, or shCK17-73 cells were injected bilaterally into opposing fourth mammary fat
pads of 8-week-old female NSG mice. Six mice were used per group. Tumors were measured weekly and
volumes estimated by the formula l(w2)/2, plotted as mean ± s.e.m. Two-way ANOVA with multiple
comparisons was used to determine statistical significance, indicated for the last timepoint. *P<0.05,
**P<0.01. B. Final tumor mass (mg) is plotted with mean ± s.e.m. One-way ANOVA/Tukey tests are
indicated. *P<0.05, **P<0.01, ****P<0.0001. C. Representative images of animals from each group at
necropsy under an Illumatool. D. The number of GFP+ metastases visible at necropsy is plotted per group. ANOVA/Tukey significance is indicated, *P<0.05, ns=not significant. CK5/VIM
CK5/VIM
CK17/VIM
shCont
shCK5
shCK17
CK5/VIM
CK17/VIM
CK17/VIM
Figure S6. Expression of CK5/CK17/vimentin in MDA-MB-468 spontaneous lung metastases. Lungs
were collected from mice bearing mammary fat pad tumors derived from MDA-MB-468 shCont, shCK5-22,
shCK17-73 cells from the experiment shown in Figure 4 (equal tumor size harvest). Paraffin section of lungs
were stained by IHC for CK5 or CK17 (green) and vimentin (VIM, red) and counterstained with DAPI. Scale
bars, 20 µm. CK5/VIM
CK5/VIM
CK17/VIM
shCont
shCK5
shCK17
CK5/VIM
CK17/VIM
CK17/VIM shCont shCK5 Figure S6. Expression of CK5/CK17/vimentin in MDA-MB-468 spontaneous lung metastases. Lungs
were collected from mice bearing mammary fat pad tumors derived from MDA-MB-468 shCont, shCK5-22,
shCK17-73 cells from the experiment shown in Figure 4 (equal tumor size harvest). Paraffin section of lungs
were stained by IHC for CK5 or CK17 (green) and vimentin (VIM, red) and counterstained with DAPI. Scale
bars, 20 µm. BT20
MDA-MB-468
BT20
MDA-MB-468
A
B
Figure S7. IC50 of doxorubicin (replicates) and paclitaxel in shCont, shCK5-22, and shCK17-73
BLBC cells. The indicated cell lines were treated with varying concentrations of doxorubicin (A) o
paclitaxel (B) in sextuplicate and growth monitored using the IncuCyte. Normalized transformed graphs are
plotted for percent cell viability vs Log(conc) drug at 72 h. Error bars represent mean ± SEM. C. Table
shows IC50 values for doxorubicin and paclitaxel in the indicated cell lines. 95% confidence interval i
shown in parentheses. 17
5
PI
ure S1. Immunocytochemistry was performed for CK5 (red), CK17 (green), and DAPI (blue) in the
cated cell lines and visualized by fluorescent microscopy. Pie charts indicate the proportion of cells that
CK17+ (green), CK5/17+ (yellow), CK5+ (red), or CK5/17−(grey). No CK5 or CK17 was detected in
D cells. Scale bars, 100 µm.
CK5−CK17−
CK5+CK17−
CK5−CK17+
CK5+CK17+
T47D
BT20
EWD8
MDA-MB-468
UCD46 MDA-MB-468
shCont
shCK5-22
shCK17-73
Doxorubicin (nM)
115.70 (97.0-138.5)
64.02 (50.58-81.72)
39.17 (33.11-46.46)
Paclitaxel (nM)
1.54 (1.44-1.65)
1.24 (1.17-1.31)
1.85 (1.67-2.04)
BT20
shCont
shCK5-22
shCK17-73
Doxorubicin (nM)
157.2 (100.1-244.1)
51.46 (33.08-79.74)
46.19 (35.48-60.01)
Paclitaxel (nM)
1.52 (1.33-1.74)
1.46 (1.22-1.75)
1.45 (1.28-1.64)
C BT20
MDA-MB-468
A MDA-MB-468
A BT20 A BT20
MDA-MB-468
B MDA-MB-468
B BT20 B MDA-MB-468
shCont
shCK5-22
shCK17-73
Doxorubicin (nM)
115.70 (97.0-138.5)
64.02 (50.58-81.72)
39.17 (33.11-46.46)
Paclitaxel (nM)
1.54 (1.44-1.65)
1.24 (1.17-1.31)
1.85 (1.67-2.04)
BT20
shCont
shCK5-22
shCK17-73
Doxorubicin (nM)
157.2 (100.1-244.1)
51.46 (33.08-79.74)
46.19 (35.48-60.01)
Paclitaxel (nM)
1.52 (1.33-1.74)
1.46 (1.22-1.75)
1.45 (1.28-1.64)
C C C Figure S7. IC50 of doxorubicin (replicates) and paclitaxel in shCont, shCK5-22, and shCK17-73
BLBC cells. The indicated cell lines were treated with varying concentrations of doxorubicin (A) or
paclitaxel (B) in sextuplicate and growth monitored using the IncuCyte. Normalized transformed graphs are
plotted for percent cell viability vs Log(conc) drug at 72 h. Error bars represent mean ± SEM. C. Table
shows IC50 values for doxorubicin and paclitaxel in the indicated cell lines. 95% confidence interval is
shown in parentheses. Figure S8. Expression of CK5/vimentin in shCont and shCK5 MDA-MB-468 tumors trea
vehicle or doxorubicin. A. MDA-MB-468 shCont and shCK5 tumors were grown to an average
mm3 then treated once week with vehicle (Veh) or doxorubicin (Doxo) for two weeks. T-tests at
point indicated. Mean final tumor mass is down on the right. T-tests are indicated. B. Tumors were
from mice bearing mammary fat pad tumors derived from MDA-MB-468 shCont and shCK5
following two weeks of Veh or Doxo (5 mg/kg) treatment. Paraffin sections of tumors were staine
for CK5 (green) and vimentin (VIM, red) and counterstained with DAPI. Scale bars, 20 µm. shCont, Veh
shCont, Doxo
shCK5, Veh
shCK5, Doxo
A
B
CK5
VIM
DAPI A A A Figure S8. Expression of CK5/vimentin in shCont and shCK5 MDA-MB-468 tumors treated w
vehicle or doxorubicin. A. MDA-MB-468 shCont and shCK5 tumors were grown to an average of ~
mm3 then treated once week with vehicle (Veh) or doxorubicin (Doxo) for two weeks. T-tests at final
point indicated. Mean final tumor mass is down on the right. T-tests are indicated. B. Tumors were colle
from mice bearing mammary fat pad tumors derived from MDA-MB-468 shCont and shCK5-22
following two weeks of Veh or Doxo (5 mg/kg) treatment. 17
5
PI
ure S1. Immunocytochemistry was performed for CK5 (red), CK17 (green), and DAPI (blue) in the
cated cell lines and visualized by fluorescent microscopy. Pie charts indicate the proportion of cells that
CK17+ (green), CK5/17+ (yellow), CK5+ (red), or CK5/17−(grey). No CK5 or CK17 was detected in
D cells. Scale bars, 100 µm.
CK5−CK17−
CK5+CK17−
CK5−CK17+
CK5+CK17+
T47D
BT20
EWD8
MDA-MB-468
UCD46 Paraffin sections of tumors were stained by
for CK5 (green) and vimentin (VIM, red) and counterstained with DAPI. Scale bars, 20 µm. shCont, Veh
shCont, Doxo
shCK5, Veh
shCK5, Doxo
B
CK5
VIM
DAPI shCont, Veh
shCK5, Veh
B
CK5
VIM
DAPI B Figure S8. Expression of CK5/vimentin in shCont and shCK5 MDA-MB-468 tumors trea
vehicle or doxorubicin. A. MDA-MB-468 shCont and shCK5 tumors were grown to an average
mm3 then treated once week with vehicle (Veh) or doxorubicin (Doxo) for two weeks. T-tests at
point indicated. Mean final tumor mass is down on the right. T-tests are indicated. B. Tumors were
shCont, Veh
shCont, Doxo
shCK5, Veh
shCK5, Doxo
B
CK5
VIM
DAPI shCK5, Veh shCK5, Doxo Figure S8. Expression of CK5/vimentin in shCont and shCK5 MDA-MB-468 tumors treated with
vehicle or doxorubicin. A. MDA-MB-468 shCont and shCK5 tumors were grown to an average of ~450
mm3 then treated once week with vehicle (Veh) or doxorubicin (Doxo) for two weeks. T-tests at final time
point indicated. Mean final tumor mass is down on the right. T-tests are indicated. B. Tumors were collected
from mice bearing mammary fat pad tumors derived from MDA-MB-468 shCont and shCK5-22 cells
following two weeks of Veh or Doxo (5 mg/kg) treatment. Paraffin sections of tumors were stained by IHC
for CK5 (green) and vimentin (VIM, red) and counterstained with DAPI. Scale bars, 20 µm. UCD46
UCD115
UCD18
UCD46
UCD115
UCD18
epithelial
mesenchymal
KRT5
KRT5
KRT5
KRT17
KRT17
KRT17
VIM
VIM
VIM
A
B
Figure S9. Single cell expression of CK5, CK17, and vimentin in BLBC PDX. A. A published g
signature for generating epithelial-mesenchymal scores was used {Taube, 2010 #2644}. This signa
contains 246 genes that are down- or upregulated during EMT, which are designated as “epithelial”
mesenchymal” respectively. The average expression for epithelial and mesenchymal genes was calcu
or each cell and the log2 of the epithelial/mesenchymal ratio calcutated. Cells with positive scores w
designated as epithelial (blue) and negative scores as mesenchymal (red). These are indicated for t
PDX on the UMAPs. B. UMAP plots for CK5 (KRT5), CK17 (KRT17), and vimentin (VIM) in each o
hree BLBC PDX. 17
5
PI
ure S1. Immunocytochemistry was performed for CK5 (red), CK17 (green), and DAPI (blue) in the
cated cell lines and visualized by fluorescent microscopy. Pie charts indicate the proportion of cells that
CK17+ (green), CK5/17+ (yellow), CK5+ (red), or CK5/17−(grey). No CK5 or CK17 was detected in
D cells. Scale bars, 100 µm.
CK5−CK17−
CK5+CK17−
CK5−CK17+
CK5+CK17+
T47D
BT20
EWD8
MDA-MB-468
UCD46 A UCD46
UCD115
UCD18
epithelial
mesenchymal
A UCD46
epithelial
mesenchymal
A UCD115 UCD18 UCD46 UCD115 UCD18 UCD46
UCD115
UCD18
epithelial
mesenchymal
KRT5
KRT5
KRT5
B UCD46
UCD115
UCD18
KRT5
KRT5
KRT5
B B UCD46
KRT5 UCD18
KRT5 UCD115
KRT5 KRT17
KRT17
KRT17 KRT17 KRT17 KRT17 KRT17 VIM VIM VIM
VIM VIM
VIM
VIM VIM VIM VIM Figure S9. Single cell expression of CK5, CK17, and vimentin in BLBC PDX. A. A published gene
signature for generating epithelial-mesenchymal scores was used {Taube, 2010 #2644}. This signature
contains 246 genes that are down- or upregulated during EMT, which are designated as “epithelial” and
“mesenchymal” respectively. The average expression for epithelial and mesenchymal genes was calculated
for each cell and the log2 of the epithelial/mesenchymal ratio calcutated. Cells with positive scores were
designated as epithelial (blue) and negative scores as mesenchymal (red). These are indicated for three
PDX on the UMAPs. B. UMAP plots for CK5 (KRT5), CK17 (KRT17), and vimentin (VIM) in each of the
three BLBC PDX.
|
https://openalex.org/W3120344196
|
https://www.frontiersin.org/articles/10.3389/fphy.2020.531662/pdf
|
English
| null |
Influence of Multiple Animal Scanning on Image Quality for the Sedecal SuperArgus2R Preclinical PET Scanner
|
Frontiers in physics
| 2,021
|
cc-by
| 8,576
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Influence of Multiple Animal Scanning
on Image Quality for the Sedecal
SuperArgus2R Preclinical PET
Scanner Nikos Efthimiou 1,2*, John D. Wright 1, Luke Clayton 1, Isaline Renard 1, Federico Zagni 3,
Paulo R.R.V. Caribé 4, Stephen J. Archibald 1,2 and Christopher J. Cawthorne 1,2* 1Positron Emission Tomography Research Centre, University of Hull, Hull, United Kingdom, 2Department of Biomedical Sciences,
University of Hull, Hull, United Kingdom, 3Medical Physics Department, University Hospital ‘S. Orsola-Malpighi’, Bologna, Italy,
4Medical Image and Signal Processing – MEDISIP, Ghent University, Gent, Belgium Background: Increased throughput in small animal preclinical studies using positron
emission tomography leads to reduced costs and improved efficiency of experimental
design, however the presence of multiple off-centre subjects, as opposed to a single
centered one, may affect image quality in several ways. Citation: Efthimiou N, Wright JD, Clayton L,
Renard I, Zagni F, Caribé PRRV,
Archibald SJ and Cawthorne CJ (2021)
Influence of Multiple Animal Scanning
on Image Quality for the Sedecal
SuperArgus2R Preclinical
PET Scanner. Edited by:
Ivo Rausch,
Medical University of Vienna, Austria Edited by:
Ivo Rausch,
Medical University of Vienna, Austria Ivo Rausch,
Medical University of Vienna, Austria
Reviewed by:
Daniele De Paula Faria,
University of São Paulo, Brazil
Peter Homolka,
Medical University of Vienna, Austria
*Correspondence:
Christopher J. Cawthorne,
christopher.cawthorne@kuleuven.be
Nikos Efthimiou
nikos.efthimiou@ieee.org Reviewed by:
Daniele De Paula Faria,
University of São Paulo, Brazil
Peter Homolka,
Medical University of Vienna, Austria *Correspondence:
Christopher J. Cawthorne,
christopher.cawthorne@kuleuven.be
Nikos Efthimiou
nikos.efthimiou@ieee.org *Correspondence:
Christopher J. Cawthorne,
christopher.cawthorne@kuleuven.be
Nikos Efthimiou
nikos.efthimiou@ieee.org Results: Count rate performance assessed with a single NEMA scatter phantom was in
line with previous literature, with simulated data in good agreement. Simulation of dual
scatter phantoms showed an increase in scatter fraction. For the NEMA Image Quality
phantom, uniformity and Recovery coefficients were degraded in the offset, and dual
phantom cases, while spillover ratios were increased, notably when the chamber was
placed nearest the gantry. Image quality metrics were comparable between the 20- and
10 min timeframes. Specialty section:
This article was submitted to
Medical Physics and Imaging,
a section of the journal
Frontiers in Physics Specialty section:
This article was submitted to
Medical Physics and Imaging,
a section of the journal
Frontiers in Physics Conclusion: Dual animal scanning results in some loss of image quality on the Sedecal
Argus PET scanner; however, this degradation is within acceptable limits. Received: 31 January 2020
Accepted: 26 November 2020
Published: 12 January 2021 Keywords: preclinical PET CT, dual phantom scanning, multiple animal scanning, Monte Carlo simulation, image
quality, NEMA NU4, Sedecal Argus 2R ORIGINAL RESEARCH
published: 12 January 2021
doi: 10.3389/fphy.2020.531662 Methods: We evaluated the count rate performance using a NEMA scatter phantom. A Monte Carlo simulation of the system was validated against this dataset and used to
simulate the count rate performance for dual scatter phantoms. NEMA NU4 image
quality phantoms were then scanned in the central and offset positions, as well as in
the offset position next to a uniform activity phantom. Uniformity, recovery
coefficients and spillover ratios were then compared, as were two time frames for
acquisition. 2.1 SuperArgus2R Scanner Aide et al. [1] went on to characterize a self-manufactured
four-animal bed on the Siemens Inveon, demonstrating an
increase in spillover ratios and a decrease in resolution for the
NEMA IQ phantom; albeit with a high correlation between PET
quantitation and biodistribution using this arrangement. Habte
et al. [6] characterized a 3- and 4-bed setup for the R4 (Concorde
Microsystems/Siemens) and Inveon (Siemens) scanners and
demonstrated comparable in vivo quantitation between single
and multiple animal scanning as long as attenuation correction
was performed. Yagi et al. [7] reported another self-assembled
multiple
animal
bed
for
the
Inveon
scanner,
evaluating
uniformity
in
mouse
phantoms
for
four
animals
and
demonstrating a comparable decrease to Habte et al. [6]. Seidel et al. [8] reported a dual mouse bed that enabled ECG-
gated imaging to be carried out on the Argus PET-CT scanner but
did not characterize effects on image quality. The Sedecal SuperArgus2R preclinical PET scanner (Figure 1)
[14–16] features 18 phoswich detector blocks in an arrangement
of 2 rings with diameter 118 mm and axial FOV 48.0 mm. In
between the two rings there is gap of 8 mm. Each block has a two
layers of 13 × 13 crystals of cerium-doped Lutetium Orthosilicate
(LSO:Ce) (7 mm length) at the inner layer and cerium-doped
Gadolinium Orthosilicate (GSO:Ce) (8 mm length) at the outer
layer. Each crystal element has size mm2. The two crystals are
joined back to back with an optically transparent method. From
the 18 blocks, 7 are in coincidence. The coincidence window was
5 ns for all crystal combinations. The phoswich detector arrangement provides Depth of
Interaction (DOI) information, allowing for more uniform
images and better radial spatial resolution [17]. As such, this
detector configuration should also reduce spatial resolution
degradation when scanning dual objects. Siepel et al. [9] studied the effects of scanning 2 and 4 animals,
axially or radially displaced, in an Inveon scanner using the
NEMA NU4 IQ phantom. They reported a decrease in
uniformity, a reduction in Recovery Coefficient (RC) and an
increase in Spill-Over ratio (SOR) for the 2 and 4 phantom
radially-displaced cases, which could be improved by axial
displacement. Rominger et al. [10] characterized an 8-mouse
bed in the Inveon scanner, demonstrating that scatter correction
and reduced overall activity in the field of view was necessary to
achieve optimum quantitation in this setting. Using the PET-
SORTEO simulation methodology, Reilhac et al. 2.2 Monte Carlo Simulation Model Due to the supplied animal bed on the scanner being too small to
accommodate
two
scatter
phantoms
simultaneously,
investigations on a single phantom in an offset bed position
and
dual
phantom
scatter
fraction
were
performed
in
simulation only. The MC simulation model was developed using the GATE
(v.8.1) [18], simulation toolkit. The model was validated over the
scanner’s acceptance tests and measurements following the
NEMA
protocol
[19]. The
energy
window
was
set
to
250–700 keV, the same as the one used in the experiments. The
coincidence
window
was
set
to
5.0 ns. The
emstandard_opt3,
physics
model,
was
used
without
any
variance reduction techniques or energy cuts. 1 INTRODUCTION [5] reported the use of a Siemens
Biograph TrueV clinical PET-CT scanner and showed that tracer
quantitation in the central and offset positions was highly
correlated, albeit with lower system sensitivity and resolution
(and higher injected activities) than commonly found in
preclinical systems. 2.1 SuperArgus2R Scanner [11] assessed the
signal degradation for dual animal scanning on the Inveon and
the impact of this on the detection of biological variation. After
demonstrating that their simulation gave comparable results to
acquired phantom data, they went on to demonstrate that
recovery coefficients and uniformity were decreased, while
SOR (in this case peak-to-valley measure) were increased. 1 INTRODUCTION Preclinical Positron Emission Tomography (PET) allows the longitudinal study of a range of
biological processes in disease models, as well as being invaluable in the screening of novel
diagnostic/theranostic PET probes. It is increasingly common for preclinical PET centers to scan
multiple animals simultaneously in order to increase the cost-efficiency and throughput of studies, January 2021 | Volume 8 | Article 531662 Frontiers in Physics | www.frontiersin.org 1 Multiple Animal Image Quality SuperArgus2R Efthimiou et al. especially with the use of short-lived PET isotopes such as [11C],
or where statistical requirements demand large study groups for
the correct powering of experiments [1, 2]. studies, they demonstrated a decrease in uniformity and
recovery coefficients, and an increase in spillover ratios, in the
multiple vs. single animal cases, though these increases were
within the recently suggested limits for bias [13]. Scanning multiple, radially displaced mice in the Field Of
View (FOV) will increase attenuation and scatter, as well as the
complexity of scatter correction. Additionally sensitivity and
resolution will be reduced, while an increase in the overall
activity in the FOV will increase dead time. As such, image
quality is likely to degrade, and it is necessary to assess these
effects on individual scanner/bed geometries as these vary
considerably [3]. Although ultimately the effect on biological
quantitation is of greatest importance, use of the NEMA NU4
image quality (IQ) phantom [4] allows direct comparison
between systems. This study seeks to investigate the effect of dual animal
scanning on the Sedecal SuperArgus2R preclinical scanner
using the NEMA IQ phantom filled with a range of activities;
also to assess the effects on IQ for common static scan frame
lengths. Initially, we validate a Monte Carlo (MC) simulation of
the scanner geometry and use it to compare the effect on count
rate performance between single and dual NEMA scatter
phantoms. We then go on to assess IQ metrics on phantoms
placed either centrally, offset, or offset alongside a 20 mL syringe
containing an equal activity, also comparing spillover ratios
where air and water chambers are displaced toward the center
of the field of view or the bore. Several groups have reported setups to achieve multiple
animal imaging. Aide et al. Frontiers in Physics | www.frontiersin.org 2.2.1 Simulations for Count Losses, Scatter Fraction
and NECR Most recently, Greenwood et al. [12] characterized a
commercial four-chamber bed on a Mediso preclinical PET
scanner, using NEMA phantoms and mice. As in previous A model of the NEMA scatter fraction phantom was simulated in
order to compare the counting performance, scatter fraction and January 2021 | Volume 8 | Article 531662 Frontiers in Physics | www.frontiersin.org 2 Efthimiou et al. Multiple Animal Image Quality SuperArgus2R FIGURE 1 | (A) Representation of the geometry of the Sedecal SuperArgus 2R detector geometry. The inner layer of crystals (red) are made of LSO:Ce and the
outer layer (gray) are GSO:Ce. (B) Sinogram with corrections, generated by Sedecal’s FORE-3D software (size 175 × 128 × 61). (C) Non corrected sinogram created
from the .tru file containing only counts (size 117 × 117 × 61), as generated in STIR. The two sinograms are from the axial position 29 of the first acquisition 33 MBq. (D)
Illustration of the position of NEMA phantom inside the FOV. The mirror transformation is with respect to the position of the cold containers. (E)–(G) photographs of
the phantom in the central, off-centre and dual acquisitions. FIGURE 1 | (A) Representation of the geometry of the Sedecal SuperArgus 2R detector geometry. The inner layer of crystals (red) are made of LSO:Ce and the
outer layer (gray) are GSO:Ce. (B) Sinogram with corrections, generated by Sedecal’s FORE-3D software (size 175 × 128 × 61). (C) Non corrected sinogram created
from the .tru file containing only counts (size 117 × 117 × 61), as generated in STIR. The two sinograms are from the axial position 29 of the first acquisition 33 MBq. (D)
Illustration of the position of NEMA phantom inside the FOV. The mirror transformation is with respect to the position of the cold containers. (E)–(G) photographs of
the phantom in the central, off-centre and dual acquisitions. Noise Equivalent Count Rate (NECR) for different placements in
the FOV, using the validated GATE model. The model phantom
matched the manufactured phantom that was used for the
validation of the simulation model; a polyethylene cylindrical
phantom 70 mm long and 25 mm in diameter (for details see
Section 2.3). Phantom data were acquired with a starting activity of
approximately 35 MBq of [18F]. 2.3 NEMA Phantom Studies All acquisitions were made using [18F] produced using an ABT
mini-cylotron (ABT, United States). The scanner’s software has the option to extract pre-corrected
(for
dead
time
and
decay)
FORE-3D
sinograms
[22]
(Figure
1B),
typically
for
Filtered
Back-Projection
reconstruction, which are not suitable for the execution of
the NEMA protocol. 2.2.1 Simulations for Count Losses, Scatter Fraction
and NECR A series of 10 min scans
followed by a 15 min of rest were acquired, lasting in total for
more than 3 half-lives (6 h), using the 250–700 keV energy
window. 2.3.2 Raw Data Processing The phantom was simulated solo in the center, offset
(±19.0 mm on the x axis, as defined in GATE) positions of the
FOV [20, 21] as well as in combination with a second phantom. The phantom was simulated solo in the center, offset
(±19.0 mm on the x axis, as defined in GATE) positions of the
FOV [20, 21] as well as in combination with a second phantom. The model of the scanner included all parts of the physical
scanner (plastic tubes, shielding etc). The NEMA NU4-2008 [4] protocol requires that uncorrected
sinograms holding only number of coincidences are processed. However, direct extraction of such data from the scanner was
not possible. By default, the scanner saves the recorded
coincidences (after application of the energy window) in the
proprietary .tru files which essentially are histograms holding
counts per detector combination with detector pair coordinates. The scanner’s software has the option to extract pre-corrected
(for
dead
time
and
decay)
FORE-3D
sinograms
[22]
(Figure
1B),
typically
for
Filtered
Back-Projection
reconstruction, which are not suitable for the execution of
the NEMA protocol. The model of the scanner included all parts of the physical
scanner (plastic tubes, shielding etc). .1 Validation of the Simulation Model 3.1 Validation of the Simulation Model
The count rates of prompt, true, random, scattered events and the
NECR of the measured phantom acquisition are presented in
Figure 2A as a function of activity in the FOV, and summarized
in Table 1. At a starting activity of 10 MBq, reflecting a suggested
maximal injected dose for a mouse, the count loses are
approximately 3.3% which increases to 9.76% when the
activity doubles to 20 MBq; representative of a dual scanning
scenario. The ratio between random and true events remains
below 21% up to activities of 20 MBq, and the NECR has a peak of
105 kcps at 26 MBq. Comparison between this measured data and
its simulated counterpart (Figure 2B) shows good agreement,
with the simulation data returning a 4.5% reduction in NECR
peak. Scatter fractions were comparable at both 10- and 20 MBq
(19.65 and 18.26% vs. 20.9 and 19.35% respectively). Images were converted into DICOM format and loaded into a
Mediso image anaylsis software (Mediso NEMA Tests, NEMA
IQ) which automatically recognized the NEMA IQ phantom
geometry and generated appropriate ROI’s for uniformity
(%Standard Deviation (%STD)), Spill-Over ratio (SOR) and
Recovery Coefficient (RC). Generated ROIs were manually
positioned over the respective regions of the phantom,
according to the protocol’s instructions, to return mean
values and the percentage standard deviation. Furthermore, the scatter rate steadily increases in the
measured centrally positioned acquisition until around 25 MBq
is reached, peaking at 45.6 kcps (Figure 2A). This closely
correlates to the simulated counterpart where the scatter rate
peaks at similar activity levels. The simulated data, however, show
that 57.9% of scattered events occur within the phantom, and
28.4% of events take place in the Delrin (polyoxymethylene) tube,
which resides in the scanner bore to protect the detector rings and
electronics. 30% of scattered events occur on the gantry shielding
and less than 1% on the animal bed. In addition we sought to investigate whether the above
figures of merit were consistent across the FOV. To assess
this, we made acquisitions on single NEMA IQ phantoms
positioned off-centre on the left- and right-hand side of the
bore. For SOR assessments we also performed left- and right-
sided offset acquisitions with the air chamber facing either
toward
or
away
from
the
scanner
bore,
in
order
to
investigate any impact made through the inherent bias of the
NEMA IQ phantom geometry. 2.3.4 Image Reconstruction PET image reconstruction was performed using Ordered Subsets-
Expectation Maximization (OSEM) 2D, with 16 subsets after 2
full iterations. The 2D sinogram was generated using 3D Fourier
Rebinning (FORE-3D) on the stored data files provided by the
manufacturer. The stored sinograms were corrected for linearity
and dead-time . In order to reduce the amount of blurring, the
maximum ring difference was set to 16 rings and a span 3; the 2.3.3 Assessment of Image Quality A NEMA NU4-2008 image quality phantom [4] containing 2.5 or
10 MBq of 18F (representing low and high activity acquisitions in
mice respectively) was scanned at the center and offset positions
and alongside a (20 mL) syringe filled with the same activity as
illustrated in (Figure 1B). The activity was within 2.5% of stated
dose at acquisition start. PET acquisitions used the 250 −700 keV
energy window, with two bed positions to fully cover the
phantom, over a period of 20 min (2 × 10 min) or 10 min
(2 × 5 min) to assess the effect of different timeframes on
image quality. CT acquisitions covered the entire PET field of
view and were performed at 40 kV and 140 µA beam current, with
360 projections and 1 shot). Attenuation maps were generated
from a segmented version of the CT image scaled to 511 keV, as
part
of
the
standard
reconstruction
process
from
the
manufacturer. .1 Validation of the Simulation Model We also investigated the impact
on dual phantom scanning when the NEMA IQ phantom is
positioned on the left or right imaging bed. 3.2 Simulation of Dual Scatter Phantoms
Simulations of the single NEMA scatter phantom in the offset bed
position shows count losses of 0.98% at 10 MBq, increasing to 13.3%
at 20 MBq (Figure 2C). Likewise, the dual scatter phantom
simulation returned count losses at 5.78% and 17.26% for both
10- and 20 MBq, respectively. The rate of random events occurring
appears to almost double from 10 to 20 MBq yet remains below 20%
(Figure 2D). In the case of simulated offset and dual phantom
acquisitions, the scatter rate continues to increase until a peak of
53.22- and 58.23 kcps at 30 MBq, respectively. The dual phantom
simulation increased in scatter rate by 17.4% compared to the
centered phantom (Table 2). Finally, we performed dynamic acquisitions at 10- and 20 min
frames on centrally positioned NEMA IQ phantoms and dual
offset positioned phantoms in order to assess the impact of
reduced scan time on the same image quality metrics. 2.3.1 Assessment of Scatter Fraction, Count Losses,
Random Coincidences and NECR The system was evaluated according to Section 4 of the NEMA
NU4-2008 standard [4]. A polyethylene cylindrical phantom
70 mm long and 25 mm in diameter was manufactured and a
3.2 mm hole was drilled 10 mm off center to fit a capillary tube
made of Borosilicate glass (World Precision Instruments, 1B200-4). The capillaries had an outer diameter 2.0 mm and inner 1.12 mm. We addressed the issue by appropriately modifying the IO
component of the STIR image reconstruction library [23] to read
the .tru files and export projection data. Besides the new input
function, the issue of the multiple detector layers, which are not
currently supported by STIR, was addressed by means of simple January 2021 | Volume 8 | Article 531662 Frontiers in Physics | www.frontiersin.org 3 Multiple Animal Image Quality SuperArgus2R Efthimiou et al. default settings recommended by the manufacturer. No post-
filtering was applied in the reconstructed images. The
reconstruction
was
performed
with
all
available
corrections applied (randoms, scatter and attenuation) and
included normalization. The reconstructed images had 175 ×
175 voxels, with 64 slices for acquisitions with a single bed
position and 116 for two bed positions. The voxel size was 0.39 ×
0.39 × 0.775 mm3. CT reconstruction was performed using the
manufacturer’s filtered back-projection algorithm. geometrical translations in order to assign the events on a single
cylindrical model. default settings recommended by the manufacturer. No post-
filtering was applied in the reconstructed images. Finally, as suggested in the NEMA protocol, Single Slice
Rebinning was performed on the projection data using the
tool provided in STIR (Figure 1A). The sum of all bins in the
sinograms we generated was equal to the sum of events in the
.tru file. The
reconstruction
was
performed
with
all
available
corrections applied (randoms, scatter and attenuation) and
included normalization. The reconstructed images had 175 ×
175 voxels, with 64 slices for acquisitions with a single bed
position and 116 for two bed positions. The voxel size was 0.39 ×
0.39 × 0.775 mm3. CT reconstruction was performed using the
manufacturer’s filtered back-projection algorithm. 3.3 NEMA NU4 Image Quality Phantom
3.3.1 Static Phantom Acquisition
3.3.1.1 Uniformity The uniformity, (%STD) at the center of the FOV, offset position
and dual scanning, for initial activities 2.5- and 10 MBq, is January 2021 | Volume 8 | Article 531662 Frontiers in Physics | www.frontiersin.org 4 Multiple Animal Image Quality SuperArgus2R Efthimiou et al. FIGURE 2 | Prompt, Trues, Random Scatter rates and NECR for the NEMA scatter phantom for (A) Experimental measurements at the central bed position. (B)
Simulated central bed position. (C) Simulated off center, and (D) dual phantom scanning. The linear fit to the Prompts was performed for the first five points. FIGURE 2 | Prompt, Trues, Random Scatter rates and NECR for the NEMA scatter phantom for (A) Experimental measurements at the central bed position. (B)
Simulated central bed position. (C) Simulated off center, and (D) dual phantom scanning. The linear fit to the Prompts was performed for the first five points. TABLE 1 | Measured and simulated trues, scattered and randoms fraction for two activities. Activity (MBq)
Trues ratio
Scattered fraction
Random ratio
NECR
Sim (%)
Real (%)
Sim (%)
Real (%)
Sim (%)
Real (%)
Sim (%)
Real (%)
10
69.45
73.22
20.9
19.65
4.00
6.78
67.46
61.9
20
64.76
68.12
19.35
18.26
11.7
13.81
97.2
95.6 presented in Figure 3A. In Figure 3J, we present the
corresponding CT images. not as apparent in the dual phantom cases. Interestingly, when
OSEM 3D reconstruction was employed, the artefact was more
pronounced (see Supplementary Data). For both low (2.5 MBq) and high (10 MBq) acquisitions, the %
STD increased when the phantom was placed at the offset
position or when dual phantoms were scanned, compared to
the single centered phantom case. Overall, %STD was higher for
the low activity acquisitions (11–12%) vs those of higher activity
(6–8%). Dual phantom acquisition returns 13% higher noise
compared to single phantom acquisitions. We observe that
line profiles placed over the uniform region of the NEMA IQ
phantom and fitted with a polynomial (Figure 4) demonstrate
asymmetrical fits for offset phantoms, with higher apparent
activities closer to the scanner gantry. This phenomenon is
not seen in the single centered phantom acquisitions and is Phantom positioning on the left- or right-hand side of the
scanner had little impact on uniformity. Similarly, placement of
the air chamber facing inwards or outwards had little effect
(Figure 3D, corresponding images Figure 3K). Frontiers in Physics | www.frontiersin.org 3.3 NEMA NU4 Image Quality Phantom
3.3.1 Static Phantom Acquisition
3.3.1.1 Uniformity Likewise, uniformity is comparable between dual phantom
acquisitions with the NEMA IQ phantom placed on the left or
right side (Figure 3G). Doubling the scan time from 10 to 20 min reduces %STD by
38.1 and 29.5% in single centered phantom acquisition for 2.5-
and 10 MBq, respectively (Figures 5A,D). Increasing the
scanning time from 10- to 20 min for dual phantom scanning January 2021 | Volume 8 | Article 531662 5 Efthimiou et al. Multiple Animal Image Quality SuperArgus2R FIGURE 3 | Assessment of NEMA NU4 Image Quality for the single centered, single offset and dual phantom cases. (A–C) Uniformity, SOR and Recovery
Coefficients for the centered, offset and dual phantom positions indicated in (J). (D–F) Uniformity, SOR and Recovery Coefficients for the single left or right offset position
with the air chamber facing the center or edge of the field of view as indicated in (K). (G–I) Uniformity, SOR and Recovery Coefficients for the dual phantom configuration,
with the NEMA IQ phantom in the left or right position as indicated in (L). (M) Variable orientation of rods in NEMA NU4 phantom. Error bars: standard deviation. FIGURE 3 | Assessment of NEMA NU4 Image Quality for the single centered, single offset and dual phantom cases. (A–C) Uniformity, SOR and Recovery
Coefficients for the centered, offset and dual phantom positions indicated in (J). (D–F) Uniformity, SOR and Recovery Coefficients for the single left or right offset position
with the air chamber facing the center or edge of the field of view as indicated in (K). (G–I) Uniformity, SOR and Recovery Coefficients for the dual phantom configuration,
with the NEMA IQ phantom in the left or right position as indicated in (L). (M) Variable orientation of rods in NEMA NU4 phantom. Error bars: standard deviation. reduces the %STD of the uniformity by 34.4 and 26.0% for 2.5-
and 10 MBq, respectively. Recovery Coefficients remained generally consistent when the
offset phantom was positioned on the left and right side of the
scanner FOV (Figure 3F). Left side, air chamber facing outwards
continued to show RC greater than 1.0 for 3- and 5 mm rods at
10 MBq, as did the 3 mm rod on the right side phantom, air
chamber facing outwards at 2.5 MBq, but to a smaller extent. 3.3 NEMA NU4 Image Quality Phantom
3.3.1 Static Phantom Acquisition
3.3.1.1 Uniformity Interestingly, left side, air chamber facing inwards show a normal
expected range of RC’s at 10 MBq, albeit with a comparatively
reduced 2 mm RC. No particular observations could be made
between 2.5- and 10 MBq acquisitions, except that the 2 mm RC
appeared to be poorer at 10 MBq, excluding the aforementioned
left side, air out acquisitions. However, these observations could
be equally explained by the variability in the exact positioning of
the rods. Adding a second activity source, as is the case for dual
phantom scanning, increases the %STD 13.1 and 16.2%
compared a single centered phantom at 2.5 MBq for both 10-
and 20 min acquisition times, respectively. In the case of 10 MBq
acquisitions, dual phantom scanning increases the %STD
uniformity by 22.8 and 26.2% for 10- and 20 min acquisitions,
respectively. Frontiers in Physics | www.frontiersin.org 3.3.1.2 Recovery Coefficients The mean values and %STD for the different phantom positions
are presented in Figure 3C. The central slices of the rods taken
from the 10 MBq acquisitions are shown in Figure 3M. With the exception of the 10 MBq offset scan, acquisitions
performed on phantoms positioned both centrally and offset
return comparable data on recovery coefficients. In the case of
the 10 MBq offset data, both the 3- and 5 mm diameter rods
return recovery coefficients greater than 1. On the other hand,
both offset acquisitions performed at 2.5- and 10 MBq show a
more favourable recovery coefficient for the 1 mm compared to
the centrally positioned scans. Finally, the dual phantom
acquisitions show progressively poorer RCs with reducing rod
size, with a reduction of 60 and 24% for the 2- and 1 mm rods,
respectively, compared to the single offset acquisitions. When the NEMA IQ phantom is positioned at the right-hand
side for dual phantom scanning, the 3 mm rod’s RC is poorer at
both 2.5- and 10 MBq (Figure 3I). No other noteworthy
difference can be seen between the RCs of the dual phantom
acquisitions when the NEMA IQ phantom is position at either the
left- or right-hand side. Increasing the scan time from 10- to 20 min makes little
difference to the overall mean values of the RC, with none of
the mean values changing greater than 5.01% across all
acquisitions,
regardless
of
activity
present
and
phantom
orientation
between
single
centered
and
dual
phantom Frontiers in Physics | www.frontiersin.org January 2021 | Volume 8 | Article 531662 6 Efthimiou et al. Multiple Animal Image Quality SuperArgus2R FIGURE 4 | Assessment of uniform region asymmetry for the NEMA IQ phantom in the single centered (A, D), offset (B, E) and dual (C, E) cases. Yellow lines
indicate selected region. Adjacent graphs indicate line profiles fitted with a polynomial function. FIGURE 4 | Assessment of uniform region asymmetry for the NEMA IQ phantom in the single centered (A, D), offset (B, E) and dual (C, E) cases. Yellow lines
indicate selected region. Adjacent graphs indicate line profiles fitted with a polynomial function. 4.1 Simulation of Multiple Phantoms 4.1 Simulation of Multiple Phantoms
Both the measured and simulated prompt counts are in good
agreement with Kehl et al. [24] and Zagni et al. [19] however, the
latter study reports a marginally higher NECR. We suggest that
this difference is due to the data processing method, as our results
are produced by processing the raw sinograms, as suggested by
the NEMA protocol. Other groups have reported the scanners’
counting performance [14, 25], however those studies used non-
standard phantoms, making direct comparison difficult. 4 DISCUSSION (Figures 5C,F). The %STD of the RC, however, reduced by 46.4,
39.3, 50.7, 52.6 and 31.8% in the 20 min scan as compared to the
10 min scan for 1-, 2-, 3-, 4- and 5 mm rods, respectively. 3.3.1.3 Spill-Over Ratio The spill-over ratios for the air and water chambers in the NEMA
IQ
phantoms
positioned
centrally,
offset
and
offset
in
combination
with
a
second
phantom
are
presented
in
Figure 3B. At central positions, the SOR in the water chamber
is 27% higher than the air chamber at both 2.5- and 10 MBq. The
SOR for both chambers increases when dual phantom data is
acquired. Compared to the centrally positioned phantom, the
chamber orientation data from single offset scans show that the
SOR in water increases by 15% when the water chamber is closest
to the edge of the bore yet reduces by 5% when the water is closer
to the center (Figure 3E). Whereas in the same data, the SOR in
air remains largely unchanged regardless of orientation. When
the same data is compared between the left- and right-hand side
of the bore, we see that the air chamber has a slight increase in
SOR on the right side than that of the left side, whereas the water
chamber remains largely unchanged. It is noticeable from Figures
3B,E that the activity levels make little difference, with 10 MBq
acquisitions increasing the SOR only slightly in each case. The offset and dual phantom simulations do not result in a
marked increase in randoms and NECR when compared to the
single centred phantom for both 10 to 20 MBq (Figure 2). However, in the simulated studies the scatter fraction increases
for both the off-centre and dual phantom compared to the single
phantom case at both activity levels (Figure 2; Table 2). Scatter
fractions for the single phantom (19.7%) were in line with
literature values (21%), [3] and are relatively high, due to
either the small size of the gantry or the increased within-
detector scattering resulting from the phoswitch design [26]. It
is not possible to assess the effect of scatter on image quality
directly as this will depend on the implemented corrections, January 2021 | Volume 8 | Article 531662 Frontiers in Physics | www.frontiersin.org 7 Efthimiou et al. Multiple Animal Image Quality SuperArgus2R FIGURE 5 | Assessment of NEMA NU4 Image Quality for the 10 and 20 min summed frames for single centered and dual phantom cases. (A–C) Uniformity, SOR
and Recovery Coefficients for the centered and dual phantom positions, 10 min summed frame. 3.3.1.3 Spill-Over Ratio (D–F) Uniformity, SOR and Recovery Coefficients for the single or dual
phantom positions, 20 min summed frame. Error bars standard deviation. FIGURE 5 | Assessment of NEMA NU4 Image Quality for the 10 and 20 min summed frames for single centered and dual phantom cases. (A–C) Uniformity, SOR
and Recovery Coefficients for the centered and dual phantom positions, 10 min summed frame. (D–F) Uniformity, SOR and Recovery Coefficients for the single or dual
phantom positions, 20 min summed frame. Error bars standard deviation. FIGURE 5 | Assessment of NEMA NU4 Image Quality for the 10 and 20 min summed frames for single centered and dual phantom cases. (A–C) Uniformity, SOR
and Recovery Coefficients for the centered and dual phantom positions, 10 min summed frame. (D–F) Uniformity, SOR and Recovery Coefficients for the single or dual
phantom positions, 20 min summed frame. Error bars standard deviation. studies, although our reported modest increase in random events
associated with higher activities suggests that imaging with higher
activities would have a relatively small impact to image quality. though these could be assessed with simulation studies Given the
high density of the Delrin gantry cover (1.41 g/cm3), our findings
suggest that replacing the Delrin tube with a lower density
material or removing altogether would significantly improve
the scatter fraction. This simple modification could improve
the NECR of the scanner and thereby provide better image
quality at higher activity levels. Frontiers in Physics | www.frontiersin.org 4.3 Recovery Coefficients Recovery coefficients generally decreased for the dual case, as
reported previously. The variation that is sometimes seen in RCs
(e.g., the 10 MBq offset case) may be explained by the placement
of the 1 mm rod being geometrically closer to the center of the
FOV in case of the offset acquisitions as the exact position of rod
geometry was not factored in our acquisition protocol (see
Figure 3M). No notable difference was observed in SOR between 10 and
20 min frames at both high and low activities, and between single
centered and dual phantom scanning. As Greenwood et al. [12] demonstrated, increasing the
number of iterations during reconstruction may improve the
SOR values. However, this may come at the expense of noise
amplification, as shown by Gaitanis et al. [34]. The above, has not
been assessed here. Similar behavior has been observed and reported on by Aide
et al. [32] and Siepel et al. [9], who attributed this to the combined
effect of ‘over-estimation of scatter correction plus the non-
negativity
constraint
in
the
image
reconstruction’. Furthermore, Greenwood et al. [12] showed that the smaller,
1 mm diameter rod was not discernible when scanning four
phantoms simultaneously, which stands by our observations
that RC diminishes as more sources of activity are introduced
into the FOV. When comparing simulated phantom data to NEMA IQ
acquisition, it is worth noting that the SuperArgus2R has an
axial FOV smaller than that of the NEMA phantom (47 vs. 50 mm), and thus NEMA IQ acquisitions used multiple bed
positions. However, data on scatter fraction and NECR were
acquired using effectively a single bed position as the NEMA
protocol does not specify a similar setting for this case. It should also be noted that the NEMA protocol derives the
%STD from the standard deviations of the line profiles along axial
directions
and
the
%STDuni
assuming
Gaussian
error
propagation. However,
this
is
not
the
correct
standard
deviation of the RC as the %STD of the maximum value of a
randomly distributed value is not the standard deviation of the
underlying distribution [33]. This results in an artificial increase
of RC for smaller sources. Image quality is also dependent on energy discrimination, and
our studies used the 250–700 keV setting as used in other studies
[3] and suggested to provide a good balance between counting
performance and scatter rejection [35]. 4.2 Uniformity Our results indicate a higher level of noise for the single phantom
case than previously reported [3]. However, this data was
acquired using a single bed position and with a different
activity (3.7 vs. 2.5 MBq). It has been previously reported that
multiple bed positions increase variability [31]. It has previously been proposed that the activity in the FOV at
the time of acquisition should be approximately 90 −95% of the
NECR peak in order to improve image standardization [27]. In
the case of the scanner being reviewed in this paper this
corresponds to 18–20 MBq. Although, NECR has a valuable
role in quantifying the statistical quality of the PET data, its value
as a metric of image quality, with iterative reconstruction, is
limited [28]. When more than one or off-centre sources of activity are
present in the scanner, our findings suggest that the Sedecal Super
Argus 2R scanner performs favourably compared to similar
studies used other scanners. Siepel et al. [9] reported an 18.4%
rise in %STD of the uniformity from a single, centrally positioned
phantom, to dual offset acquisition, similar to our study. However, a 50 mL centrifuge tube was used compared to our
20 mL syringe, which was chosen to more accurately reflect the
volume of a mouse. Reilhac et al. [11] reported a higher increase
of 43% in the %STD of the uniformity when scanning a Derenzo The image quality strongly depends on the many parameters
of the reconstruction algorithm [29]. An increase in starting
activity would allow for shorter imaging times, however this may
result in an increase in absorbed dose to the animal above the
threshold for biological effects [30], an issue for longitudinal January 2021 | Volume 8 | Article 531662 8 TABLE 2 | Scatter fraction for simulated off-centre and dual phantom scanning. MBq
Single centre (actual)
Single centre (simulated)
Scatter faction
Single offset (simulated)
Dual (simulated)
10
19.65%
18.26%
22.52%
23.71% (21.36% @ 20 MBq total)
20
20.9%
19.35%
20.42%
21.36% (18.53% @ 40 MBq total)
Efthimiou et al. Multiple Animal Image Quality SuperArgus2R Multiple Animal Image Quality SuperArgus2R Efthimiou et al. Efthimiou et al. TABLE 2 | Scatter fraction for simulated off-centre and dual phantom scanning. 4.3 Recovery Coefficients The primary application of preclinical PET at the University of
Hull PET Research center is to assess the pharmacokinetics and
biodistribution of novel imaging probes [36–39], for this
application the degradation in image quality predicted for the
two animal case is acceptable. For the application of existing
tracers to provide physiological readouts in disease models, our
data suggest that higher injected doses provide better image
quality overall if dual animal scanning is warranted for
logistical reasons [40]. The phoswich detector arrangement inherently provides DOI
information, allowing for an improved and more uniform spatial
resolution in the images [17]. This detector configuration should
naturally reduce
the spatial resolution degradation when
scanning dual objects, allowing for reconstruction methods
without Point Spread Function (PSF) correction to perform
well under these conditions. This is in contrast to the reported
performance of OSEM 2D reconstruction for the Inveon [9, 32]. Our data shows that RC values do not significantly change
between 10- and 20 min imaging frames. However, as expected,
the %STD of the RCs is markedly higher on the shorter imaging
time frames regardless of activity or scan orientation. The phoswich detector arrangement inherently provides DOI
information, allowing for an improved and more uniform spatial
resolution in the images [17]. This detector configuration should
naturally reduce
the spatial resolution degradation when
scanning dual objects, allowing for reconstruction methods
without Point Spread Function (PSF) correction to perform
well under these conditions. This is in contrast to the reported
performance of OSEM 2D reconstruction for the Inveon [9, 32]. Our data shows that RC values do not significantly change
between 10- and 20 min imaging frames. However, as expected,
the %STD of the RCs is markedly higher on the shorter imaging
time frames regardless of activity or scan orientation. 4.2 Uniformity MBq
Single centre (actual)
Single centre (simulated)
Scatter faction
Single offset (simulated)
Dual (simulated)
10
19.65%
18.26%
22.52%
23.71% (21.36% @ 20 MBq total)
20
20.9%
19.35%
20.42%
21.36% (18.53% @ 40 MBq total) could be a result either of limitations of the scatter correction
algorithm, or by photons scattered by the Delrin tube, in close
proximity at the edge of the gantry. phantom alongside a NEMA IQ phantom. Finally, Greenwood
et al. [12] reported a decrease in uniformity (an increase of 55% in
%STD) when assessing four phantoms simultaneously vs the
single centered case. It may be expected to see such a significant
increase in %STD when there are four sources of activity present,
which was not assessed in our study. When scanning two subjects, the SOR increases by 17 and 12%
for water and air, respectively. Siepel et al. [9] reported an approx. 32% increase in the SOR values for water and approx. 40% for air,
albeit using a larger non-NEMA phantom in a scanner with a
wider bore. The position dependency that we show for the single
offset subject was not apparent in the dual case. As the SOR did
not increase markedly between 2.5 and 10 MBq in the single
centered case this suggests the effect is not due to the increased
activity in the FOV. 5 CONCLUSION In this study, we present the impact of dual subject scanning on
the PET signal and image quality using the Sedecal SuperArgus
2R preclinical scanner. In brief, our experiments demonstrate a
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to help found the PET Research Center at the University of Hull
and for their continued support. We acknowledge the Viper High
Performance Computing facility of the University of Hull and its
support team. The authors would like to thank D. P. Roberts (PET
Research Center, Univ. of Hull) for his valuable support and
training. The datasets generated for this study are available upon
reasonable request to the corresponding authors. AUTHOR CONTRIBUTIONS NE and JW contributed equally to this work and share first
authorship. LC performed the scans of the NEMA scatter
phantom for the characterisation of the scanner. NE and LC
processed the raw data acquired from the scanner, and did the
image analysis for the image quality phantom. NE and PC
performed the GATE simulations, validated the simulations to
the
measured
data,
and
performed
the
Monte
Carlo 4.4 Spillover Ratios Our data indicated a dependence of SOR on gantry proximity. The apparent increase on the water container in the offset case January 2021 | Volume 8 | Article 531662 Frontiers in Physics | www.frontiersin.org 9 Multiple Animal Image Quality SuperArgus2R Efthimiou et al. cases, however of less magnitude than similar decreases reported
for other scanners. investigation. JW and IR performed the acquisition and
analysis of the NEMA IQ phantom. FZ developed the
original GATE Monte Carlo simulation model for the Argus
scanner and advised in the processing of the data acquired
from the scanner. CC contributed to the conception and design
of the study. CC and SA are the grant holders and were
involved in the organization and planning of the work and
discussions on the results. All authors contributed in the
drafting of the paper. We conclude that the Sedecal AuperArgus 2R preclinical
scanner is, therefore,
suitable for dual animal scanning,
particularly in biodistribution studies of novel radiotracers - as
is a key operational objective of our facility. Regarding
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dose should be carefully considered to ensure that image quality is
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1026–36. doi:10.1118/1.2558115 Copyright © 2021 Efthimiou, Wright, Clayton, Renard, Zagni, Caribé, Archibald
and Cawthorne. 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
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https://openalex.org/W2932046773
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https://europepmc.org/articles/pmc6524104?pdf=render
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English
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Incidence of postoperative complications in transabdominal preperitoneal repair for groin hernia is influenced by poor performance status rather than by old age
|
Annals of gastroenterological surgery
| 2,019
|
cc-by
| 5,312
|
O R I G I N A L A R T I C L E O R I G I N A L A R T I C L E Abstract Aim: The present study was designed to evaluate the safety and feasibility of transab-
dominal preperitoneal (TAPP) repair for very old patients with groin hernia and to
identify the risk factors predicting perioperative complications. Correspondence
Hirokazu Noshiro, Department of Surgery,
Faculty of Medicine, Saga University, Saga,
Japan. Email: noshiro@cc.saga-u.ac.jp Methods: A total of 140 patients treated by TAPP were reviewed retrospectively. They were divided into two groups: patients ≥80 years of age (≥80 years group;
n = 26) and those <80 years of age (<80 years group; n = 114). Patient characteris-
tics and surgical outcomes were then statistically compared between the two
groups. Results: Number of patients with any comorbidities was significantly higher in the
≥80 years group than in the <80 years group (96.2% vs 61.4%, P = 0.003). There were
no significant differences in surgical outcomes between the two groups. In the uni-
variate analysis of perioperative complications, poor performance status (PS)
(P = 0.014), lower hemoglobin level (P = 0.038) and lower albumin level (P = 0.016)
were significantly associated with the occurrence of postoperative complications,
and multivariate analysis showed that only poor PS was an independent factor (PS
0-2 vs 3-4: P = 0.034, OR 5.192 [95% CI; 1.137 to 23.71]). Conclusions: This is the first report to show that the incidence of postoperative com-
plications in TAPP repair for groin hernia is influenced by poor PS rather than old age. TAPP can be a safe surgical procedure for very old patients with a good PS, with
benefits that are equal to those in young patients. 318 |
Ann Gastroenterol Surg. 2019;3:318–324. www.AGSjournal.com
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. © 2019 The Authors. Annals of Gastroenterological Surgery published by John Wiley & Sons Australia, Ltd on behalf of The Japanese Society of
Gastroenterological Surgery
1 | INTRODUCTION
Laparoscopic repair of groin hernia by the totally extraperito-
neal (TEP) or transabdominal preperitoneal (TAPP) approach is an
established surgical method for treating groin hernia and has been
carried out widely in recent years.1,2 Previous studies have reported
that laparoscopic hernia surgery (LHS) results in a lower incidence of
wound infection, chronic pain and numbness and an earlier return Noriyuki Egawa
| Jun Nakamura | Tatsuya Manabe | Hironori Iwasaki |
Hirokazu Noshiro Noriyuki Egawa
| Jun Nakamura | Tatsuya Manabe | Hironori Iwasaki |
Hirokazu Noshiro Department of Surgery, Faculty of
Medicine, Saga University, Saga, Japan Department of Surgery, Faculty of
Medicine, Saga University, Saga, Japan Department of Surgery, Faculty of
Medicine, Saga University, Saga, Japan Ann Gastroenterol Surg. 2019;3:318–324. Received: 27 November 2018 | Revised: 18 February 2019 | Accepted: 10 March 2019
DOI: 10.1002/ags3.12247 Received: 27 November 2018 | Revised: 18 February 2019 | Accepted: 10 March 2019
DOI: 10.1002/ags3.12247 Received: 27 November 2018 | Revised: 18 February 2019 | Accepted: 10 March 2019 Received: 27 November 2018 | Revised: 18 February 2019 | Accepted: 10 March 2019
DOI: 10.1002/ags3.12247 to work than open hernia surgery (OHS).3–5 However, LHS requires
general anesthesia and a longer operation time than OHS. In addi-
tion, LHS has a higher incidence of postoperative complications than
OHS, and TAPP, in particular, seems to be associated with serious
complications, such as port-site hernia and visceral injury.3,6,7 Based
on these findings, current guidelines recommend LHS as an option
for the treatment of primary unilateral groin hernia when carried out
by a sufficiently experienced surgeon.8,9 were as follows: contraindication for general anesthesia as a result of
severe comorbidities (n = 4); severe intra-abdominal adhesion as a re-
sult of multiple laparotomies (n = 6); and emergency cases as a result
of intestinal obstruction and/or necrosis (n = 10). Furthermore, among
143 patients who underwent TAPP, three who received additional sur-
gery at the same time were excepted. Therefore, this study retrospec-
tively reviewed 140 consecutive groin hernia patients who received
TAPP. These 140 patients were divided into two groups: patients
≥80 years of age (≥80 years group; n = 26) and those <80 years of age
(<80 years group; n = 114) (Figure 1). Patient characteristics and surgi-
cal outcomes including operation time, blood loss, hospital stay and
postoperative complications were statistically compared between the
two groups. Details of comorbidity are as follows: pulmonary dysfunc-
tion was defined as % vital capacity (%VC) ≤80% or forced expiratory
volume in 1-second (FEV1.0%) ≤70%; renal dysfunction was defined
as estimated glomerular filtration rate (eGFR) ≤60 mL/min. Circulatory
disease included myocardial infarction, arrhythmia and valvular heart
disease. Postoperative complications were classified according to the
Clavien-Dindo classification of surgical complications,19 and complica-
tions classified as grade 2 or higher were reviewed. Because the incidence of groin hernia is higher in the elderly
than in younger individuals due to the weakening of tissue with
age,1,10–12 the number of groin hernia surgeries for elderly patients
is expected to increase as the population progressively ages in de-
veloped countries. Furthermore, postoperative morbidity and mor-
tality rates in major surgery are also higher in elderly patients than
in young patients.13–15 Elderly patients often receive many medica-
tions and have multiple comorbidities and histories of laparotomy
that can hamper laparoscopic surgery. Therefore, older age may be
a risk factor predicting postoperative complications in LHS. 2.1 Data analyses were carried out using JMP Pro 13 (SAS Institute
Inc., Cary, NC, USA) statistical software. All data are expressed
as mean ± standard deviation. Fisher's exact test or a two-tailed
Student's t-test was used to evaluate group differences. Variables
with P values ≤0.05 on univariate analysis were included in a
multivariate logistic regression model. All P values <0.05 were
accepted as statistically significant. The purpose of the present study was to evaluate the safety and
feasibility of TAPP repair for very old patients with groin hernia and
to identify the risk factors predicting postoperative complications in
this population. 2 | PATIENTS AND METHODS From September 2011 to May 2016, 163 patients underwent groin
hernia repair at Saga University Hospital. During this period, almost all
groin hernia patients were treated by TAPP repair when the patient's
condition allowed for laparoscopic surgery under general anesthesia. Among 163 groin hernia patients, 20 were treated by OHS, whereas
the remaining 143 were treated by TAPP. Selection criteria for OHS Several
studies have reported comparable outcomes between LHS and OHS
in elderly patients;16–18 however, whether older age influences the
incidence of postoperative complications in LHS has been unclear. Therefore, objective parameters regarding the selection of laparo-
scopic surgery for elderly groin hernia patients are needed. 1 | INTRODUCTION 318 |
Ann Gastroenterol Surg. 2019;3:318–324. www.AGSjournal.com
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. © 2019 The Authors. Annals of Gastroenterological Surgery published by John Wiley & Sons Australia, Ltd on behalf of The Japanese Society of
Gastroenterological Surgery
1 | INTRODUCTION
Laparoscopic repair of groin hernia by the totally extraperito-
neal (TEP) or transabdominal preperitoneal (TAPP) approach is an
established surgical method for treating groin hernia and has been
carried out widely in recent years.1,2 Previous studies have reported
that laparoscopic hernia surgery (LHS) results in a lower incidence of
wound infection, chronic pain and numbness and an earlier return Ann Gastroenterol Surg. 2019;3:318–324. 319 EGAWA et al. and steroid therapy were observed between the two groups. The
proportion of patients with any comorbidities (96.2% vs 61.4%,
P = 0.003), especially those having renal dysfunction (57.8%
vs 20.2%, P = 0.003), pulmonary dysfunction (%VC ≤80% or
FEV1.0% ≤70%) (53.9% vs 15.8%, P < 0.001), and cerebrovascular
infarction (23.1% vs 3.5%, P = 0.003) was significantly higher in
the ≥80 years group than in the <80 years group. It is noteworthy
that over 95% of the patients in the ≥80 years group had some
comorbidities. The proportion of patients with comorbidities of
circulatory disease, liver disease, diabetes, and dementia did not
differ to a statistically significant extent. who received TAPP. Mean age was 83.6 ± 3.1 years old in the
≥80 years group and 64.3 ± 13.4 years old in the <80 years
group (P < 0.001). Body mass index (BMI), American Society
of Anesthesiologists (ASA) class, performance status (PS),
type of groin hernia and size of hernia orifice were similar be-
tween the two groups. Lower hemoglobin (Hb) (12.8 ± 1.6 mg/
dL vs 13.7 ± 2.7 mg/dL, P = 0.038) and albumin (Alb) levels
(3.7 ± 0.4 mg/dL vs 4.0 ± 0.5 mg/dL, P = 0.016) were observed
in the ≥80 years group. No significant differences regarding the
proportions of emergency operations, recurrent cases and the
histories of laparotomy, prostatectomy, antithrombotic therapy who received TAPP. Mean age was 83.6 ± 3.1 years old in the
≥80 years group and 64.3 ± 13.4 years old in the <80 years
group (P < 0.001). Body mass index (BMI), American Society
of Anesthesiologists (ASA) class, performance status (PS),
type of groin hernia and size of hernia orifice were similar be-
tween the two groups. Lower hemoglobin (Hb) (12.8 ± 1.6 mg/
dL vs 13.7 ± 2.7 mg/dL, P = 0.038) and albumin (Alb) levels
(3.7 ± 0.4 mg/dL vs 4.0 ± 0.5 mg/dL, P = 0.016) were observed
in the ≥80 years group. p
p
aIncluding bilateral cases. Described according to European Hernia Society classification. bIn bilateral cases, the larger size of defect was listed. Board Certified Surgeon in Gastroenterology.
c Excluding female cases.
eBoard-certified surgeon in gastroenterology of the Japanese Society of Gastroenterological Surgery. No significant differences regarding the
proportions of emergency operations, recurrent cases and the
histories of laparotomy, prostatectomy, antithrombotic therapy TA B LE 1 Characteristics of TAPP repair patients
≥80 y group (n = 26)
<80 y group (n = 114)
P value
Gender, male/female
22/4
98/16
0.766
Age (y, mean ± SD)
83.6 ± 3.1
64.3 ± 13.4
<0.001
BMI (kg/m2, mean ± SD)
21.6 ± 2.7
22.5 ± 2.7
0.101
Performance status (0-2/3-4)
23/3
109/5
0.167
ASA classification (1-2/3-4)
25/1
111/3
0.565
Blood test
Hb (mg/dL, mean ± SD)
12.8 ± 1.6
13.7 ± 2.7
0.038
Alb (mg/dL, mean ± SD)
3.7 ± 0.4
4.0 ± 0.5
0.016
Diseased side (unilateral/bilateral)
25/1
100/14
0.304
Type of groin herniaa (lateral/
medial/femoral/combined)
21/5/1/0
93/28/2/5
0.635
Size of hernia orificeb
(<3 cm/≥3 cm)
20/6
98/16
0.392
Emergency operation, n (%)
2 (7.7)
2 (1.8)
0.157
Recurrent case, n (%)
3 (11.5)
11 (9.7)
0.724
History of other side inguinal
hernia repairc, n (%)
3 (12.0)
17 (17.0)
0.762
History of abdominal surgery, n (%)
14 (53.9)
41 (36.0)
0.12
History of prostatectomyd, n (%)
2 (9.1)
9 (9.2)
1.000
Antithrombotic therapy, n (%)
9 (34.6)
27 (23.7)
0.319
Steroid therapy, n (%)
0 (0.0)
4 (3.5)
1.000
Comorbidity, n (%)
25 (96.2)
70 (61.4)
0.003
Circulatory disease, n (%)
9 (34.6)
26 (22.8)
0.218
Renal dysfunction, n (%)
15 (57.7)
23 (20.2)
0.003
Pulmonary dysfunction, n (%)
14 (53.9)
18 (15.8)
<0.001
Liver disease, n (%)
2 (7.7)
12 (10.5)
1.000
Cerebral infarction, n (%)
6 (23.1)
4 (3.5)
0.003
Diabetes, n (%)
7 (26.9)
18 (15.8)
0.254
Dementia, n (%)
2 (7.7)
2 (1.8)
0.157
Surgeon (resident/experte)
17/9
64/50
0.385
Alb, albumin; ASA, American Society of Anesthesiologists; BMI, body mass index; Hb, hemoglobin; PS, performance status; TAPP, transabdominal
it
l cases. Described according to European Hernia Society classification. Alb, albumin; ASA, American Society of Anesthesiologists; BMI, body mass index; Hb, hemoglobin; PS, performa
preperitoneal. 3.1 | Patient characteristics Table 1 compares the characteristics between the ≥80 years
group (n = 26) and the <80 years group (n = 114) among patients FI G U R E 1 Study flow chart. OHS, open hernia surgery; TAPP,
transabdominal preperitoneal FI G U R E 1 Study flow chart. OHS, open hernia surgery; TAPP,
transabdominal preperitoneal 320 EGAWA et al. ize of defect was listed. Board Certified Surgeon in Gastroenterology. es.
on in gastroenterology of the Japanese Society of Gastroenterological Surgery. Excluding bilateral cases.
dExcluding female cases. 3.3 | Identification of risk factors predicting
perioperative complications of TAPP Univariate and multivariate analyses regarding the perioperative
complications of TAPP repair are shown in Table 3. In the univari-
ate analysis, poor PS was significantly associated with the occur-
rence of postoperative complications (P = 0.014). Furthermore,
Hb level (12.9 ± 1.9 mg/dL vs 13.7 ± 1.8 mg/dL, P = 0.036) and
Alb level (3.8 ± 0.4 mg/dL vs 4.0 ± 0.5 mg/dL, P = 0.035) were sig-
nificantly lower in the patients with complications. Perioperative
complications also tended to be more frequent among elderly
and male patients (≥80 years vs <80 years; P = 0.116, male vs
female; P = 0.076), although not to a statistically significant de-
gree. There were no correlations between the perioperative com-
plications and other factors, such as BMI (P = 0.511), ASA class
(P = 1.000), size of hernia orifice (P = 0.419), emergency opera-
tion (P = 1.000), history of laparotomy (P = 0.835), antithrombotic
therapy (P = 0.646), steroid therapy (P = 0.213) and presence of
comorbidities (P = 0.126). Incidence of perioperative complica-
tions in procedures carried out by residents and experts did not
differ to a statistically significant extent (P = 0.385). In addition,
multivariate analysis showed that only poor PS was an independ-
ent factor predicting the occurrence of perioperative complica-
tions (PS 0-2 vs 3-4: P = 0.034, odds ratio: 5.192 [95% confidence
interval: 1.137-23.71]). Rate of overall perioperative complications was similar between
the two groups (34.6% vs 19.3%, P = 0.116). were no significant differences in the rates of both intraoperative
complications (0% vs 0.9%, P = 1.000) and postoperative compli-
cations (34.6% vs 18.4%, P = 0.109). Although the incidence of he-
matoma was significantly higher in the ≥80 years group (11.5% vs
0%, P = 0.006), the incidences of other complications, such as se-
roma, wound infection and visceral injury, were similar between the
two groups. There were no mortalities in either group. Pain and/or
discomfort that lasted more than 1 month after surgery was more
frequent in the <80 years group, although not to a statistically sig-
nificant degree (0% vs 6.1%, P = 0.348). 3.2 | Surgical outcomes and perioperative
complications of TAPP Surgical outcomes and perioperative complications of TAPP repair
were compared between the ≥80 and <80 years groups (Table 2). There were no significant differences in the respective surgical
outcomes between the ≥80 and <80 years groups, including opera-
tion time (125.8 ± 40.4 minutes vs 137.5 ± 45.7 minutes, P = 0.230),
rate of conversion to open surgery (0% vs 0.9%, P = 1.000), blood
loss (0.9 ± 2.1 g vs 1.5 ± 4.5 g, P = 0.510) and postoperative hospi-
talization duration (3.8 ± 1.4 days vs 3.3 ± 1.6 days, P = 0.140). Of all
140 patients who underwent TAPP, only one case (0.7%) had to be
converted to open surgery. This patient was diagnosed with an ir-
reducible inguinal hernia and received TAPP electively; however, the
incarcerated omentum was unable to be returned into the abdomi-
nal cavity laparoscopically. Although the hernia defect was able to
be repaired laparoscopically, an additional incision at the groin was
needed to excise the strangulated omentum. 3.3 | Identification of risk factors predicting
perioperative complications of TAPP In addition, there TA B LE 2 Surgical outcomes and perioperative complications of
TAPP repair patients TA B LE 2 Surgical outcomes and perioperative complications of
TAPP repair patients
≥80 y group
(n = 26)
<80 y group
(n = 114)
P value
Operation time (min),
mean ±SD
125.8 ± 40.4
137.5 ± 45.7
0.230
Conversion to open
surgery, n (%)
0 (0)
1 (0.9)
1.000
Blood loss (g),
mean ± SD
0.9 ± 2.1
1.5 ± 4.5
0.510
Postoperative
hospitalization (d),
mean ± SD
3.8 ± 1.4
3.3 ± 1.6
0.140
Perioperative
complicationsa, n (%)
9 (34.6)
22 (19.3)
0.116
Intraoperative
complications, n (%)
0 (0)
1 (0.9)
1.000
Other organ injury
0 (0)
1 (0.9)
1.000
Postoperative
complications, n (%)
9 (34.6)
21 (18.4)
0.109
Seroma
5 (19.2)
19 (16.7)
0.775
Hematoma
3 (11.5)
0 (0)
0.006
Wound infection
0 (0)
0 (0)
–
Pneumonia
0 (0)
1 (0.9)
1.000
Cystitis
0 (0)
1 (0.9)
1.000
Other
1 (3.8)
0 (0)
0.186
Inguinal pain and/or
discomfortb
0 (0)
7 (6.1)
0.348
TAPP, transabdominal preperitoneal. aComplications grade 2 or higher according to the Clavien-Dindo
classification. bObservation at 1 mo after surgery. TA B LE 1 Characteristics of TAPP repair patients TA B LE 1 Characteristics of TAPP repair patients EGAWA et al. 321 bObservation at 1 mo after surgery. aComplications grade 2 or higher according to the Clavien-Dindo
classification. 4 | DISCUSSION Groin hernia repair is one of the most common surgical procedures
carried out worldwide.19 According to a population-based study by
Zendejas et al,20 the number of patients who undergo inguinal her-
nia repair increases with age, resulting in a cumulative incidence of
18.9% by age 70, 27.7% by age 80, 35.1% by age 90 and 42.5% for
the entire lifetime for men. Currently, individuals over the age of 80
years are not unusual in Japan, where individuals aged over 65 years
comprise 27.3% of the population.21 Some reports allowed for non-
surgical management (watchful waiting) of asymptomatic or mini-
mally symptomatic groin hernias in elderly patients.22–25 However,
it is generally considered that symptomatic groin hernias should
be treated by surgery to prevent incarceration and reduce mortal-
ity,8,22,23 and strangulated hernias should be urgently operated, 322 EGAWA et al. EGAWA et al. TA B LE 3 Univariate and multivariate analyses of perioperative complications in TAPP repair patients
Univariate analysis
Multivariate analysis
Complicationsa (+)
(n = 31)
Complications
(−) (n = 109)
P value
OR
95% CI
P value
Age (≥80 y/<80 y)
9/22
17/92
0.116
Gender (male/female)
30/1
90/19
0.076
BMI (kg/m2, mean ± SD)
22.1 ± 2.4
22.4 ± 2.8
0.511
Performance status (0-2/3-4)
26/5
106/3
0.014
5.192
1.137-23.71
0.034
ASA classification (1-2/3-4)
30/1
106/3
1.000
Blood test
Hb (mg/dL, mean ± SD)
12.9 ± 1.9
13.7 ± 1.8
0.036
1.130
0.864-1.479
0.371
Alb (mg/dL, mean ± SD)
3.8 ± 0.4
4.0 ± 0.5
0.035
1.821
0.604-5.493
0.287
Type of groin herniab (lateral/medial/
femoral/combined)
25/10/0/0
89/23/3/5
0.420
Diseased side (unilateral/bilateral)
27/4
98/11
0.742
Size of hernia orificec (<3 cm/≥3 cm)
24/7
92/17
0.419
Emergency operation (yes/no)
1/30
3/106
1.000
Recurrent case (yes/no)
3/28
11/98
1.000
History of laparotomy (yes/no)
13/18
42/67
0.835
Antithrombotic therapy (yes/no)
9/22
27/82
0.646
Steroid therapy (yes/no)
2/29
2/107
0.213
Comorbidity (yes/no)
25/6
70/39
0.126
Circulatory disease (yes/no)
9/22
26/83
0.639
Renal dysfunction (yes/no)
8/23
30/79
1.000
Pulmonary dysfunction (yes/no)
10/21
22/87
0.224
Liver disease (yes/no)
4/27
10/99
0.511
Cerebral infarction (yes/no)
2/29
8/101
1.000
Diabetes (yes/no)
4/27
21/88
0.596
Dementia (yes/no)
2/29
2/107
0.213
Surgeon, (resident/expertd)
20/11
61/48
0.392
Alb, albumin; ASA, American Society of Anesthesiologists; BMI, body mass index; Hb, hemoglobin; TAPP, transabdominal preperitoneal. 4 | DISCUSSION TA B LE 3 Univariate and multivariate analyses of perioperative complications in TAPP repair patients TA B LE 3 Univariate and multivariate analyses of perioperative complications in TAPP repair patients Alb, albumin; ASA, American Society of Anesthesiologists; BMI, body mass index; Hb, hemoglobin; TAPP, transabdominal preperitoneal. aComplications grade 2 or higher according to the Clavien-Dindo classification. and urinary retention in elderly patients compared with non-elderly
patients. Mayer et al also showed that the rate of perioperative com-
plications in LHS tends to increase with age, in particular from the
age of 80 years.12 However, to our knowledge, no studies have com-
pared the outcomes of TAPP repair between elderly and non-elderly
patients in consecutive cases of groin hernia. regardless of the patient's age. However, the current guidelines
make no recommendations regarding the optimum age-specific sur-
gical procedures for groin hernias. Previous studies have described similar surgical outcomes be-
tween LHS and OHS in patients over 80 years of age and concluded
that LHS is safe and can be an alternative procedure to OHS even
in very elderly patients.17,18 However, these two studies included a
very small number of patients who received LHS, and the selection
criteria for LHS or OHS were not described. Recently, Vigneswaran
et al evaluated the surgical outcomes of inguinal hernia repair in de-
tail for each age group.16 In their study, 380 of 471 patients were
treated by the TEP approach, and 13 of 380 patients who received
TEP were older than 80 years. The results suggested that patient-
reported outcomes may be better in LHS than in OHS, regardless of
a greater risk of minor postoperative complications such as seroma In the present study, despite the higher rate of underlying dis-
ease in elderly patients, there were no significant differences in
the surgical outcomes of TAPP repair between the elderly group
and the non-elderly group. Although postoperative hematoma was
more frequently observed in elderly patients, overall morbidity
was similar between the two groups as reported in the previous
studies. Low Hb and Alb levels were associated with periopera-
tive complications in univariate analyses; however, multivariate
analyses showed that only poor PS was an independent risk factor EGAWA et al. 323 that predicted perioperative complications. Other factors includ-
ing elderly age were not associated with perioperative complica-
tions. REFERENCES Chung RS, Rowland DY. Meta-analyses of randomized controlled
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Conze J, et al. European Hernia Society guidelines on the treatment
of inguinal hernia in adult patients. Hernia. 2009;13(4):343–403. The major limitations of the present study are its retrospective
design and small sample size. In addition, many surgeons from resi-
dent to expert carried out operations in this study. The preoperative
complication rate did not differ between procedures carried out by
residents and experts; however, experts may have carried out more
difficult operations such as hernia after total prostatectomy, recur-
rent hernia, and hernia of high-risk patients including the very elderly. 9. Bittner R, Arregui ME, Bisgaard T, Dudai M, Ferzli GS, Fitzgibbons
RJ, et al. Guidelines for laparoscopic (TAPP) and endoscopic (TEP)
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P, et al. Quality assessment of 26,304 herniorrhaphies in Denmark:
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a prospective nationwide study. Lancet. 2001;358(9288):1124–8. 3. Voyles CR, Hamilton BJ, Johnson WD, Kano N. Meta-analysis of
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of the open tension-free mesh-plug, transabdominal preperitoneal
(TAPP), and totally extraperitoneal (TEP) laparoscopic techniques
for primary unilateral inguinal hernia repair: a prospective random-
ized controlled trial. Surg Endosc. 2011;25(1):234–9. In the present study, there were no patients with chronic post-
operative inguinal pain requiring long-term follow up or drug treat-
ment. However, there was a tendency for young people to feel
postoperative inguinal pain or discomfort in the first month after
surgery, although there was no significant difference. Young age was
regarded as a risk factor for postoperative pain in previous reports;
however, these reports were mainly based on research on OHS.28,29
In reports on LHS, some studies noted that age did not have a sig-
nificant influence on postoperative inguinal pain;30,31 however, this
point remains controversial. 6. Chung RS, Rowland DY. Meta-analyses of randomized controlled
trials of laparoscopic vs conventional inguinal hernia repairs. Surg
Endosc. 1999;13(7):689–94. 6. ORCID Noriyuki Egawa
https://orcid.org/0000-0003-4229-6324 Generally, when TAPP is carried out for patients with groin her-
nia, general anesthesia and intraperitoneal manipulation become
major concerns. In other words, if general anesthesia is possible,
then it seems that TAPP can be carried out safely, regardless of the
patient's age. However, this study did not conclude that the surgi-
cal outcomes of TAPP for elderly patients were superior to those
of TAPP for young patients. Furthermore, it did not show that TAPP
was superior to OHS for elderly patients with groin hernia. To obtain
a consensus regarding the optimal surgical procedure for elderly pa-
tients and/or poor PS, comparison between LHS and OHS in such
patients will be needed. 4 | DISCUSSION These results suggest that TAPP can be one of the optimal
treatment options even for very old patients as well as for young
patients when the patient's condition allows LHS under general an-
esthesia and PS is 0 to 2. On the contrary, even for young patients,
TAPP may provide less advantage compared with OHS when the
patient's PS is poor. This can lead to the occurrence of periopera-
tive complications and delay the return to daily life. Generally, the
risk assessment of LHS was based on patient's age, underlying dis-
eases, type of hernia, size of hernia orifice, ASA class etc;12,16,26,27
however, no studies investigated the association between patient's
PS and the incidence of perioperative complications. This study for
the first time showed that poor PS is an independent predictive
factor of perioperative complications after TAPP. More careful pre-
operative assessment is needed to decide the surgical procedure
for poor PS patients. can be one of the optimal surgical procedures even for very elderly
patients over 80 years of age, as it is for young patients. Very elderly
patients with a good PS can enjoy the benefits of TAPP equally to
young people. DISCLOSURE This study was carried out in agreement with the guidelines of the
institutional ethics committee and was conducted in accordance
with the Declaration of Helsinki. Conflicts of Interest: Authors declare no conflicts of interest for this
article. Conflicts of Interest: Authors declare no conflicts of interest for this
article. in minimally symptomatic men: a randomized clinical trial. JAMA. 2006;295(3):285–92. 14. Hamel MB, Henderson WG, Khuri SF, Daley J. Surgical outcomes for
patients aged 80 and older: morbidity and mortality from major non-
cardiac surgery. J Am Geriatr Soc. 2005;53(3):424–9. 26. Muschalla F, Schwarz J, Bittner R. Effectivity of laparoscopic ingui-
nal hernia repair (TAPP) in daily clinical practice: early and long-term
result. Surg Endosc. 2016;30(11):4985–94. 15. Manku K, Bacchetti P, Leung JM. Prognostic significance of postop-
erative in-hospital complications in elderly patients. I. Long-term sur-
vival. Anesth Analg. 2003;96(2):583–9. 27. Jacob DA, Hackl JA, Bittner R, Kraft B, Köckerling F. Perioperative
outcome of unilateral versus bilateral inguinal hernia repairs in
TAPP technique: analysis of 15,176 cases from the Herniamed
Registry. Surg Endosc. 2015;29(12):3733–40. 16. Vigneswaran Y, Gitelis M, Lapin B, Denham W, Linn J, Carbray J,
et al. Elderly and octogenarian cohort: comparable outcomes with
nonelderly cohort after open or laparoscopic inguinal hernia repairs. Surgery. 2015;158(4):1137–43. 28. Bay-Nielsen M, Nilsson E, Nordin P, Kehlet H, Swedish Hernia Data
Base the Danish Hernia Data Base. Chronic pain after open mesh
and sutured repair of indirect inguinal hernia in young males. Br J
Surg. 2004;91(10):1372–6. 17. Hernandez-Rosa J, Lo CC, Choi JJ, Colon MJ, Boudourakis L, Telem
DA, et al. Laparoscopic versus open inguinal hernia repair in octoge-
narians. Hernia. 2011;15(6):655–8. 29. Franneby U, Sandblom G, Nordin P, Nyren O, Gunnarsson U. Risk factors for long-term pain after hernia surgery. Ann Surg. 2006;244(2):212–9. 18. Hope WW, Bools L, Menon A, Scott CM 3rd, Adams A, Hooks WB
3rd. Comparing laparoscopic and open inguinal hernia repair in octo-
genarians. Hernia. 2013;17(6):719–22. 19. Rutkow IM. Demographic and socioeconomic aspects of hernia repair
in the United States in 2003. Surg Clin North Am. 2003;83(3):1045–51. 30. Liem MS, van Duyn EB, van der Graaf Y, van Vroonhoven TJ, Coala
Trial Group. Recurrences after conventional anterior and laparo-
scopic inguinal hernia repair: a randomized comparison. Ann Surg. 2003;237(1):136–41. 20. Zendejas B, Ramirez T, Jones T, Kuchena A, Ali SM, Hernandez-Irizarry
R, et al. Incidence of inguinal hernia repairs in Olmsted County, MN: a
population-based study. Ann Surg. 2013;257(3):520–6. 31. Bakker WJ, van Hessen CV, Verleisdonk EJMM, Clevers GJ,
Davids PHP, Schouten N, et al. Is young age a risk factor for
chronic postoperative inguinal pain after endoscopic totally ex-
traperitoneal (TEP) repair? Hernia. 2019. https://doi.org/10.1007/
s10029-019-01882-3. 21. Statistics Japan. 5 | CONCLUSION 12. Mayer F, Lechner M, Adolf D, Öfner D, Köhler G, Fortelny R, et al. Is the age of > 65 years a risk factor for endoscopic treatment of
primary inguinal hernia? Analysis of 24,571 patients from the
Herniamed Registry. Surg Endosc. 2016;30(1):296–306. The present study for the first time showed that the incidence of
perioperative complications in TAPP repair for groin hernia is influ-
enced by poor PS rather than old age. In conclusion, when the con-
dition of the patient allows general anesthesia and PS is 0-2, TAPP 13. Goldman L, Caldera DL, Nussbaum SR, Southwick FS, Krogstad D,
Murray B, et al. Multifactorial index of cardiac risk in noncardiac
surgical procedures. N Engl J Med. 1977;297(16):845–50. 324 | 324 | 324 324 EGAWA et al. Current Population Estimates as of October 1, 2017. http://www.stat.go.jp/english/index.htm. Accessed 24 November
2018. 22. INCA Trialist Collaboration. Operation compared with watchful wait-
ing in elderly male inguinal hernia patients: a review and data analysis. J Am Coll Surg. 2011;212(2):251–9. How to cite this article: Egawa N, Nakamura J, Manabe T,
Iwasaki H, Noshiro H. Incidence of postoperative
complications in transabdominal preperitoneal repair for groin
hernia is influenced by poor performance status rather than by
old age. Ann Gastroenterol Surg. 2019;3:318–324. https://doi.org/10.1002/ags3.12247 23. O'Dwyer PJ, Norrie J, Alani A, Walker A, Duffy F, Horgan P. Observation
or operation for patients with an asymptomatic inguinal hernia. Ann
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Iwasaki H, Noshiro H. Incidence of postoperative
complications in transabdominal preperitoneal repair for groin
hernia is influenced by poor performance status rather than by
old age. Ann Gastroenterol Surg. 2019;3:318–324. https://doi.org/10.1002/ags3.12247 24. Fitzgibbons RJ Jr, Ramanan B, Arya S, Turner SA, Li X, Gibbs JO, et al. Long-term results of a randomized controlled trial of a nonoperative
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McCarthy M Jr, et al. Watchful waiting vs repair of inguinal hernia
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Effect of oral administration of Bacillus coagulans B37 and Bacillus pumilus B9 strains on fecal coliforms, Lactobacillus and Bacillus spp. in rat animal model
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Veterinary world/Veterinary World
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Copyright: Haldar and Gandhi. 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 RESEARCH ARTICLE
Open Access Veterinary World, EISSN: 2231-0916
Available at www.veterinaryworld.org/Vol.9/July-2016/15.pdf Abstract Aim: To investigate the effect of oral administration of two Bacillus strains on fecal coliforms, Lactobacillus and Bacillus
spp. in rat animal model. Materials and Methods: An in vivo experiment was conducted for 49-day period on 36 adult male albino Wister rats
divided equally into to four groups. After 7-day adaptation period, one group (T1) was fed on sterile skim milk along with
basal diet for the next 28 days. Second (T2) and (T3) groups received spore biomass of Bacillus coagulans B37 and Bacillus
pumilus B9, respectively, suspended in sterilized skim milk at 8-9 log colony-forming units/ml plus basal diet for 28 days,
while control group (T4) was supplied with clean water along with basal diet. There was a 14-day post-treatment period. A total of 288 fecal samples (8 fecal collections per rat) were collected at every 7-day interval starting from 0 to 49 days and
subjected to the enumeration of the counts of coliforms and lactobacilli and Bacillus spores using respective agar media. In vitro acid and bile tolerance tests on both the strains were performed. Results: The rats those (T2 and T3) received either B. coagulans B37 or B. pumilus B9 spore along with non-fermented
skim milk showed decrease (p<0.01) in fecal coliform counts and increase (p<0.05) in both fecal lactobacilli and Bacillus
spore counts as compared to the control group (T4) and the group fed only skim milk (T1). In vitro study indicated that both
the strains were found to survive at pH 2.0 and 3.0 even up to 3 h and tolerate bile up to 2.0% concentration even after 12 h
of exposure. Conclusions: This study revealed that oral administration of either B. coagulans B37 or B. pumilus B9 strains might
be useful in reducing coliform counts accompanied by concurrent increase in lactobacilli counts in the intestinal flora
in rats. Keywords: acid salt tolerance, antibacterial activity, Bacillus coagulans, Bacillus pumilus, bile salt tolerance, probiotics. Keywords: acid salt tolerance, antibacterial activity, Bacillus coagulans, Bacillus pumilus, bile salt tolerance, probiotics. Keywords: acid salt tolerance, antibacterial activity, Bacillus coagulans, Bacillus pumilus, bile salt tolerance, probiotics. Introduction
bile [3]. Lactic acid bacteria (LAB) have been used Lopamudra Haldar1 and D. N. Gandhi2 Lopamudra Haldar1 and D. N. Gandhi2 1. Department of Basic Sciences, Faculty of Science and Technology, ICFAI University, Kamalghat - 799 210, Tripura,
India; 2. Dairy Microbiology Division, National Dairy Research Institute, Karnal - 132 001, Haryana, India. Corresponding author: Lopamudra Haldar e-mail: mohor7@gmail com y
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,
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,
,
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,
Corresponding author: Lopamudra Haldar, e-mail: mohor7@gmail.com, DNG: dngandhi@rediffmail.com g
Received: 18-03-2016, Accepted: 16-06-2016, Published: 26-07-2016 g
Received: 18-03-2016, Accepted: 16-06-2016, Published: 26-07-2016 doi: 10.14202/vetworld.2016.766-772 How to cite this article: Haldar L, Gandhi DN (2016) Effect of oral administration
of Bacillus coagulans B37 and Bacillus pumilus B9 strains on fecal coliforms, Lactobacillus and Bacillus spp. in rat animal
model, Veterinary World, 9(7): 766-772. Experimental animals 36 adult male albino Wister rats were divided
equally into to four groups (9 rats in each group) on
the basis of their body weight and age so that the mean
body weight and mean age of four groups did not dif
fer (p>0.10) at the beginning of the experiment. Each
group having 9 animals were again divided into three
subgroups (3 animals in each subgroup) as replicates. The animals were 75±5 days old with mean body weight
of 210±10 g. The animals were housed in aluminum
cages under controlled temperature (22°C±2°C) and
humidity (56±5%) at Small Animal House of National
Dairy Research Institute, Karnal, Haryana, India. Each
group having 9 animals were again divided into three
subgroups (3 animals in each subgroup) as replicates. Bacterial counts All bacterial enumerations were done within
2 h of sampling. The homogenized samples in PBS
were used for enumerating coliforms and lactobacilli
counts using Eosin Methylene Blue agar and MRS
agar (named by its inventors: DeMan, Rogosa, and
Sharpe), respectively. For enumeration of Bacillus
spore count in fecal samples, the homogenized sam
ples in PBS were put into a water bath at 80°C for
10 min, and then cooled immediately to favor spore
germination. The samples were serially diluted in Fecal sample collection The fecal samples were collected from all exper
imental rats to evaluate the concentration of bacterial
spores. The fecal samples were collected at every
7-day interval starting from 0 to 49 days. A total of
288 fecal samples (8 fecal collections per rat) were
collected in sterile container squeezing of the rectal
area of the rat. Just after collection, the samples were
weighed and immediately diluted in phosphate buff
ered saline (PBS) and then transported immediately
in the laboratory and kept at 4°C in a refrigerator until
analysis, performed on the same day. The fecal sam
ples were pooled from 3 animals of each subgroup to
obtain a total of 12 subgroups (3 from each group of
T1, T2, T3, and T4). Each subgroup’s fecal sample
was homogenized in PBS, and then, serial dilutions
were made in the sterile physiological saline solution,
and thereafter, subjected to enumeration of bacterial
counts. Ethical approval An experiment was designed to investigate the
effect of Bacillus strains on fecal coliforms, Lactobacillus
and Bacillus count in rat animal model at the National
Dairy Research Institute, Karnal, Haryana, India. The
experimental animals were treated in accordance with
the guidelines of the Ethical Committee for Animal
Experiments of National Dairy Research Institute. Bacillus strains for the feeding trail B. coagulans B37 and B. pumilus B9 strains used
for oral administration in rats in the current study were
isolated from buffalo milk and subsequently charac
terized morphologically, biochemically, and geneti
cally based on single strand conformational polymor
phism banding patterns and partial 16S rRNA gene
sequencing and also found on hemolysis on blood agar
plates after incubation at 37°C for 18 h as previously
reported [15]. Both Bacillus strains were maintained
on B. coagulans agar (BCA) slants [16] and subcul
tured after every 25-30 days period. The stocks of both
Bacillus strains were preserved in 20% glycerol stock
medium at −76°C. The cultures were activated before
use by twice subculturing in BCA broth. The spore
biomass of B. coagulans B37 and B. pumilus B9 was
produced using a whey-based medium supplemented
with suitable nutrients and minerals [17]. Introduction bile [3]. Lactic acid bacteria (LAB) have been used
extensively as starter cultures in food fermentation
and found to have many health benefits [4]. In recent
years, research, development, and use of spore-form-
ing bacteria as probiotic have brought a breakthrough
in probiotic world. Probiotics are defined by the World Health
Organization as “live microorganisms which, when
administered in adequate amounts, confer a health
benefit on the host” [1]. The global trend in the food
sector is to incorporate probiotic microorganisms into
foods for imparting its functional attributes that can
provide some health-promoting component(s) beyond
its traditional nutrients [2]. Worldwide, there are
numerous strains of probiotics used in dietary supple-
ments and foods, but most are unstable at room tem-
perature and need to be freeze dried or encapsulated
via special processes to remain viable during manu-
facturing, storage, and exposure to stomach acid and p
The use of bacterial spore formers, Bacillus spp. in particular, as probiotics could provide practical
advantages for both humans and animals [5]. The abil-
ity to form spores endows probiotics with higher resis-
tance to technological stresses during production and
storage processes and also higher resistance to gastric
(pH, digestive enzymes) and intestinal environmental
conditions [6]. Indeed, a number of products containing
spores of Bacillus spp. are currently commercialized as
probiotics for human [7] as well as for farm animals,
particularly in cattle [8], swine [9], and poultry [10]. Besides many benefits, such as reduction of irritable
bowel syndrome in human patients [11,12] and Bacillus
subtilis is a use for vaccine production host and deliv-
ery vector as it is safe for human consumption and the
production of spores exerting adjuvant effects [13]. Veterinary World, EISSN: 2231-0916 766 Available at www.veterinaryworld.org/Vol.9/July-2016/15.pdf testing regimen. All groups were fed on standard basal
diet (containing starch - 57.46%, casein - 20.0%,
sucrose - 5.0%, vegetable oil - 10.0%, cellulose pow
der - 1.91%, mineral mixture - 4.62%, and vitamin
mixture - 1.01%) 7 days (adaptation period) before
and 14 days after the treatment period. During the
experimental period, the rats were fed twice, at morn
ing (08:00 h) and evening (20:00 h) at 12 h interval. After the adaptation period, one group (T1) was fed
on sterile skim milk along with basal diet for the next
28 days. The two experimental groups (T2 and T3)
received spore biomass of B. coagulans B37 and
B. Introduction pumilus B9, respectively, suspended in sterilized
skim milk at 8-9 log colony-forming unit (cfu)/ml
for 28 days as the previous report indicated that oral
administration of a proprietary preparation of B. coag
ulans - GanedenBC(30)™ - a novel probiotic up to
9.38×10 (10) cfu/day for 1-year period caused no
signs of toxicity in the parental generation (male or
female) nor the F1 offspring of Wister rats [18]. No
water was supplied to these three groups. After the
feeding of Bacillus organisms for 28 days, all the ani
mals were fed on the basal diet along with water for
further 14 days. The last 14 days was considered as
post-treatment period. The fourth group (T4) was sup
plied with clean water along with basal diet during the
whole 49-day experimental period. This group served
as control group. There is limited information on the antimicro
bial activity of Bacillus probiotics [14] in vivo ani
mal model. Hence, we have conducted a study with
the objectives (i) to elucidate the antimicrobial effect
of Bacillus coagulans B37 and Bacillus pumilus B9
strains on fecal coliforms, (ii) to investigate their
effect on fecal Lactobacillus as well as Bacillus spp. in rat animal model, and (iii) to examine functional
potential in regards to acid and bile tolerance of these
Bacillus strains under in vitro conditions. Effects on fecal coliform count The acid and bile tolerance of B. coagulans B37
and B. pumilus B9 strains were assessed as per the
methods described previously [20,21]. Different low
pH solutions were prepared to stimulate gastric acidic
conditions by adding reagent grade HCl (35.8%) in
distilled water drop by drop in a beaker and solutions
having pH of 1.0, 2.0, and 3.0 were obtained. Sterile
distilled water (pH adjusted to 6.5) was served as con
trol. High bile salt solutions (1.0% and 2.0%) were
prepared by dissolving bile salts (Hi-media, India)
in distilled water. 1 ml of fresh culture containing
approximately 107-108 cfu/ml was added to the dif
ferent pH solutions (1.0, 2.0, 3.0, and 6.5) as well as
bile solutions (1.0% and 2.0%) and mixed thoroughly. 1 ml of each solution was taken from each tube imme
diately (0 h) and 10-fold serial dilutions were prepared
in 0.1% peptone water. Pour plating was done on BC
agar media. The inoculated pH solutions were incu
bated at 37°C for 3 h, and 1 ml of culture was taken
hourly from each tube after an interval of 1, 2, and 3 h
of incubation followed by plating. The bile solutions
containing cultures were incubated at 37°C for 12 h,
and 1 ml of bile solution containing culture was taken
from each tube after 1, 3, and 12 h of incubation and
plated on BCA. All plates were incubated at 37°Cfor
24-72 h and the cfu were counted. The effect of feeding of B. coagulans B37 and
B. pumilus B9 on changes in the counts of fecal
coliforms is presented in Figure-1. The number
of fecal coliform counts decreased by more than
1 log cycle (p<0.01) for rats in both the groups
received non-fermented skim milk supplemented
with Bacillus organisms than the counts for control
rats as well as the rats received only skim milk (T1)
during the 49-day experimental period. However,
the fecal coliform counts between the two groups
fed Bacillus organisms did not differ (p>0.05). Fecal coliform counts for the control (T4) and the
rats fed skim milk (T1) remained unchanged during
the experimental period. In the present study, the
feeding of Bacillus spores decreased the counts
of fecal coliform in rats as reported previously in
piglets [26]. Bacillus probiotic mixtures might be
allowed for tailoring strategies to prevent infec
tious diseases [27,28]. Our earlier study showed the
surface hydrophobicity of B. Effect on fecal Lactobacillus count The diets were supplemented with control - water ();
T1 - Skim milk (); T2 - non-fermented skim milk with B37
isolate (×); T3- non-fermented skim milk with B9 isolate
from 15th day up to 35th day (). indicates fecal coliform
count among different groups differ significantly (p<0.01). Effects on fecal coliform count coagulans B37 and
B. pumilus B9 were 42.88% and 37.43%, respec
tively, under in vitro condition [29]. In the pres
ent study, feeding of Bacillus organisms probably
exerts an antagonistic effect on intestinal coliform
leading to displacement of a considerable popula
tion of coliforms from the intestinal flora. Feeding schedule The experimental rats were allowed a 7-day
adjustment period to remove the effect of stress pos
sibly experienced by the animals due to separation
from the main stock and to become accustomed to the Veterinary World, EISSN: 2231-0916 767 Available at www.veterinaryworld.org/Vol.9/July-2016/15.pdf physiological saline after holding 2 h at 37°C, and
plating was performed using BCA media and incu
bated aerobically at 37°C for 24-72 h after which the
Bacillus spores were identified on the basis of their
characteristic morphology [19] and then counted and
recorded. and B. pumilus B9 on fecal coliform, Lactobacillus,
and Bacillus in rat animal model. Despite the infor
mation on the antimicrobial effect of probiotic
Lactobacillus and Bifidobacterium [22-24], there is
a dearth of knowledge on the antimicrobial effect of
Bacillus spp. in animal models [25]. Effect on fecal Lactobacillus count T-test was employed to test the significance of
differences between various groups of animals (rats)
on age and body weight at the beginning of the exper
iment using Microsoft® Excel 2000 Software package,
Microsoft Corporation, USA. General Linear Model
(GLM) procedure with post-hoc test was also done on
each set of data of fecal Coliform, Lactobacillus, and
Bacillus count to determine whether significant differ
ences existed among different groups from SYSTAT
6.0.1 Statistical Software Package, 1996, SPSS, Inc.,
USA. Fisher’s least significant difference test was
applied to determine the statistical significance of the
difference in effects of treatments among different
groups in the animal study, from matrix of pairwise
comparison probabilities of different means. A p<0.05
or <0.01 was considered to be statistically significant. The mean±standard error of mean different parame
ters studied was graphically presented using GraphPad
Prism 3.02, 1999, GraphPad Software Inc., San Diego
CA. Figure-2 shows the effect of dietary treatment
of Bacillus organisms on changing pattern in the
counts of fecal lactobacilli. The rats in the group Figure-1: Trend in mean±standard error of mean fecal
coliform count (colony-forming units/g fecal sample) in
different groups of rat. Rats were fed with cholesterol-
enriched diet from 0 up to 35th day cholesterol-enriched
diet. The diets were supplemented with control - water ();
T1 - Skim milk (); T2 - non-fermented skim milk with B37
isolate (×); T3- non-fermented skim milk with B9 isolate
from 15th day up to 35th day (). indicates fecal coliform
count among different groups differ significantly (p<0.01). Figure-1: Trend in mean±standard error of mean fecal
coliform count (colony-forming units/g fecal sample) in
different groups of rat. Rats were fed with cholesterol-
enriched diet from 0 up to 35th day cholesterol-enriched
diet. The diets were supplemented with control - water ();
T1 - Skim milk (); T2 - non-fermented skim milk with B37
isolate (×); T3- non-fermented skim milk with B9 isolate
from 15th day up to 35th day (). indicates fecal coliform
count among different groups differ significantly (p<0.01). Figure-1: Trend in mean±standard error of mean fecal
coliform count (colony-forming units/g fecal sample) in
different groups of rat. Rats were fed with cholesterol-
enriched diet from 0 up to 35th day cholesterol-enriched
diet. Effect on fecal Bacillus count The effect of feeding of Bacillus organisms
on changing pattern in the counts of fecal Bacillus
is presented in Figure-3. The fecal Bacillus counts
in rats fed non-fermented skim milk plus B. coagu
lans B37 (T2) as well as rats fed non-fermented skim
milk plus B. pumilus B9 (T3) increased immediately
just after day 14 (days when the feeding of Bacillus
organisms was started). The rate of increment was
higher in group T3 than that of group T2. Average
of 4 log cycle increment was evidenced on the
35th day in both cases. As the probiotic feeding was
stopped from 36th day onward, the fecal spore count
of group T2 decreased sharply within 7 days by more
than 1 log cycle. However, the fecal Bacillus counts
in groups T2 remained quite stable in post-feeding
period. The Bacillus counts in feces of the control
rats as well as the rats fed only skim milk (T1) were
found to be very low or sometimes non-detectable. The significant increment of the Bacillus counts after
feeding of B. coagulans B37 and B. pumilus B9 in
rats strongly supports the previous reports on use of
gut-colonizing B. subtilis spores as a new platform
for the mucosal delivery of vaccine antigens [32] as
well as survivability of Bacillus clausii spores in the
human gastrointestinal (GI) tract, and thereafter ger
mination, outgrowth, and multiplication as vegetative
forms following oral administration as spore-based
probiotic formulation [33]. In the present study, the
feeding of B. coagulans B37 revealed that the fecal
Bacillus count remained stable for 10 days after with
drawal of feeding of B. coagulans B37. This finding
suggests that B. coagulans B37 probably adhere to
the intestine of the rat and help to exert beneficial
effects. Figure-2: Trend in mean±standard error of mean fecal
lactic acid bacteria (LAB) count (colony-forming units/g
fecal sample) in different groups of rat. Rats were fed with
cholesterol-enriched diet from 0 up to 35th day cholesterol-
enriched
diet. The
diets
were
supplemented
with
control - water (); T1 - skim milk (); T2 - non-fermented
skim milk with B37 isolate (×); T3 - non-fermented skim
milk with B9 isolate from 15th up to 35th day (). Fecal LAB
count among different groups differ significantly (p<0.05). «Indicates fecal lactobacilli count among different groups
differ significantly (p<0.05). Results and Discussion The present investigation was undertaken to
explore the antibacterial effect of B. coagulans B37 Veterinary World, EISSN: 2231-0916 768 Available at www.veterinaryworld.org/Vol.9/July-2016/15.pdf in the gut [34,35]. In vitro acid and bile tolerance
abilities of the selected Bacillus isolates are pre
sented in Tables-1 and 2. In the present study, both
B. coagulans B37 and B. pumilus B9 strains were
shown to survive at pH 2.0 and 3.0 even up to 3 h
under in vitro experiment. B. pumilus B9 strain,
specially, could survive at pH 1.0 up to 3 h, but
B. coagulans B37 strain showed tolerance at pH 1.0
up to only 1 h. Resisting exposure to pH 3.0 for 1.5
to 2 h is considered one standard for low pH tol
erance of probiotic bacteria [36]. Surviving bac
teria from the stomach would then contact bile in
the small intestine. Multiplication of B. coagulans
in the small intestine should not be expected since
the bacteria are strictly aerobic. Tolerance to bile is
thus a criterion for B. coagulans to survive passage fed non-fermented skim milk plus B. coagulans
B37 exhibited significantly increase (p<0.05) in the
numbers of lactobacilli (more than 1 log cycle) in
their feces than that of the other groups during the
49-day experimental period. However, rats those
received non-fermented milk along with B. pumilus
B9 (T3) showed significantly higher (p<0.05) fecal
lactobacilli counts than had the control group and
the group fed only skim milk (T1) at day 42 and 49
also. The present result also depicted that there was
no variation (p>0.05) in fecal lactobacilli counts
between the two groups fed Bacillus probiotic at 42
and 49 days. The present finding of increase in the
count of fecal lactobacilli agrees well with the ear
lier observations reflected large bowel colonization
of LAB for biological effects [10,30,31]. Probably,
the Bacillus organisms act synergistically with lac
tobacilli in the gut of rats, by producing acid and
other metabolites essential for the growth of the
lactobacilli. Figure-2: Trend in mean±standard error of mean fecal
lactic acid bacteria (LAB) count (colony-forming units/g
fecal sample) in different groups of rat. Rats were fed with
cholesterol-enriched diet from 0 up to 35th day cholesterol-
enriched
diet. Effect on fecal Bacillus count Figure-3: Trend in mean±standard error of mean fecal
Bacillus count (colony-forming units/g fecal sample) in
different groups of the rat. Rats were fed with cholesterol-
enriched diet from 0 up to 35th day cholesterol-enriched
diet. The diets were supplemented with control - water ();
T1 - skim milk (); T2 - non-fermented skim milk with B37
isolate (×); T3 - non-fermented skim milk with B9 isolate
from 15th day up to 35th day (). ««indicates fecal Bacillus
count among different groups differ significantly (p<0.01). Figure-3: Trend in mean±standard error of mean fecal
Bacillus count (colony-forming units/g fecal sample) in
different groups of the rat. Rats were fed with cholesterol-
enriched diet from 0 up to 35th day cholesterol-enriched
diet. The diets were supplemented with control - water ();
T1 - skim milk (); T2 - non-fermented skim milk with B37
isolate (×); T3 - non-fermented skim milk with B9 isolate
from 15th day up to 35th day (). ««indicates fecal Bacillus
count among different groups differ significantly (p<0.01). Results and Discussion The
diets
were
supplemented
with
control - water (); T1 - skim milk (); T2 - non-fermented
skim milk with B37 isolate (×); T3 - non-fermented skim
milk with B9 isolate from 15th up to 35th day (). Fecal LAB
count among different groups differ significantly (p<0.05). «Indicates fecal lactobacilli count among different groups
differ significantly (p<0.05). Conclusions As Bacillus spp. is not considered resident
members of the gut microflora, controversies are
there whether it can survive the transit within GI
tract and can be able to exert health protecting
effects followed by germination of spores within
the gut. In vivo as well as in vitro study clearly
indicated that B. coagulans B37 and B. pumilus B9
spores could withstand the adverse environment of
the GI. tract. The decreasing counts in fecal coliform
with the increasing counts in both lactobacilli and
Bacillus counts in the feces of the treated rats sug
gested that both B. coagulans B37 and B. pumilus
B9 probably inhibited the proliferation of coliform
in rats and exerted synergistic effects with lactoba
cilli. Thus, both B. coagulans B37 and B. pumilus B9
could be used as an adjunct probiotic in combination
with established strains of probiotic lactobacilli to
achieve combined and more pronounced health-pro
moting effects. Since Food and Drug Administration
has some restrictions for granting GRAS (Generally
Regarded as Safe) status for any Bacillus species, two
indigenous strains, B. coagulans B37 and B. pumilus
B9 could be considered for probable probiotics after
necessary clinical studies and safety assessment in
animals and human. Acknowledgments The authors acknowledge the financial assis
tance and necessary supports provided by the
Director of National Dairy Research Institute,
Karnal, Haryana, India, to carry out the study. The first author wishes to express her sincere
thanks to Dr. Rameshwar Singh, Project Director,
ICAR-Directorate of Knowledge Management in
Agriculture, Indian Council of Agricultural Research,
Krishi Anusandhan Bhawan-I, Pusa, New Delhi,
India, for his constant help and suggestions during
the investigation at the Dairy Microbiology Division,
National Dairy Research Institute, Karnal, Haryana,
India. In vitro acid and bile tolerance Surviving passage through the GI tract is
believed to be important for probiotics to function Veterinary World, EISSN: 2231-0916 769 Available at www.veterinaryworld.org/Vol.9/July-2016/15.pdf Available at www.veterinaryworld.org/Vol.9/July-2016/15.pdf
Table-1: In vitro acid tolerance of the selected Bacillus isolates. Isolate
Bacterial count (cfu/ml)
At pH 6.4
At pH 1.0
At pH 2.0
At pH 3.0
0 h
1 h
2 h
3 h
0 h
1 h
2 h
3 h
0 h
1 h
2 h
3 h
0 h
1 h
2 h
3 h
Bacillus coagulans (B37) 8.64 8.11 8.34 8.49 3.69 2.23
‑
‑
6.18 5.85 5.28 5.36 6.81 6.73 6.53 6.11
Bacillus pumilus (B9)
8.86 8.79 8.18 7.96 6.32 5.67 3.51 2.11 7.86 6.36 6.23 5.85 7.96 7.59 7.18 6.87
cfu=Colony‑forming units
Table-2: In vitro bile tolerance of the selected Bacillus isolates. Isolate
Bacterial count (cfu/ml)
At 0.0% bile
At 1.0% bile
At 2.0% bile
0 h
1 h
3 h
12 h
0 h
1 h
3 h
12 h
0 h
1 h
3 h
12 h
Bacillus coagulans (B37)
8.15
7.60
8.15
7.95
7.46
6.11
6.86
6.81
6.28
5.62
5.48
5.53
Bacillus pumilus (B9)
8.75
8.15
7.99
7.85
6.43
5.95
5.64
6.28
5.70
5.36
4.59
4.87
cfu=Colony‑forming units Table-1: In vitro acid tolerance of the selected Bacillus isolates. Bacillus coagulans (B37) 8.64 8.11 8.34 8.49 3.69 2.23
‑
‑
6.18 5.85 5.28 5.36 6.81 6.73 6.53 6.11
Bacillus pumilus (B9)
8.86 8.79 8.18 7.96 6.32 5.67 3.51 2.11 7.86 6.36 6.23 5.85 7.96 7.59 7.18 6.87
cfu=Colony‑forming units
Table-2: In vitro bile tolerance of the selected Bacillus isolates. Isolate
Bacterial count (cfu/ml)
At 0.0% bile
At 1.0% bile
At 2.0% bile
0 h
1 h
3 h
12 h
0 h
1 h
3 h
12 h
0 h
1 h
3 h
12 h
Bacillus coagulans (B37)
8.15
7.60
8.15
7.95
7.46
6.11
6.86
6.81
6.28
5.62
5.48
5.53
Bacillus pumilus (B9)
8.75
8.15
7.99
7.85
6.43
5.95
5.64
6.28
5.70
5.36
4.59
4.87
cfu=Colony‑forming units Table-2: In vitro bile tolerance of the selected Bacillus isolates. Authors’ Contributions through the small intestine. Both B. coagulans B37
and B. pumilus B9 strains were found to possess
a strong tolerance to bile up to 2.0% concentra
tion even after 12 h of exposure. The significantly
higher (p<0.01) counts of Bacillus in feces of rats
fed with Bacillus spores than the counts in feces in
rats that did not receive Bacillus spores, supports the
previous finding of potential probiotic functions of
Bacillus Species under simulated GI condition [37]. Hence, the present study suggests that consumption
of non-fermented milk plus either B. coagulans B37
or B. pumilus B9 strains might be useful in reducing
coliform counts accompanied by concurrent increase
in lactobacilli counts in the intestinal flora in rats. Both the authors contributed to conception and
design of the study. LH carried out the research work,
collected data, analyzed data, interpreted the results,
and drafted the article critically for important intellec
tual content. DNG monitored the whole research pro
gram. Both authors have read and approved the final
manuscript. Competing Interests The authors declare that they have no competing
interests. 2.
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Dahl, W.J. (2015) Evaluation of Bacillus subtilis R0179
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109 Suppl 2: S35-S50. Veterinary World, EISSN: 2231-0916 772
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Ways to improve the anticorrosive properties of motor oils used in vehicles
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E3S web of conferences
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Ways to improve the anticorrosive properties of
motor oils used in vehicles Ways to improve the anticorrosive properties o
motor oils used in vehicles
Zebo Alimova1*[10000-0002-6711-5318], Ravshanjon Akhmatjanov1, Nargiza Kholikova2, and
Kamola Karimova3
1Tashkent State Transport University, Tashkent, Uzbekistan
2Tashkent Institute of Irrigation and Agricultural Mechanization Engineers, Tashkent, Uzbekistan
3Jizzakh Polytechnic Institute, Jizzakh, Uzbekistan Zebo Alimova1*[10000-0002-6711-5318], Ravshanjon Akhmatjanov1, Nargiza Kholikova2, and
Kamola Karimova3
1Tashkent State Transport University, Tashkent, Uzbekistan
2Tashkent Institute of Irrigation and Agricultural Mechanization Engineers, Tashkent, Uzbekistan
3Jizzakh Polytechnic Institute, Jizzakh, Uzbekistan 1Tashkent State Transport University, Tashkent, Uzbekistan
2Tashkent Institute of Irrigation and Agricultural Mechanization Engineers, Tashkent, Uzbekistan
3Jizzakh Polytechnic Institute, Jizzakh, Uzbekistan 1Tashkent State Transport University, Tashkent, Uzbekistan
2Tashkent Institute of Irrigation and Agricultural Mechanization Engineers, Tashkent, Uzbekistan
3Jizzakh Polytechnic Institute, Jizzakh, Uzbekistan Abstract. In recent years, increased requirements for protective properties
have been imposed on petroleum oils for various purposes. One of the
functions of the oil is to protect the surface of parts from corrosion. Corrosion becomes especially intense when the engine is operated in hot,
humid climates. In this case, oil plays a double role: on the one hand, it
protects the surfaces of parts from the aggressive influence of the external
environment; on the other hand, the oil itself is corrosive due to the
presence of corrosive substances in it. Corrosion is especially intensified after the engine is stopped. When it
cools, moisture condenses on the parts, the lubricating oil, flowing down
from the lubricated surface, cannot protect the metal from corrosion. The
reason for the corrosive properties of oils is that they contain organic and
inorganic acid peroxides and other oxidation products and sulfur
compounds, alkalis, and water. The purpose of this work is to study and improve the anti-corrosion
properties of lubricants. With an increase in temperature, the combined
action of oxygen in the air and water present in the lubricating oil causes
rusting of the crankshaft, liner walls, cylinders of the internal combustion
engine. We have studied anticorrosive additives as an additive to improve the anti-
corrosion properties of oils. Dialkylphenyl orthophosphoric acid additive
was used as such additives. The anticorrosive activity of these substances
is related to their ability to orient themselves on the oil-water surface so
that hydrophilic groups are firmly bound to water while the hydrocarbon
radical remains in the oil. *Corresponding author: zeboalimova7841@mail.ru E3S Web of Conferences 264, 05004 (2021)
CONMECHYDRO - 2021 E3S Web of Conferences 264, 05004 (2021)
CONMECHYDRO - 2021 https://doi.org/10.1051/e3sconf/202126405004 1 Introduction In recent years, increased requirements for protective properties have been imposed on
petroleum oils for various purposes. One of the functions of the oil is to protect the surface
of parts from corrosion. The high protective effect is based on the ability of oils to quickly
displace active compounds from the metal surface, keep it in the bulk of the lubricant, and © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons
Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). E3S Web of Conferences 264, 05004 (2021)
CONMECHYDRO - 2021 https://doi.org/10.1051/e3sconf/202126405004 form strong adsorption and chemisorption films on it which prevent the development of
electrochemical processes. Petroleum-based oils are not capable of long-term protection of
metals from electrochemical corrosion. Corrosion can be related to external and internal
factors. The main internal factor is the chemical composition of the oil. Active sulfur
compounds directly cause corrosion of metals; therefore, they must be removed during the
refining of oil products. The content of active sulfur compounds is determined by a sample
on a copper plate (3 hours at 50 ° C). Sulfur oxides SOx, nitrogen oxides NOx, and water resulting from the combustion of
hydrogen, which form mineral acids H2SO3, H2SO4, HNO3, enter the crankcase from the
combustion chamber of the engine together with the exhaust gases into the crankcase,
which causes corrosion of the engine lubrication system parts. Corrosion of metals,
especially non-ferrous ones, is also caused by organic acids formed due to the oxidation of
hydrocarbons. It seems most realistic to combat corrosion of engine parts and corrosion-mechanical
wear by introducing corrosion inhibitors into engine oils and creating working conservation
oils. Corrosion inhibitors are designed to protect the surface of engine parts from corrosion
caused by organic and mineral acids formed during the oxidation of oil and additives. The
mechanism of their action is the formation of a protective film on the surface of parts and
the neutralization of acids. 2 Materials and Methods In addition to temperature, the nature of oil oxidation is influenced by the specific
conditions of its operation in the engine: large areas of contact between oil and air (oil
films, fog, oil foaming in the crankcase). Iron, copper, lead have a catalytic effect on the
oxidation process. The most intense oxidation is observed in relatively thin layers of oil on
highly heated metal surfaces. Particularly dangerous is the corrosion of non-ferrous metal
bearing shells, which acidic oxidation products and sulfur compounds can cause. At high
temperatures, sulfur compounds become especially aggressive towards silver and lead. The corrosiveness of oils increases significantly when they contain water, entering the
crankcase from the atmosphere or the engine cooling system. Corrosion becomes especially intense when the engine is operated in hot, humid
climates. In this case, oil plays a double role: on the one hand, it protects the surfaces of
parts from the aggressive influence of the external environment; on the other hand, the oil
itself is corrosive due to the presence of corrosive substances in it. The combined action of oxygen in the air and water present in the lubricating oil causes
rusting of the crankshaft, liner walls, cylinders of an internal combustion engine, etc. Corrosion increases, especially after stopping the engine. When it cools, moisture
condenses on the parts, lubricating oil flowing down from the lubricated surface cannot
protect the metal from corrosion. The corrosive aggressiveness of oils about lead bronze from which the crankshaft
bearing shells are made is assessed by the breakdown on the lead plate. In this case, the loss
of its mass is determined under the conditions of its presence in oil for 50 hours at 140°C. The temperature factor especially affects the rate of general corrosion. An increase in
temperature from 20 to 80°C at 5% by volume of water causes an increase in corrosion
from 0.1 to 0.25 mm/year. Suppose we assume that the permissible corrosion rate is 0.2
mm/year. In that case, this value is achieved at a temperature of 80°C by 2%, and a further
increase in the water content in the oil leads to a significant increase in the corrosion rate
(Table 1). 2 https://doi.org/10.1051/e3sconf/202126405004 E3S Web of Conferences 264, 05004 (2021)
CONMECHYDRO - 2021 Table 1. 2 Materials and Methods Dependence of the rate of general corrosion of metal (St-20) on the amount of water in oil
M-10V2 and on temperature
Temperatura,
°С
General corrosion rate, mm / year at water content,% vol. 0%
0.5%
1.0%
2.0%
5.0%
10.0%
50%
20
0.050
0.060
0.065
0.078
0.104
0.204
0.496
40
0.065
0.078
0.098
0.122
0.188
0.466
0.850
60
0.065
0.104
0.098
0.124
0.204
0.504
1.244
80
0.100
0.107
0.135
0.196
0.253
0.608
1.723 Table 1. Dependence of the rate of general corrosion of metal (St-20) on the amount of water in oil
M-10V2 and on temperature Table 1. Dependence of the rate of general corrosion of metal (St-20) on the amount of water in oil
M-10V2 and on temperature To combat corrosion, special additives are added to the oils. Anti-corrosion additives
are mainly polar substances that easily adsorb to metal surfaces. The mechanism of their action is to create a protective monomolecular layer on the metal,
which prevents acidic and other active agents from acting on the metal. There is a relationship between the value of adsorption and surface tension at constant
temperature and pressure: 𝐺𝑎𝑑𝑑 𝐺𝐺= −𝑎𝑎
𝑅𝑅𝑅𝑅
𝑑𝑑𝑑𝑑
𝑑𝑑𝑑𝑑 𝐺𝐺= −𝑎𝑎
𝑅𝑅𝑅𝑅
𝑑𝑑𝑑𝑑
𝑑𝑑𝑑𝑑 𝐺𝐺= −𝑎𝑎
𝑅𝑅𝑅𝑅
𝑑𝑑𝑑𝑑
𝑑𝑑𝑑𝑑 Where: G is the surface concentration (the amount of substance accumulated per unit of
interface); a is activity of the solute; dy is change in surface tension; R is universal gas
constant; Т is absolute temperature. If an increase in the solute concentration leads to a decrease in surface tension, then the
solute accumulates at the interface (positive adsorption). Conversely, if the surface tension
increases with increasing the solute concentration, then the solute is removed from the
interface (negative adsorption). Polar groups capable of hydrating, dissolving and orienting in polar solvents are called
hydrophilic; they may have sour or main character: -COOH, -OSO3H, -SO3H, -OH, -NH2, -NHR, -N (R)2-N + (R)3, R2O, R-CONHR Hydrophobic non-polar or weakly polar structural elements, soluble in non-polar
organic solvents, are most often alkyl chains (straight or branched), aromatic mono-
polycyclic groups, or alkyl aromatic radicals. In the oil-water system, the molecules of compounds having hydrophilic and
hydrophobic groups are oriented as follows: 3 3 https://doi.org/10.1051/e3sconf/202126405004 E3S Web of Conferences 264, 05004 (2021)
CONMECHYDRO - 2021 The physical characteristics of these systems are very important in terms of the
effectiveness of surfactants used as anti-corrosive agents. The physical characteristics of these systems are very important in terms of the
effectiveness of surfactants used as anti-corrosive agents. Such substances include high molecular weight fatty acids, salts of fatty and naphthenic
acids, hydroxy acids, amines, etc. The introduction of surfactants into hydrocarbon media
should, therefore, first of all, increase the wettability of metals in the oil-water system and
create conditions for inhibitors to show the main functional property. The wetting ability of
surfactants can manifest itself due to the formation of strong hydrogen bonds with water
and displacement of water from the metal surface. Before giving recommendations on the use of any additives, it is necessary to study
their mechanism of action, without knowledge of which their effective use is impossible. The chemical composition of petroleum products and the additives they contain determine
the composition and corrosiveness of the electrolyte formed during operation. When the oil burns, high molecular weight organic acids are formed, which have a
detrimental effect on metals in the presence of oxygen. Oxygen is part of peroxides;
therefore, in the presence of oxygen and water, the metal undergoes electrochemical
dissolution. In this case, the corrosion process occurs in the form of a chemical reaction: Ме + Н2О +0,5О2 = Ме(ОН)2
Ме(ОН)2+2RСООН = Ме(RСОО)2+2Н2О Ме + Н2О +0,5О2 = Ме(ОН)2 Ме(ОН)2+2RСООН = Ме(RСОО)2+2Н2О Ме(ОН)2+2RСООН = Ме(RСОО)2+2Н2О where Me is metal. where Me is metal. where Me is metal. The indicator of the corrosion resistance of the oil is the acid number, which should not
exceed 0.4 mg of caustic potassium KOH (potassium hydroxide) per 1 g of oil. The indicator of the corrosion resistance of the oil is the acid number, which should not
exceed 0.4 mg of caustic potassium KOH (potassium hydroxide) per 1 g of oil. In corrosive terms, this concentration is practically not dangerous. Due to the high
molecular weight, the acids in the fresh oil dissociate weakly, and the acids formed during
the oxidation of the oil become the most dangerous since their low molecular weight has
increased corrosiveness due to good solubility in water and better dissociation. Anti-corrosion additives are mainly polar substances that easily adsorb to metal
surfaces. Such substances include high molecular weight fatty acids, salts of fatty and
naphthenic acids, hydroxy acids, amines, etc. The most active additives of this type are 4 4 E3S Web of Conferences 264, 05004 (2021)
CONMECHYDRO - 2021 https://doi.org/10.1051/e3sconf/202126405004 surface-active compounds such as sodium salts of petroleum sulfonic acids, esters of stearic
and other fatty acids, as well as dibasic fatty acids, some nitrogen- and phosphorus-
containing compounds, for example, compounds of the RSО2NHCOO С4H9 type,
dicyclohexylamine nitrite. Additives obtained by oxidation of ceresin and petrolatum,
containing esters and internal esters, are very effective. surface-active compounds such as sodium salts of petroleum sulfonic acids, esters of stearic
and other fatty acids, as well as dibasic fatty acids, some nitrogen- and phosphorus-
containing compounds, for example, compounds of the RSО2NHCOO С4H9 type,
dicyclohexylamine nitrite. Additives obtained by oxidation of ceresin and petrolatum,
containing esters and internal esters, are very effective. Substances used as anticorrosive additives are salts of organic acids, metal phenates,
various thiophosphoric compounds, and some other surface-active compounds. The
introduction of surface-active compounds into hydrocarbon media should, therefore, first of
all, increase the wettability of metals in the oil-water system and create conditions for the
inhibitors to display their main functional properties. The following additives have practical
application. pp
Calcium Alkyl Salicylate: pp
Calcium Alkyl Salicylate: Calcium alkylaryl sulfonate: Calcium alkylaryl sulfonate: The action of alkyl sulfates depends on the structure and length of the carbon chain and
the position of the sulfo-ether group. where Me is metal. Thus, they are greatly reduced with the branching of
the carbon chain due to which alcohols and olefins with a straight chain of carbon atoms are
used for the synthesis of alkyl sulfates. The ability is observed in alkyl sulfates with a
terminal position of the sulfoester group and gradually decreases as this group is further
from the end of the chain. Nitrogen-containing compounds, aromatic amines С6Н5NH2, which are derivatives of
ammonia, deserve considerable attention as an anticorrosive additive. Nitrogen-containing compounds, aromatic amines С6Н5NH2, which are derivatives
ammonia, deserve considerable attention as an anticorrosive additive. Amines displace water from the metal surface by the following mechanism: ammonia, deserve considerable attention as an anticorrosive additive. Amines displace water from the metal surface by the following mechanism: Amines displace water from the metal surface by the following mechanism: In this case, the primary reaction between the ROO⋅ or R⋅ radical and the amine
molecule is the addition of this radical to the nitrogen atom, which has a pair of free
electrons: 5 5 https://doi.org/10.1051/e3sconf/202126405004 E3S Web of Conferences 264, 05004 (2021) CONMECHYDRO - 2021 This complex radical reacts with another radical ROO⋅ or R⋅, forming stable products,
leading to the termination of the oxidative chain. In this case, the activity of water molecules adsorbed on the metal surface will be
significantly reduced. The displacement of water from the metal surface can occur as a
result of its binding: due to solvation by metal cations, the inclusion of hydrophilic
additives in the hydration shells, and solubilization or emulsification and stabilization in the
form of water - oil product emulsions. The disadvantage of these additives is their poor
solubility in oils. E3S Web of Conferences 264, 05004 (2021)
CONMECHYDRO - 2021 E3S Web of Conferences 264, 05004 (2021)
CONMECHYDRO - 2021 With these additives, it was possible to double the oil life. To achieve the desired effect,
it is required to apply it in quantities of 1–2%. We have analyzed M-10B2 motor oils and anti-corrosive additives of dialkylphenyl
dithiophosphoric acid. After the introduction of this concentration of the additive into the
oil, we observed its dissolution. Having determined the dissolution of the additives in the
engine oil, we determined the physicochemical parameters of the engine oil for various
concentrations of additives. According to the results of laboratory studies, when the additive was introduced into the
engine oil, they gave a positive result. The results showed a fairly high level of required
properties for lubricants. In addition to the generally accepted properties, the speed of the
water-displacing, protective and antiwear properties of the oil solutions of the obtained
compounds were evaluated. From the analysis results, we selected an additive content of 1.4%, which shows the
optimal value of the base number. The alkaline properties of the oil are characterized by the
base number, determined by the amount of potassium hydroxide - KOH (caustic
potassium), which is equivalent to the amount of hydrochloric acid HCl consumed to
neutralize all the basic compounds contained in 1 g of oil. The alkaline number is expressed
in mg KOH per 1 g of oil, and, according to GOST, it should not exceed 26 mg. Fig. 1. Piston ring wear versus base number Fig. 1. Piston ring wear versus base number Fig. 1. Piston ring wear versus base number Fig. 1. Piston ring wear versus base number Fig. 1. Piston ring wear versus base number Figure 1 shows that with an increase in the neutralizing (alkaline) ability of the oil, the
wear of the piston rings sharply decreases. The base number decreases with decreasing
additive concentration. At the same time, acidic products accumulate in the oil, which
increases the corrosive wear of parts. Based on the studies carried out, it can be assumed that the obtained corrosion
inhibitors, displacing water from the metal surface, form strong adsorption-chemisorption
films on it, which prevent the metal from contacting the electrolyte. 3 Results and Discussion As an anticorrosive additive, we studied a mixture of barium alkylphenolate and a zinc salt
of dialkylphenyl dithiophosphoric acid-containing complex and internal esters. These
products, unlike other additives, dissolve well in petroleum products, possessing several
valuable properties necessary for lubricants. Research has shown that this compound has several advantages over other additives. Research has shown that this compound has several advantages over other additives. The anticorrosive activity of these additives is related to their ability to orient
themselves on the oil-water surface so that hydrophilic groups are firmly bound to water
while the hydrocarbon radical remains in the oil. The wetting ability of such additives can
be manifested through the formation of strong hydrogen bonds with water and the
displacement of water from the metal surface. The displacement of water from the metal
surface can occur as a result of its binding: due to solvation by metal cations, the inclusion
of hydrophilic additives in the hydrate shells, as well as due to solubilization or
emulsification and stabilization in the form of water - oil product emulsions. 6 https://doi.org/10.1051/e3sconf/202126405004 E3S Web of Conferences 264, 05004 (2021)
CONMECHYDRO - 2021 E3S Web of Conferences 264, 05004 (2021)
CONMECHYDRO - 2021 Thus, the mechanism of action of the proposed corrosion inhibitors based on
dialkylphenyl dithiophosphoric acid is reduced to their ability to orient themselves on the
oil-water surface so that hydrophilic groups are firmly bound to water, and the hydrocarbon
radical remains in the oil. In this case, the activity of inhibitors is the greater, the more
hydrocarbon atoms the radical contains. 7 7 E3S Web of Conferences 264, 05004 (2021)
CONMECHYDRO - 2021 https://doi.org/10.1051/e3sconf/202126405004 From the analysis results, we selected an additive content of 2.5%, which shows the
optimal value of the base number. At the same time, the base number increased to 5.3. From the analysis results, we selected an additive content of 2.5%, which shows the
optimal value of the base number. At the same time, the base number increased to 5.3. With a further increase in concentration, the viscosity increases strongly, leading to
increased frictional losses. Fig. 2. Change in oil viscosity at 1000C depending on the concentration of additives. Fig. 2. Change in oil viscosity at 1000C depending on the concentration of additives. Therefore, the main function of alkaline oil additives is to neutralize acids and protect the
metal from corrosion. Therefore, one of the signs indicating the need to change the oil may
decrease the base number. Therefore, the main function of alkaline oil additives is to neutralize acids and protect the
metal from corrosion. Therefore, one of the signs indicating the need to change the oil may
decrease the base number. With a further increase in concentration, the viscosity increases strongly, leading to
increased frictional losses. With a further increase in concentration, the viscosity increases strongly, leading to
increased frictional losses. References 1. Dzherikhov V.B. Automotive operational materials: textbook. St. Petersburg:
SPGASU, (2009) 2. Magerramov A.M., Akhmedova R.A., Akhmedova N.F. Petrochemistry and Oil
Refining: A Textbook for Higher Educational Institutions, Baku: Baki Universiteti,
(2009) (
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3. Ostrikov V.V., Kleimenov O.A., Bautin V.M. Lubricants and their quality control in
the agro-industrial complex - M.: Rosinformatekh, p. 172, (2008) 4. Alimova Z. Research of change of quality of motor oils when operating the engine
and improving their. Industrial Technology and Engineering. 3 (36), pp.11-17, (2020) 4. Alimova Z. Research of change of quality of motor oils when operating the engine
and improving their. Industrial Technology and Engineering. 3 (36), pp.11-17, (2020)
5
Danilov V F Oils lubricants and special fluids Study guide Elabuga: publishing and improving their. Industrial Technology and Engineering. 3 (36), pp.11 17, (2020)
5. Danilov V.F. Oils, lubricants and special fluids. Study guide Elabuga: publishing
house of the K (P) FU branch. (2013) 5. Danilov V.F. Oils, lubricants and special fluids. Study guide Elabuga: publishing
house of the K (P) FU branch. (2013) 6. Alimova Z. Improvement of properties of oils used in hydraulic systems of road-
construction equipment/ IOP Conference Series: Materials Science and Engineering
883, p. 012167, (2020) 6. Alimova Z. Improvement of properties of oils used in hydraulic systems of road-
construction equipment/ IOP Conference Series: Materials Science and Engineering
883, p. 012167, (2020) 7. Alimova Z. Ways to improve the properties of lubricants used in vehicles
“VNESHINVESTROM”, (2020) 8. Alimova Z. The Influence of the Process Off Oxidation of Engine Oils on Engine
Performance and Improving Antioxidant Properties, Acta of Turin Polytechnic
University in Tashkent. 8, №. 1, pp. 17, (2018) 9. Imomov S., Alimova, Z., Nuritov, I., and Temirkulova, N. Oil Purification Devices
Used in Internal Combustion Engines. International Journal of Innovative Technology
and Exploring Engineering, 9(1), pp. 3103-3107, (2019) 10. Alimova, Z., Kholikova N., and Xolova S. Ways to improve the performance of
hydraulic oils for agricultural machinery Research Archive (30). (2020) 11. Imomov, S., Kholikova, N., Alimova, Z., Nuritov, I., and Temirkulova N. Oil
Purification Devices Used in Internal Combustion Engines, Scientific Research
Archive, 30, (2020). 12. Alimova, Z. The Influence of the Process Off Oxidation of Engine Oils on Engine
Performance and Improving Antioxidant Properties. Acta of Turin Polytechnic
University in Tashkent, 8(1), p. 17, (2018) 13. Alimova, Z. 4 Conclusions 1. From the studies carried out, the following conclusion can be drawn: the corrosion
inhibitors we have obtained displace water from the metal surface and, at the same time,
form strong adsorption-chemisorption films that prevent the metal from contacting the
electrolyte. y
2. The proposed corrosion inhibitors based on dialkylphenyl dithiophosphoric acid can
orient themselves on the oil-water surface so that hydrophilic groups are firmly bound to
water, and the hydrocarbon radical remains in the oil. From the analysis results, we selected
an additive content of 2.5%, which shows the optimal value of the base number of 5.3. 2. The proposed corrosion inhibitors based on dialkylphenyl dithiophosphoric acid can
orient themselves on the oil-water surface so that hydrophilic groups are firmly bound to
water, and the hydrocarbon radical remains in the oil. From the analysis results, we selected
an additive content of 2.5%, which shows the optimal value of the base number of 5.3. 8 8 https://doi.org/10.1051/e3sconf/202126405004 E3S Web of Conferences 264, 05004 (2021)
CONMECHYDRO - 2021 References The Influence of The Process Off Oxidation of Engine Oils on Engine
Performance and Improving Antioxidant Soust, Acta of Turin Polytechnic University
in Tashkent, 8(2), pp. 50-53, (2018) ( ) pp
(
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14. Kubaymurat, I., and Gulomovna, K.K. The Impact of Automobile Tires on the
Environment from the Period of Raw Materials to the Disposal of Them, International
Journal of Recent Technology and Engineering, 8(3), (2019). 15. Ismayilov, K., and Karimova, K. Application of used automobile tires granules for
road construction in Uzbekistan. Journal of Critical Reviews, 7(12), pp. 946-948,
(2020) 16. Grigoriev M.A. Engine oil quality and engine reliability, Publishing house of
standards, p. 232, (2009) 17. Kirichenko N.B. Automotive Maintenance Materials Textbook for secondary
vocational education, Moskva, Iz.Centre "Academy", (2012) 18. Rudnik L. R. Additives for lubricants, Properties and applications, In A. M. Danilova
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Ul'yanovsk, USKHI, (1986) 9
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БМТ барқарор ривожланиш мақсадлари дастурини амалга ошириш чоғида давлат бюджети ижросида парламент назоратининг ўрни ва аҳамияти
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ARTICLE INFO The article analyzes the international legal foundations of
parliamentary control over the state budget, the participation of
national parliaments in the implementation of the UN
Sustainable Development Goals. Universal standards of effective
parliamentary oversight of the state budget are considered. Scientific and theoretical proposals on increasing the role of
parliaments of various states in controlling the national budget
in the context of the COVID-19 pandemic are presented. Article history:
Received October 2021
Received in revised form
15 October 2021
Accepted 15 November 2021
Available online
25 December 2021 Keywords:
“parliamentary diplomacy”,
state budget,
parliamentary control,
inter-parliamentary
institutions,
BRM indicators,
Political Forum,
global sustainable policy
rating,
digital revolution. 2181-1415/© 2021 in Science LLC. DOI: https://doi.org/10.47689/2181-1415-vol2-iss11/S-pp56-62
This is an open access article under the Attribution 4.0 International
(CC BY 4.0) license (https://creativecommons.org/licenses/by/4.0/deed.ru) 2181-1415/© 2021 in Science LLC. DOI: https://doi.org/10.47689/2181-1415-vol2-iss11/S-pp56-62
This is an open access article under the Attribution 4.0 International
(CC BY 4.0) license (https://creativecommons.org/licenses/by/4.0/deed.ru) Sardorbek YUSUPOV1 Tashkent State Law University БМТ барқарор ривожланиш мақсадлари дастурини амалга
ошириш чоғида давлат бюджети ижросида парламент
назоратининг ўрни ва аҳамияти БМТ барқарор ривожланиш мақсадлари дастурини амалга
ошириш чоғида давлат бюджети ижросида парламент
назоратининг ўрни ва аҳамияти 1 D.F.L (PhD), Tashkent State Law University. Tashkent, Uzbekistan. E-mail: s.yusupov020@gmail.com. The role and importance of parliamentary control in the
execution of the state budget during the implementation of the
unsustainable development goals program Жамият ва инновациялар – Общество и инновации – Society and innovations
Issue – 2 № 6 (2021) / ISSN 2181-1415 АННОТАЦИЯ Ключевые слова:
«парламентская
дипломатия»,
государственный бюджет,
парламентский контроль,
межпарламентские
институты,
индикаторы BRM,
Политический форум,
рейтинг глобальной
устойчивой политики,
цифровая революция. Ключевые слова:
«парламентская
дипломатия»,
государственный бюджет,
парламентский контроль,
межпарламентские
институты,
индикаторы BRM,
Политический форум,
рейтинг глобальной
устойчивой политики,
цифровая революция. Ключевые слова:
«парламентская
дипломатия»,
государственный бюджет,
парламентский контроль,
межпарламентские
институты,
индикаторы BRM,
Политический форум,
рейтинг глобальной
устойчивой политики,
цифровая революция. В
статье
анализируются
международно-правовые
основы парламентского контроля над государственным
бюджетом,
участие
национальных
парламентов
в
реализации Целей устойчивого развития ООН. Рассмотрены
универсальные стандарты эффективного парламентского
надзора за государственным бюджетом. Представлены
научно-теоретические предложения о повышении роли
парламентов различных государств в контроле над
национальным бюджетом в контексте пандемии COVID-19. АННОТАЦИЯ Калит сўзлар:
“парламент
дипломатияси”,
давлат бюджети,
парламент назорати,
парламентлараро
институтлар,
БРМ кўрсаткичлари,
сиёсий форум,
глобал барқарор сиёсат
рейтинги,
рақамли инқилоб. Мазкур мақолада давлат бюджети устидан парламент
назоратининг халқаро ҳуқуқий асослари, БМТ барқарор
ривожланиш мақсадларини амалга оширишда миллий парла-
ментларнинг иштироки таҳлил қилинган. Давлат бюджети
устидан самарали парламент назоратини амалга оширишда
универсал стандартлар кўриб чиқилган. COVID-19 панде-
мияси шароитида давлат бюджети устидан назоратда парла-
ментларнинг ролини ошириш бўйича илмий-назарий фикр-
лар баён қилинган. рейтинги, р
рақамли инқилоб. L (PhD), Tashkent State Law University. Tashkent, Uzbekistan. E-mail: s.yusupov020@gmail.com. SUMMARY The national budget system of each state indicates its political position and the
factors of their implementation. Today, against the background of new real problems faced
by the international community, especially in the context of the COVID-19 pandemic, there
are new trends in increasing the role of parliaments in budget control. When exercising parliamentary control, control over the State budget is one of the
most important functions. Standards and principles of international law are of great
importance in effective governance, socio-economic development of the country, ensuring
human rights and freedoms by the State and its officials, fulfilling obligations to citizens, as
well as in fulfilling international obligations. At the same time, ensuring cooperation of the Oliy Majlis of the Republic of
Uzbekistan
with
international
organizations,
inter-parliamentary
institutions,
implementation of advanced international trends in the field of budget control into
national legislation are among the topical issues. International parliamentary cooperation,
which is the main factor in the development of the institute of “parliamentary diplomacy”,
is of great importance for achieving this goal. It is known from international legal norms that the parliamentary institute as the
highest representative institution and its members are obliged not only to meet the needs
of voters, but also to ensure the rational spending of public funds of the executive branch
and their correct accounting. Ҳар бир давлат миллий бюджет тизими унинг сиёсий позициясини ва уларни
амалга ошириш омилларини кўрсатиб беради. Бугунги кунда халқаро ҳамжамият
янги реал муаммолар фонида, айниқса, COVID-19 пандемияси шароитида бюджет
назоратида парламентларнинг ролини ошириш бўйича янги тенденциялар
кузатилмоқда. Парламент назоратини амалга оширишда давлат бюджетига оид назорат энг
муҳим функцияларидан бири ҳисобланади. Мамлакатни самарали бошқариш,
ижтимоийиқтисодий ривожланиши бевосита давлат ва унинг мансабдор шахс-
ларининг инсон ҳуқуқ ва эркинликларини таъминлаш, фуқаролар олдидаги
мажбуриятларини бажариш, шунингдек, халқаро мажбуриятларни адо этишда
халқаро ҳуқуқ стандартлари ва принциплари ҳам муҳим аҳамиятга эга. 57 Жамият ва инновациялар – Общество и инновации – Society and innovations
Issue – 2 № 6 (2021) / ISSN 2181-1415 доирадаги мутахассисларнинг билим ва кўникмалари талаб этилади. Мазкур
ташкилотларнинг аъзолари давлатлар, парламентлар, бюджет назоратига масъул
давлат ташкилотлари бўлиши мумкин. Махсус компетенцияли халқаро ташкилотларни ҳам, ўз ўрнида, қуйидагича
таснифлаш мумкин: Парламентлараро Иттифоқ. Парламентлараро Иттифоқ. Молиявий назорат органларининг халқаро ташкилотлари. Молиявий назорат органларининг халқаро ташкилотлари. Минтақавий ташкилотлар Парламентлараро Иттифоқлари. Минтақавий ташкилотлар Парламентлараро Иттифоқлари. Давлатлар. Давлат институти ҳам халқаро ҳуқуқ субъекти сифатида олий
вакиллик органи тимсолида бюджет назоратининг самарали тизимини юритиш
орқали иқтисодий хавфсизликни таъминлашга эришишга ҳаракат қилади. Давлатлар. Давлат институти ҳам халқаро ҳуқуқ субъекти сифатида олий
вакиллик органи тимсолида бюджет назоратининг самарали тизимини юритиш
орқали иқтисодий хавфсизликни таъминлашга эришишга ҳаракат қилади. р
ф
р
р
БМТ барқарор ривожланиш мақсадлари кўлами ва аҳамияти нуқтаи назари-
дан халқаро ҳамжамиятнинг энг долзарб стратегик мақсадларидан бирига
айланган. Бирлашган Миллатлар Ташкилоти расмий веб-сайтида Барқарор
ривожланиш мақсадлари институтига қуйидагича таъриф берилган: “Барқарор
ривожланиш мақсадлари (кейинги ўринларда БРМ) камбағал, бой ва ўрта ривож-
ланган мамлакатларни фаровонликни яхшилаш ва сайёрамизни ҳимоя қилишга
оид амалий ҳаракатга чақиришдир. Давлатлар иқтисодий ўсишни таъминлаш,
таълим, соғлиқни сақлаш, ижтимоий ҳимоя ва бандлик соҳасидаги қатор масала-
ларни ҳал этиш, шунингдек, иқлим ўзгаришига қарши кураш ва атроф-муҳитни
муҳофаза
қилиш
борасидаги
саъй-ҳаракатлар
билан
мутаносиб
равишда
қашшоқликни бартараф этиш чоралари кўрилишини эътироф этмоқда” [2]. Бирлашган Миллатлар Ташкилоти ва парламентлараро иттифоққа парла-
ментлар билан миллий даражада, жумладан, парламентлар имкониятларини
кенгайтириш, хусусан, 2030-йилгача бўлган даврда барқарор ривожланиш соҳаси-
даги кун тартибини амалга ошириш учун бюджет маблағларини ажратиш, қонун
устуворлигини мустаҳкамлаш ва миллий қонунчиликни халқаро мажбуриятларга
мувофиқлаштиришга кўмаклашиш мақсадида янада яқин ҳамкорликни йўлга
қўйишни тавсия қилади [3]. Ушбу дастурий ҳужжатда белгиланган мақсад ва устувор вазифаларга
эришишда ҳар бир иштирокчи давлатнинг олий вакиллик органлари самарали
бюджет назоратини ўрнатиши ва Ўзбекистон Республикасининг Ихтиёрий миллий
шарҳини тақдим этган ҳолда бу борада шаффоф ҳисобдорлик тизимини яратиш,
норма ижодкорлиги жараёнига мазкур мақсад ва вазифаларни интеграциялаш
долзарб вазифалар сирасига киради. Давлат бюджети лойиҳасини ишлаб чиқишда миллий БРМни ҳисобга олиш
бюджетнинг барқарорлигини таъминлаш ва унинг устувор йўналишларини
ҳисобга олган ҳолда харажатларни оптималлаштиришга қаратилади. БМТ
барқарор
ривожланиш
мақсадларининг
мамлакатлар
бюджети
назоратидаги
ўрни
Е. Хеж,
Л. Бримонт,
Ф. Паньон
[4],
С. Кумар,
Н Кумар,
С. Вивелкадиш [5], Й. Кабанье [6], В. Белобрагин, Т. Салимова, Л. Бирюкова [7],
Т.А. Ланьшина [8] каби бир қатор олимлар томонидан тадқиқ этилган. БМТ
барқарор
ривожланиш
мақсадларининг
мамлакатлар
бюджети
назоратидаги
ўрни
Е. Хеж,
Л. Бримонт,
Ф. Паньон
[4],
С. Кумар,
Н Кумар,
С. Вивелкадиш [5], Й. Жамият ва инновациялар – Общество и инновации – Society and innovations
Issue – 2 № 6 (2021) / ISSN 2181-1415 Жамият ва инновациялар – Общество и инновации – Society and innovations
Issue – 2 № 6 (2021) / ISSN 2181-1415 Шу билан бирга, Ўзбекистон Республикаси Олий Мажлисининг халқаро
ташкилотлар, Парламентлараро институтлар билан ҳамкорлигини таъминлаш,
бюджет назоратига оид илғор халқаро тенденцияларни миллий қонунчиликка
имплементация қилиш долзарб масалалар сирасига киради. Мазкур мақсадга эришиш
“парламент дипломатияси” институтини ривожлантиришнинг асосий омили
ҳисобланган халқаро парламентнинг халқаро ҳамкорлиги муҳим аҳамиятга эга. Бундан ташқари, демократик ўзгаришлар, сиёсий тизим модернизацияси
жараёнини кўрсатиб берувчи муҳим кўрсаткич “Эзгу бошқарув” концепциясини
амалга ошириш ҳамда БМТнинг 2030-йилгача эришилиши лозим бўлган 17 та
барқарор ривожланиш мақсадлари ва 169 вазифаларни бажаришда парламент-
ларнинг бюджет жараёнида янада фаол рол ўйнаши талаб этилади. Халқаро ҳуқуқий нормалардан шу нарса маълумки, олий вакиллик
институтлари сифатида парламент институти ва унинг аъзолари нафақат
сайловчиларнинг эҳтиёжларини қондириш ва ижро ҳокимиятининг давлат
маблағларини оқилона сарфлаш ва уларни тўғри ҳисобга олишни таъминлаши
шарт. Бу борада Ўзбекистон Республикаси Президенти Ш. Мирзиёевнинг БМТ
75 йиллик юбилей саммитида қилган маърузасида барқарор тараққиёт мақсад-
ларига эришиш ва инсон ҳуқуқларини таъминлашда парламентлар ролини ошириш
тўғрисидаги Бирлашган Миллатлар Ташкилоти Бош Ассамблеясининг махсус
резолюциясини қабул қилиш ҳамда Бирлашган Миллатлар Ташкилоти шафелигида
пандемиялар даврида давлатларнинг ихтиёрий мажбуриятлари тўғрисидаги
халқаро кодексни ишлаб чиқиш [1] таклифлари билдирилган эди. Давлат бюджети устидан парламент назоратининг халқаро ҳуқуқий асослари
жуда мураккаб бўлиб, улар ўз характерига кўра биртурлилик касб этмайди. Халқаро
ҳуқуқ субъектлари (халқаро ва ихтисослашган ташкилотлар) бошқа субъектлар,
хусусан, давлат институти хулқ-атворига мақсадли таъсир кўрсатади. Бундай
таъсир ҳуқуқ ижодкорлиги ва ҳуқуқни қўллаш, марказлаштирилган ва номарказ-
лаштирилган тартибга солиш, тарафлар манфаатларининг мувофиқлаштирилиши
ва субординацияси, қаттиқ ва юмшоқ тартибга солиш методлари орқали амалга
оширилади. Мазкур нормалар, бошқа нормалар каби тақиқ, мажбурият юклаш ёки
руҳсат бериш характерида бўлиши мумкин. Давлат бюджети устидан парламент назоратига оид халқаро ҳуқуқий
муносабатлар субъектларини қуйидагича келтириш мумкин: Умумий компетенцияли халқаро ташкилотлар. Мазкур ташкилотлар умумий
халқаро ҳуқуқ ҳамда халқаро ташкилот ўз зиммасига олган мақсад ва вазифалардан
келиб чиқиб, давлат бюджети устидан парламент назоратига тегишли сифатларни
ўзида уйғунлаштиради. Улар давлат бюджетининг мақсадли сарфланиши учун
халқаро ҳамкорликнинг мувофиқлаштирувчиси функциясини бажаради. Бу борада
Бирлашган Миллатлар Ташкилоти ва унинг ихтисослаштирилган ташкилотлари
муҳим ўринни эгаллайди. Махсус компетенцияли халқаро ташкилотлар. Бизга маълумки, умумий
компетенцияли халқаро ташкилотларнинг фаолияти биз тадқиқ этаётган соҳанинг
халқаро ҳуқуқий асосларини яратиш учун етарли эмас. Бюджет назорати устидан
парламент назоратига оид услубий кўрсатмалар, модель қонунлар, халқаро
стандарт ва принциплар каби халқаро ҳуқуқий нормаларни яратиш учун тор 58 Кабанье [6], В. Белобрагин, Т. Салимова, Л. Бирюкова [7],
Т.А. Ланьшина [8] каби бир қатор олимлар томонидан тадқиқ этилган. 2018-йил 22-майда БМТ Бош Ассамблеясининг Бирлашган Миллатлар
Ташкилоти миллий парламентлар ва парламентлараро иттифоқ ўртасидаги
ҳамкорлик тўғрисидаги 72/278-сон резолюцияси [9] қабул қилинди. Мазкур
ҳужжат билан Бош Aссамблея миллий режалар ва стратегияларни амалга ошириш, 2018-йил 22-майда БМТ Бош Ассамблеясининг Бирлашган Миллатлар
Ташкилоти миллий парламентлар ва парламентлараро иттифоқ ўртасидаги
ҳамкорлик тўғрисидаги 72/278-сон резолюцияси [9] қабул қилинди. Мазкур
ҳужжат билан Бош Aссамблея миллий режалар ва стратегияларни амалга ошириш, 59 Жамият ва инновациялар – Общество и инновации – Society and innovations
Issue – 2 № 6 (2021) / ISSN 2181-1415 ишлайдиган ҳукуматнинг аниқ белгиланган функцияларини амалга оширишни
талаб қиладиган устувор инвестициялар ва тартибга солиш муаммоларини
белгилайди. ишлайдиган ҳукуматнинг аниқ белгиланган функцияларини амалга оширишни
талаб қиладиган устувор инвестициялар ва тартибга солиш муаммоларини
белгилайди. Мазкур фикрни Ж. Ву қўллаб-қувватлаган ҳолда, БРМга қаратилган тадқи-
қотлар ва ишланмалар турли фанлар бўйича тадқиқотчилар ўртасида ҳамкорликни,
шунингдек, саноат, ҳукумат ва ташкилотлар билан кенг алоқалар ва ҳамкорликни
талаб қилиши, бу эса, ўз ўрнида, янги тадқиқот маданиятини яратишга ёрдам
беришини таъкидлайди [14]. Фикримизча, юқорида келтирилган олимларнинг доктринал қарашларидан
келиб чиқиб, парламент бюджетга оид ваколатларини амалга оширишда халқаро ва
миллий илмий ҳамжамиятлар билан мулоқот платформаларини йўлга қўйиш
даркор. Бугунги кунда ахборот-коммуникация тадқиқотлари, хусусан, сақлаш
ҳажми, ҳисоблаш тезлиги, алоқа воситалари ва тармоқларни ривожлантириш каби
технологик муаммоларни ҳал этиш аксарият ривожланаётган давлатларда мавжуд
муаммолар – иқтисодий-ижтимоий муаммоларни бартараф этишнинг асосий омили
сифатида қаралмоқда. Бундан ташқари, парламентлар бюджет устидан назоратни амалга оширишда
умумий компетенцияли халқаро ташкилотлар, парламентларо институтлар,
молиявий назорат органларининг халқаро ташкилотларидан ташқари халқаро
ноҳукумат ташкилотлари билан ҳамкорлик қилиш бўйича алоҳида стратегик
дастур ишлаб чиқиши мақсадга мувофиқ. Боиси, ушбу ташкилотлар томонидан
юритиладиган рейтинг ва кўрсаткичлар ҳам бевосита БМТнинг БРМга таъсир
кўрсатади. Жамият ва инновациялар – Общество и инновации – Society and innovations
Issue – 2 № 6 (2021) / ISSN 2181-1415 шунингдек, миллий ва глобал миқёсда шаффофлик ва ҳисобдорликни таъминлаш
бўйича миллий парламентларнинг ролини эътироф этди. Резолюция Бирлашган
Миллатлар Ташкилоти ва Парламентлараро Иттифоққа барқарор ривожланиш,
инсон ҳуқуқлари, гендер тенглиги ва хотин-қизларнинг ҳуқуқ ва имкониятларини
кенгайтириш, демократия ва “эзгу бошқарув”, ахборот-коммуникация технология-
ларини ривожлантириш ва табиий офатлар хавфини камайтириш каби турли
соҳаларда яқин ҳамкорликни давом эттиришни тавсия қилди[10]. БМТ БРМнинг тадқиқ этган олимлар Е. Хеж, Л. Бримонт, Ф. Паньон фикрича,
БРМ кўрсаткичларининг давлат бюджети устидан назоратга таъсирининг
3 хусусиятини ажратиб кўрсатади. Биринчидан, мазкур кўрсаткичлар бошқарув воситаси сифатида ишлатилади. Мазкур фикрга асос сифатида О.Р. Янгнинг [11] БРМда белгиланган бошқаришнинг
ўрта ва узоқ муддатли устувор йўналишларини белгилаш орқали миллий
бюджетларни ўз ичига олган ресурсларни тақсимлашга қаратилган жамоавий
ҳаракатларга раҳбарлик қилишни асосий омил сифатида кўрсатиб ўтади. Иккинчидан,
БРМ
давлат
бошқарув
органларининг
ҳисобдорлигини
оширишга хизмат қилади. Тарихий жиҳатдан олиб қаралганда, кўрсаткичлар ва
баҳолаш демократик мунозараларда муҳим рол ўйнайди. Аниқ статистик кўрсат-
кичларга асосланган баҳолаш ҳукуматлар ва мансабдор шахсларни ўз ҳаракат-
ларини масъулиятли ва ошкора қилишга ундаши мумкин. Учинчидан, БРМ миллий бюджетларнинг бир-бирига яқин ва стандартлаш-
тиришга ёрдам бериши ва бу эса ,ўз навбатида, глобал барқарор сиёсат рейтингига
ҳисса қўшиши мумкин. Бунда аъзо давлатлар барқарор ривожланиш бўйича юқори
даражадаги сиёсий форумга тақдим этиладиган курс ҳисоботларида БРМга
мувофиқ ўз бюджетини таҳлил қилиш имконияти яратилади [12]. Фикримизча, биринчи омил бўйича БРМ кўрсаткичлари оммавий ҳаракатлар
воситаси бўлиши мумкин. Шубҳасиз, бунда давлат бошқарув органлари БРМ
кўрсаткичларини амалга оширишга қаратилган муайян мақсадлар ёки лойиҳаларга
лойиқ бюджет эҳтиёжларини аниқлаш ҳамда уларга ечим топишга қодирми ёки
йўқми, деган саволни кўтаради. Хусусан, таълим соҳаси БРМ таснифига қанчалик
мос келади? Иккинчи омил бўйича давлатлар миллий бюджетлар ва БРМ кўрсаткичлар
тизими ўртасидаги алоқаларни ўрнатиш, мамлакатнинг БРМга эришишдаги
тараққиётини кўрсатиши ва ҳукумат фаолиятини баҳолашга ёрдам бериши талаб
этилади. Учинчи омил бўйича барқарор ривожланиш бўйича юқори даражадаги сиёсий
форумга ҳисобот тақдим этган давлатлар парламентлари, шунингдек, сиёсатчилар
ва экспертлар ўртасида фикр алмашиш, миллий бюджетнинг сарфланиши бўйича
жамоавий ақлий мунозараларнинг халқаро даражада ташкил этилиши глобал
барқарор ривожланишга ўтишда муҳим аҳамиятга эга. Бундан ташқари, демократик
институтлар, фуқаролик жамияти институтлари жамоатчилик назорати асосида
мазкур жараёнда иштирок этишлари мумкин. БРМ кўрсаткичларининг миллий бюджетда акс этишини тадқиқ этган
олимлар жамоаси Ҳ. Шмидт, Л. Гостин, Э. Эмануэл[13] глобал миқёсда барқарор
ривожланиш учун “рақамли инқилоб” талаб этилиши хусусидаги назарияни илгари
суради. Ҳар бир соҳадаги трансформация бизнес ва фуқаролик жамияти билан 60 ФОЙДАЛАНИЛГАН АДАБИЁТЛАР РЎЙХАТИ: ФОЙДАЛАНИЛГАН АДАБИЁТЛАР РЎЙХАТИ: 1. Ўзбекистон Республикаси Президенти Шавкат Мирзиёевнинг Бирлашган
Миллатлар Ташкилоти Бош Ассамблеясининг 75-сессиясидаги нутқи // URL:
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the Sustainable Development Goals: lessons for policy in Commonwealth countries //
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целей устойчивого развития ООН // Стандарты и качество. – 2019. – Т. 985. – №. 7. 7. Белобрагин В., Салимова Т., Бирюкова Л. Стандартизация в достижении
целей устойчивого развития ООН // Стандарты и качество. – 2019. – Т. 985. – №. 7. 8. Ланьшина Т.А. Опыт локализации и внедрения Целей устойчивого
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организаций: образование, наука, новая экономика. – 2019. – Т. 14. – №. 1. 8. Ланьшина Т.А. Опыт локализации и внедрения Целей устойчивого
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Measuring High Levels of Total Suspended Solids and Turbidity Using Small Unoccupied Aerial Systems (sUAS) Multispectral Imagery
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Received: 27 July 2020; Accepted: 4 September 2020; Published: 8 September 2020 Abstract: Due to land development, high concentrations of suspended sediment are produced from
erosion after rain events. Sediment basins are commonly used for the settlement of suspended
sediments before discharge. Stormwater regulations may require frequent sampling and monitoring
of these basins, both of which are time and labor intensive. Potential remedies are small, unoccupied
aerial systems (sUAS). The goal of this study was to demonstrate whether sUAS multispectral
imagery could measure high levels of total suspended solids (TSS) and turbidity in a sediment basin. The sediment basin at the Auburn University Erosion and Sediment Control Testing Facility was
used to simulate a local 2-year, 24-h storm event with a 30-min flow rate. Water samples were
collected at three depths in two locations every 15 min for six hours with corresponding sUAS
multispectral imagery. Multispectral pixel values were related to TSS and turbidity in separate
models using multiple linear regressions. TSS and turbidity regression models had coefficients
of determination (r2) values of 0.926 and 0.851, respectively. When water column measurements
were averaged, the r2 values increased to 0.965 and 0.929, respectively. The results indicated that
sUAS multispectral imagery is a viable option for monitoring and assessing sediment basins during
high-concentration events. Keywords: remote sensing; sUAS; multispectral imagery; sediment basin; suspended sediment;
total suspended solids; turbidity Measuring High Levels of Total Suspended Solids
and Turbidity Using Small Unoccupied Aerial
Systems (sUAS) Multispectral Imagery Elizabeth M. Prior 1,2,*
, Frances C. O’Donnell 2
, Christian Brodbeck 3, Wesley N. Donald 2,
George Brett Runion 4 and Stephanie L. Shepherd 5 1
Department of Biological Systems Engineering, Virginia Tech, Blacksburg, VA 24061, USA
2
Department of Civil and Environmental Engineering, Auburn University, Auburn, AL 36849, USA;
fco0002@auburn.edu (F.C.O.); donalwn@auburn.edu (W.N.D.)
3
Department of Biosystems Engineering, Auburn University, Auburn, AL 36849, USA; brodbcj@auburn.edu
4
USDA-ARS National Soil Dynamics Laboratory, Auburn, AL 36832, USA; brett.runion@usda.gov
5
Department of Geosciences, Auburn University, Auburn, AL 36849, USA; slshepherd@auburn.edu
*
Correspondence: eprior@vt.edu 1
Department of Biological Systems Engineering, Virginia Tech, Blacksburg, VA 24061, USA
2
Department of Civil and Environmental Engineering, Auburn University, Auburn, AL 36849, USA;
fco0002@auburn.edu (F.C.O.); donalwn@auburn.edu (W.N.D.) drones drones drones drones Drones 2020, 4, 54; doi:10.3390/drones4030054 Keywords: remote sensing; sUAS; multispectral imagery; sediment basin; suspended sediment;
total suspended solids; turbidity 1. Introduction TSS is a more direct
measure of the amount of solids suspended and is usable for sediment mass balance considerations
and sedimentation rate calculations. Remote sensing could aid in water quality monitoring of sediment basins, but high spatial
resolution and financial viability are necessary for practical use. Acoustic doppler current profilers
(ADCP) along with satellite remote sensing are not viable options. ADCPs are not widely used, can only
be utilized at the scale of large river reaches and are expensive [18]. Satellite remote sensing has been
used to roughly estimate TSS concentrations, however, it requires long-term in situ TSS measurements
and has coarse spatial resolution that can only be applied to coastal areas, oceans, large rivers or large
lakes [18,19]. Satellite remote sensing for water quality purposes also fails to address the inherent
variability within a low-resolution pixel due to various factors, such as unsteady flows, sediment
transport dynamics and wind influence [20]. Capturing images from a fixed camera would prove to be
difficult and cumbersome for several reasons. The initial setting up of the system would delay data
collection as well as potentially make the surrounding space unsafe for construction efforts by adding
new overhead obstructions. A new setup would be required at each sediment basin. It is essential that
the camera angle be nadir to the ground to ensure pixels are not warped which would misrepresent
the water quality measurements. Additionally, these systems would be required to stay standing
through any type of weather. Wind could potentially move the camera offnadir or push down the
structure completely. Rain could damage the sensor, and rain or high humidity could produce droplets
of water on the lens, thus preventing sound data collection. Lastly, cameras would need to capture
images during overcast days or when minimum shadows are obstructing the sediment basin to ensure
excessive shadowing and sun glint are avoided. This would either require manual operation or setting
up a timer and manually selecting the optimum images. One data collection method that would allow
the user flexibility for data collection at any time along with control of image resolution would be the
use of small unoccupied aerial systems (sUAS). As a fairly new tool, sUAS have become popular remote sensing platforms due to their
high-resolution data, low cost, and maneuverability [21–23]. However, there have been few sUAS
studies on water quality monitoring. 1. Introduction Stormwater runofffrom urban development and construction sites is and will continue to be an
increasing threat to water quality [1–4]. These flows are often laden with high sediment concentrations
that cause siltation and sedimentation of downstream waterways. Higher amounts of suspended solids
negatively affect aquatic life, transfer other contaminants and require more treatment for potability [5–7]. Further siltation and sedimentation can be expected with the acceleration of urbanization, increases in
population and global climate change [8–10]. In response to water quality issues, the United States of America (USA) established the National
Pollutant Discharge Elimination System (NPDES) through the Clean Water Act of 1972 [11]. In turn,
various stormwater regulations were established, specifically the Construction General Permit for land Drones 2020, 4, 54; doi:10.3390/drones4030054 www.mdpi.com/journal/drones www.mdpi.com/journal/drones 2 of 15 Drones 2020, 4, 54 development which requires construction sites to implement stormwater pollution prevention plans
(SWPPP) that will lessen erosion, sedimentation/siltation and the pollution of stormwater [12,13]. p
The requirement to develop SWPPPs and increased societal concern about water quality have led
to the emergence of technologies and best management practices that address water quality needs. For construction sites specifically, erosion and sediment control (ESC) techniques, such as hydroseeding,
erosion control blankets and silt fences, are being used [14]. One of the most used ESC methods is the
sediment basin which collects stormwater and allows sediment to settle before water is discharged
into streams [13]. There is a lack of agency guidance and few studies that detail monitoring protocols
for performance analysis and design configuration testing [15,16]. Sediment basin monitoring may be
necessary to demonstrate compliance with NPDES permits. Traditional sediment basin monitoring
involves a turbidity analysis of manually collected water samples. Many states use turbidity to ensure
construction sites are meeting the maximum water quality requirement or effluent limitation guidelines. Most states hold their own permit and are in charge of ensuring that water quality within their state
meet federal NPDES requirements. Turbidity is often used as the main water quality parameter
since it is less labor intensive to measure than total suspended solids (TSS), but it is not a direct
measurement since cloudiness is a function of suspended solids. Turbidity only considers the amount
of light scattered by suspended solids. Turbidity, which is reliant on stream velocity and flow, should
only be compared to water samples that were taken at similar flow events [17]. 1. Introduction Thermal imagery has been used for contamination detection
and monitoring [24–27]. sUAS multispectral imagery has been used for turbidity monitoring in
small reservoirs with little success because of model overfit [28] and on small lakes with coarser
resolution [29]. Additionally, sUAS multispectral imagery, along with a spectroradiometer, were used
for TSS monitoring at different depths in the Maumee River, Ohio, USA [30]. Models were developed
for each depth, but achieved limited coefficients of determination (r2) values and significance. A similar 3 of 15 Drones 2020, 4, 54 Drones 2020, 4, 54 study was conducted in a stream, measuring turbidity along with multispectral flights before and
after a human-induced sediment plume [31]. No multi-variable modeling was performed; the focus
was only to analyze single bands or single-band ratios. Sampling with respect to exact location and
depth was not achieved with no account for how the streambed might influence results. Despite these
limitations, results agreed with similar previous studies such as [29,32]. Prior et al. [32] also used
sUAS multispectral imaging for TSS and turbidity modeling, but in a small stream setting along with
cross-site model testing. Low and high flow events were modeled separately because different single
bands and band ratios were significant. Cross-site model testing achieved no correlation, indicating that
site-specific models are required. Depth and streambed reflectance were also suggested to influence
results when shallow, clear water was present. This study discovered that one main limitation of sUAS
multispectral remote sensing was that streambed reflectance will interfere when suspended sediment
concentrations are relatively low. sUAS multispectral monitoring of TSS and turbidity might be a better
application for sediment basin monitoring since high levels of suspended sediments are expected. To the best of our knowledge, there has been no sUAS multispectral imagery study involving high
sediment concentrations and sampling within a sediment basin. The goal of this study was to develop statistical models that relate high levels of TSS and turbidity
to sUAS multispectral imagery for sediment basin monitoring. The study was conducted using
an experimental sediment basin at the Auburn University Erosion and Sediment Control Testing
Facility (AU-ESCTF) [14]. At two locations in the basin, automatic samplers collected water samples at
three depths in the water column every 15 min (min) during a 30-min inflow that simulated runoff
from a local 2-year (y), 24-hour (h) storm event. Concurrently, sUAS flights collected corresponding
multispectral imagery. 1. Introduction This testing was done for a total of six hours. Water samples were processed for
TSS and turbidity. Models were developed using linear regressions that related pixel values back to
TSS and turbidity. 2.1. Study Site A large-scale experimental sediment basin was designed and constructed at the AU-ESCTF to
understand basin performance under various conditions and configurations and to test sediment
control devices. The basin measured 13.4 m × 4.9 m along the bottom, with a total excavated footprint
of 17.1 m × 8.5 m. The depth of the basin was approximately 1.07 m and total storage, as built,
was 79.0 m3. The sediment basin and inflow channel were lined with a tied concrete block map and an
8-ounce (0.23 kg) nonwoven filter fabric. Three rows of wire-backed coir baffles attached to metal posts
divided the basin into four sections. The baffle installation was done following recommendations from
the Alabama Department of Transportation. A lamella plate settler, along with a fence near one of
the baffles, was located in the second to last downstream section (brown rectangle in basin; Figure 1). The design details and layout of the basin were previously described in Perez et al. [33]. 2.2. sUAS Setup For this study, the sUAS platform (Table 1) consisted of a DJI Phantom 4 (DJI Ltd., Shenzhen,
China) with a mounted Parrot Sequoia (Parrot Drone SAS, Paris, France) multispectral sensor package. A solar radiation sensor was also included in the sensor package for autocalibration along with four
band sensors capable of receiving green (G), red (R), red edge (RE) and near-infrared (NIR) spectral
bands. One-point calibration plus the solar radiation sensor were used for radiometric calibration
using a test pattern with known reflectance [34]. 4 of 15
Figure Drones 2020, 4, 54
of the baffles, w Figure 1. Map of the Auburn University Erosion and Sediment Control Testing Facility sediment basin
h
h
d
d
h
l
l
d
l
f
ll Figure 1. Map of the Auburn University Erosion and Sediment Control Testing Facility sediment basin
where the experiment was conducted. The sampling locations, ground control points for spatially
referencing the drone flights and the outlet of the sediment basin are shown. Figure 1. Map of the Auburn University Erosion and Sediment Control Testing Facility sediment basin
where the experiment was conducted. The sampling locations, ground control points for spatially
referencing the drone flights and the outlet of the sediment basin are shown. Table 1. Small, unoccupied aerial systems (sUAS) platform specifications [35,36]. Table 1. Small, unoccupied aerial systems (sUAS) platform specifications [35,36]. Aircraft
Sensor
Band Span (nm)
Weight (g)
1380
72
Green:
480 to 520
Red:
640 to 680
Size (mm)
350
59 × 41 × 28
Red Edge:
730 to 810
Near-infrared:
770 to 810 2.3. Data Collection This sampling pattern was repeated for a
total of six hours, which resulted in 24 flight samples from each of two locations in the sediment basin. 76 m altitude. These parameters were selected to ensure high resolution along with a quick flight that
could be easily done within the 15 min sampling window. This sampling pattern was repeated for a
total of six hours, which resulted in 24 flight samples from each of two locations in the sediment basin. 2.4. Data Processing Water samples were processed in the laboratory for turbidity and TSS. TSS was measured following
the U.S. EPA Method 160.2 [38]. Each filter (Whatman® glass microfiber filters, Grade 934-AH® RTU)
was pre-rinsed, dried and weighed. Each sample was shaken to resuspend sediments and 250 mL
was suctioned through a filter, which was then removed from the filtering apparatus and placed in a
drying oven (103 ◦C) until a constant weight was achieved. Turbidity was measured following the
U.S. EPA Method 180.1 [39] using a Hach 2100Q turbidimeter (Hach, Loveland, CO, USA) calibrated
according to manufacturer’s recommendations. Each sample was thoroughly mixed, and 5 mL of
sample was diluted such that turbidity readings were below 40 nephelometric turbidity units (NTU). Four turbidity measurements were averaged for each water sample. For multispectral imagery processing, Pix4Dmapper (Pix4D, Prilly, Switzerland) was used for
radiometric calibration, importing and locating GCPs and orthomosaic construction. Atmospheric
correction was not applied since the sUAS was flown at 76 m above the ground which resulted in a
small atmospheric column, especially when compared to satellite atmospheric columns. Because of the
low flight altitude, the discrepancy of radiance at the sensor and at the water surface was negligible
and was disregarded [40]. Pixel values for each band were then determined using the recorded GPS
locations for each water sample and the zonal statistics tool in ArcGIS (ESRI, Redlands, CA, USA). 2.3. Data Collection Data collection was conducted on 27 May 2019. Before initiating testing, three ground control
point (GCPs) targets were placed at various locations around the sediment basin (Figure 1). GCPs were
used to ensure the geospatial accuracy of the imagery [37]. The GCP locations were selected to ensure
even, horizontal distribution while accounting for vertical undulations and confirming a clear view
of the sky. GCP targets and water sampling locations were measured and recorded using a Trimble
Geo7x Global Positioning System running TerraSync (Trimble Navigation Limited, Westminster, CO,
USA) rated at 1 cm accuracy. A total of six ISCO automatic samplers (Teledyne ISCO, Lincoln, NE,
USA) were used to collect water samples. Three samplers were used to collect water from the furthest
upstream section and three were used to collect from the furthest downstream section of the sediment
basin. Water samples were collected at the water surface and at 46 cm and 69 cm below the water
surface. Once initiated, the samplers collected 500 mL of water at fifteen minutes intervals for a total of
six hours. This resulted in 24 samples per depth location (i.e., 144 samples total). The sediment basin was filled at a flow rate of 0.042 m3/s for 30 min which simulated runofffrom
a local Alabama 2-y, 24-h storm event for a 979.3 m2 drainage area. Sediment was introduced at a rate
of 20.5 kg/min which simulated bare soil conditions based on the modified universal soil loss equation
with a soil erodibility factor of 0.085 and cover practice factor of 1.0 (i.e., worst-case scenario of no
vegetation cover). Stockpile soil, classified as sandy loam (59.5% sand, 24.0% silt, 16.5% clay) by the
USDA soil classification system, was sieved using a 1.3-cm screen to remove any large organic material
and particles. All procedures related to calculating and maintaining consistent flow and sediment
introduction rates, system setup, and sediment basin specifics can be found in Perez et al. [33]. Filling
the basin took approximately 35 min. Once filled, flow and sediment introduction were stopped. Flights and automatic water collection
were conducted every fifteen min. The sUAS was then flown with a side and front overlap of 80% at 5 of 15 Drones 2020, 4, 54 Drones 2020, 4, 54 76 m altitude. These parameters were selected to ensure high resolution along with a quick flight that
could be easily done within the 15 min sampling window. 3.1. Turbidity and TSS Turbidity and TSS results show the settling of particles over the six-h time period and also
throughout the water column (Figure 2). The TSS results (Figure 2a) showed a slightly more rapid
decline at the beginning and flatten out towards the end compared to turbidity (Figure 2b). This can
also be seen in the graphs showing space and time (Figure 2c,d). The first few measurements in the
water column closest to the inlet were similar, but rapidly diverge, indicating settlement over time. The first measurement in the water column furthest from the inlet was much lower than the initial
measurement in the first water column. This is due to larger solids settling quickly upon entry into
the basin, resulting in higher measurements near the front of the basin. The water column was more
uniform near the outlet, demonstrating that TSS and turbidity reached equilibrium towards the rear of
the sediment basin. Figure 2. TSS and turbidity from the Auburn University Erosion and Sediment Control Testing Facility
(AU-ESCTF): (a) averaged TSS of water column from the two sampling locations over time, (b) averaged
turbidity of water column from the two sampling locations over time, (c) TSS over time and space with
0 m being furthest upstream and closest to the inlet, (d) turbidity over time and space with 0 m being
furthest upstream and closest to the inlet. Figure 2. TSS and turbidity from the Auburn University Erosion and Sediment Control Testing Facility
(AU-ESCTF): (a) averaged TSS of water column from the two sampling locations over time, (b) averaged
turbidity of water column from the two sampling locations over time, (c) TSS over time and space with
0 m being furthest upstream and closest to the inlet, (d) turbidity over time and space with 0 m being
furthest upstream and closest to the inlet. Figure 2. TSS and turbidity from the Auburn University Erosion and Sediment Control Testing Facility
(AU-ESCTF): (a) averaged TSS of water column from the two sampling locations over time, (b) averaged
turbidity of water column from the two sampling locations over time, (c) TSS over time and space with
0 m being furthest upstream and closest to the inlet, (d) turbidity over time and space with 0 m being
furthest upstream and closest to the inlet. 2.5. Statistical Analysis RMSE =
sPn
i = 1(Pi −Oi)2
n
(1)
RPD =
σO
RMSEP
(2)
MNB = 1
n
n
X
i = 1
(Pi −Oi)
Oi
× 100%
(3) (1) RPD =
σO
RMSEP
MNB = 1
n
n
X
i = 1
(Pi −Oi)
Oi
× 100% (2) MNB = 1
n
n
X
i = 1
(Pi −Oi)
Oi
× 100%
(3) (3) 6 of 15 Drones 2020, 4, 54 Drones 2020, 4, 54 where:
RMSE = root mean square error;
n = number of observations;
i = a value in a dataset;
Pi = predicted value;
Oi = observed value;
RPD = residual prediction deviation;
σO = standard deviation of the observed variable;
RMSEP = root mean square error of the predicted value;
MNB = mean normalized bias. 2.5. Statistical Analysis To develop unique models for the sediment basin, correlation tests between turbidity and band
pixel values and between TSS and band pixel values were conducted for all single bands and possible
band ratios: G, R, RE, NIR, G/R, G/RE, G/NIR, R/G, R/RE, R/NIR, RE/G, RE/R, RE/NIR, NIR/G,
NIR/R and NIR/RE. Correlation tests were conducted considering all water samples and the averaged
water column values. The averaged water column value was calculated as the mean of water sample
measurements from the surface, middle and bottom of the water column. From the correlation analyses, four single bands and band ratios were identified that had the
highest and most significant correlation coefficients (r) for turbidity and TSS. These four band variables
were then used in a stepwise linear regression procedure to determine the combination that produced
the highest r2 value while including only model variables with probability (p) values less than 0.05. Once the best models were selected for each data type, each model was tested for overfitting by
randomly excluding 20% of the data followed by reprocessing this dataset through the stepwise linear
regression. The revised model was fit to the removed data points and was deemed not to be overfit if
the r2 value for the removed data was within 0.1 of the r2 of the original model. Additionally, the root
mean square error (RMSE), residual prediction deviation (RPD) and mean normalized bias (MNB) were
calculated (Equations (1)–(3), respectively). All statistical analyses were performed in SAS (SAS, Cary,
NC, USA) [41,42] and Matlab 2020a (Mathworks, Natick, MA, USA). The above methods of developing
unique TSS and turbidity models from multispectral data followed procedures from Prior et al. [32]. 3.1. Turbidity and TSS Table 2 shows the progressive decrease in suspended sediment through TSS and turbidity
measurements along with the response of single-band spectral data at the two sampling locations in
the basin. From Table 2, a decreased spectral reflectance can be seen over time as turbidity and TSS 7 of 15
8
8 Drones 2020, 4, 54
0.7
1.0 decreases. This was expected since there were fewer particles suspended to reflect light at lower TSS
levels [30,43,44]. Additionally, Table 2 shows that the red band had consistently higher reflectance
compared to the other bands. This was also expected since relatively high concentrations of sediment
were present during this study. Water with high levels of suspended sediment has been found to
exhibit higher reflectances in all wavelengths, especially in the red and NIR channels [45,46]. 1.50
0.1141
0.2355
0.1608
0.1514
204.4
315.00
208.8
333.90
212.8
373.80
1.75
0.1071
0.2196
0.1434
0.1310
172.8
295.58
190.8
319.20
212.4
352.80
2.00
0.1157
0.2293
0.1497
0.1401
155.6
284.55
184.4
322.88
201.6
347.55
2.25
0.1146
0.2235
0.1439
0.1388
143.2
270.90
178.0
307.13
188.8
345.45
2.50
0.1123
0.2198
0.1422
0.1312
128.0
253.58
163.2
281.93
187.2
358.05
2.75
0.1071
0.2055
0.1341
0.1273
102.0
233.10
158.8
287.18
162.0
353.85 Table 2. Band and water quality measurements shaded to indicate percentile relative to full dataset. 3.1. Turbidity and TSS Sampling Location Closest to the Inlet
Time (h)
Green
Red
RE
NIR
TSS
Surface (mg/L)
Turbidity
Surface (NTU)
TSS
Middle(mg/L)
Turbidity
Middle (NTU)
TSS
Bottom(mg/L)
Turbidity
Bottom (NTU)
0.25
0.1067
0.2428
0.2503
0.2193
748.4
803.78
716.8
746.55
699.6
769.65
0.75
0.1274
0.2648
0.1880
0.1714
278.4
417.38
309.2
441.53
329.2
522.38
1.00
0.1275
0.2595
0.1858
0.1708
225.6
392.70
250.8
391.13
294.0
442.58
1.25
0.1162
0.2407
0.1632
0.1505
175.2
379.58
228.0
374.33
232.4
411.60
1.50
0.1141
0.2355
0.1608
0.1514
204.4
315.00
208.8
333.90
212.8
373.80
1.75
0.1071
0.2196
0.1434
0.1310
172.8
295.58
190.8
319.20
212.4
352.80
2.00
0.1157
0.2293
0.1497
0.1401
155.6
284.55
184.4
322.88
201.6
347.55
2.25
0.1146
0.2235
0.1439
0.1388
143.2
270.90
178.0
307.13
188.8
345.45
2.50
0.1123
0.2198
0.1422
0.1312
128.0
253.58
163.2
281.93
187.2
358.05
2.75
0.1071
0.2055
0.1341
0.1273
102.0
233.10
158.8
287.18
162.0
353.85
3.00
0.1079
0.2069
0.1309
0.1230
110.0
217.35
142.8
270.38
178.0
323.93
3.25
0.1023
0.1980
0.1268
0.1222
98.0
206.64
130.4
250.95
170.0
302.93
3.50
0.1058
0.2082
0.1293
0.1270
86.8
193.04
106.4
260.40
168.8
308.18
3.75
0.1084
0.2143
0.1323
0.1294
85.2
186.17
134.0
250.43
158.0
318.15
4.00
0.1066
0.2137
0.1293
0.1268
66.4
171.36
129.6
254.63
137.6
301.88
4.75
0.1427
0.2729
0.1787
0.1503
78.0
162.28
112.8
225.75
141.6
266.70
5.00
0.1055
0.2089
0.1276
0.1182
77.6
163.96
110.4
215.78
144.0
273.00
5.50
0.1231
0.2352
0.1386
0.1370
73.6
169.42
105.2
215.25
136.0
260.93
5.75
0.1152
0.2465
0.1393
0.1364
72.4
161.12
101.2
209.58
133.2
269.33
6.00
0.1200
0.2020
0.1271
0.1201
68.0
169.58
101.6
195.72
128.4
270.38
Sampling Location Furthest from the Inlet
Time (h)
Green
Red
RE
NIR
TSS
Surface (mg/L)
Turbidity
Surface (NTU)
TSS
Middle(mg/L)
Turbidity
Middle (NTU)
TSS
Bottom(mg/L)
Turbidity
Bottom (NTU)
0.25
0.1215
0.2659
0.2111
0.1888
296.8
399.00
371.2
486.68
348.4
488.00
0.75
0.1259
0.2669
0.1716
0.1588
182.4
322.35
244.8
404.78
270.0
405.60
1.00
0.1297
0.2631
0.1694
0.1575
164.0
277.73
218.4
364.35
225.6
348.80
1.25
0.1154
0.2335
0.1402
0.1287
135.2
267.75
189.2
343.35
209.6
340.00
1.50
0.1157
0.2363
0.1488
0.1404
130.4
246.75
162.8
300.83
186.8
317.20
1.75
0.1097
0.2174
0.1345
0.1236
125.6
251.48
142.4
270.90
182.8
322.00
2.00
0.1161
0.2260
0.1393
0.1303
117.6
224.70
132.0
254.10
165.6
299.60
2.25
0.1169
0.2240
0.1391
0.1354
114.8
240.45
114.0
241.50
153.2
309.60
2.50
0.1161
0.2204
0.1318
0.1229
104.0
218.40
112.8
217.88
142.0
278.80
2.75
0.1142
0.2188
0.1287
0.1188
86.8
206.59
104.0
217.88
127.6
268.00
3.00
0.1100
0.2052
0.1238
0.1162
84.8
211.37
100.0
202.65
117.2
229.60
3.25
0.1056
0.2041
0.1205
0.1125
94.8
191.99
101.2
197.66
104.8
209.20
3.50
0.1105
0.2165
0.1252
0.1172
82.4
183.02
98.0
198.35
102.8
224.40
3.75
0.1114
0.2125
0.1248
0.1189
80.4
195.98
95.6
202.97
100.0
213.20
4.00
0.1101
0.2132
0.1203
0.1137
90.0
185.69
92.4
179.81
95.2
200.00
4.25
0.1725
0.3314
0.1628
0.1636
82.4
187.32
87.6
194.46
84.4
191.60
5.00
0.1098
0.2090
0.1163
0.1070
78.0
177.40
89.2
181.76
83.2
186.80
5.50
0.1271
0.2303
0.1266
0.1183
76.0
183.02
80.4
174.62
83.2
186.80
5.75
0.1295
0.2630
0.1432
0.1274
71.2
167.84
78.8
170.73
79.6
178.00
6.00
0.1385
0.2136
0.1319
0.1241
70.0
161.39
78.4
170.10
78.4
170.00
3.00
0.1079
0.2069
0.1309
0.1230
110.0
217.35
142.8
270.38
178.0
323.93
3.25
0.1023
0.1980
0.1268
0.1222
98.0
206.64
130.4
250.95
170.0
302.93
3.50
0.1058
0.2082
0.1293
0.1270
86.8
193.04
106.4
260.40
168.8
308.18
3.75
0.1084
0.2143
0.1323
0.1294
85.2
186.17
134.0
250.43
158.0
318.15
4.00
0.1066
0.2137
0.1293
0.1268
66.4
171.36
129.6
254.63
137.6
301.88
4.75
0.1427
0.2729
0.1787
0.1503
78.0
162.28
112.8
225.75
141.6
266.70
5.00
0.1055
0.2089
0.1276
0.1182
77.6
163.96
110.4
215.78
144.0
273.00
5.50
0.1231
0.2352
0.1386
0.1370
73.6
169.42
105.2
215.25
136.0
260.93
5.75
0.1152
0.2465
0.1393
0.1364
72.4
161.12
101.2
209.58
133.2
269.33
6.00
0.1200
0.2020
0.1271
0.1201
68.0
169.58
101.6
195.72
128.4
270.38
Sampling Location Furthest from the Inlet
Time
(h)
Green
Red
RE
NIR
TSS
Surface
(mg/L)
Turbidity
Surface
(NTU)
TSS
Middle
(mg/L)
Turbidity
Middle
(NTU)
TSS
Bottom
(mg/L)
Turbidity
Bottom
(NTU)
0.25
0.1215
0.2659
0.2111
0.1888
296.8
399.00
371.2
486.68
348.4
488.00
0.75
0.1259
0.2669
0.1716
0.1588
182.4
322.35
244.8
404.78
270.0
405.60
1.00
0.1297
0.2631
0.1694
0.1575
164.0
277.73
218.4
364.35
225.6
348.80
1.25
0.1154
0.2335
0.1402
0.1287
135.2
267.75
189.2
343.35
209.6
340.00
1.50
0.1157
0.2363
0.1488
0.1404
130.4
246.75
162.8
300.83
186.8
317.20
1.75
0.1097
0.2174
0.1345
0.1236
125.6
251.48
142.4
270.90
182.8
322.00
2.00
0.1161
0.2260
0.1393
0.1303
117.6
224.70
132.0
254.10
165.6
299.60
2.25
0.1169
0.2240
0.1391
0.1354
114.8
240.45
114.0
241.50
153.2
309.60
2.50
0.1161
0.2204
0.1318
0.1229
104.0
218.40
112.8
217.88
142.0
278.80
2.75
0.1142
0.2188
0.1287
0.1188
86.8
206.59
104.0
217.88
127.6
268.00
3.00
0.1100
0.2052
0.1238
0.1162
84.8
211.37
100.0
202.65
117.2
229.60
3.25
0.1056
0.2041
0.1205
0.1125
94.8
191.99
101.2
197.66
104.8
209.20
3.50
0.1105
0.2165
0.1252
0.1172
82.4
183.02
98.0
198.35
102.8
224.40
3.75
0.1114
0.2125
0.1248
0.1189
80.4
195.98
95.6
202.97
100.0
213.20
4.00
0.1101
0.2132
0.1203
0.1137
90.0
185.69
92.4
179.81
95.2
200.00
4.25
0.1725
0.3314
0.1628
0.1636
82.4
187.32
87.6
194.46
84.4
191.60
5.00
0.1098
0.2090
0.1163
0.1070
78.0
177.40
89.2
181.76
83.2
186.80
5.50
0.1271
0.2303
0.1266
0.1183
76.0
183.02
80.4
174.62
83.2
186.80
5.75
0.1295
0.2630
0.1432
0.1274
71.2
167.84
78.8
170.73
79.6
178.00
6.00
0.1385
0.2136
0.1319
0.1241
70.0
161.39
78.4
170.10
78.4
170.00
0th percentile
100th percentile
3 2 Regression Model Development
0th percentile
100th percentile Table 2. 3.1. Turbidity and TSS Band and water quality measurements shaded to indicate percentile relative to full dataset. 3.00
0.1079
0.2069
0.1309
0.1230
110.0
217.35
142.8
270.38
178.0
323.93
3.25
0.1023
0.1980
0.1268
0.1222
98.0
206.64
130.4
250.95
170.0
302.93 er quality measurements shaded to indicate percentile relative to full dataset. 0.2069
0.1309
0.1230
110.0
217.35
142.8
270.38
178.0
323.93
0.1980
0.1268
0.1222
98.0
206.64
130.4
250.95
170.0
302.93 g
The initial analysis w
3.2. Regression Model Development g
The initial analysis w
3.2. Regression Model Development The first set of models considered all water column measurements, while the second set of models
The initial analysis was conducted with TSS and turbidity values from all sampling locations. The first set of models considered all water column measurements, while the second set of models
considered the water column averages. By testing the correlations between multispectral parameters
versus turbidity and TSS, the four single bands and/or band ratios with the highest r values and
lowest p values were identified (i.e., band, r and p value rows in Table 2). All possible combinations
(ranging from one variable to all four variables) of the selected four bands and/or band ratios were
then inputted as predictive variables in a stepwise linear regression procedure. The best r2 results
were those that included all four variables. Linear regression model coefficients and statistics can be
found in Table 3. The strongest and most significant correlations were all with band ratios and were
consistent between models. Turbidity models had lower r2 values than corresponding TSS models due
to the lower r values of NIR/R and RE/R. Turbidity r values for NIR/R and RE/R increased to greater 8 of 15 Drones 2020, 4, 54 than 0.9 when averaged values were used. These findings were reinforced by linear regression values
and comparisons of modeled predictions and measurements (Figure 3). In Figure 3, the linear fit is
consistent across a wide range of measurements with the mid-water column measurements tending
to be closest to the linear fit. Sediment maps were generated by applying the models to the entire
sediment basin. Sediment maps using the average TSS and turbidity models were applied at various
times to the sediment basin area (Figure 4). Sediment maps using the total TSS and turbidity models
were applied at various times to the sediment basin area (Figure 5). Note that the blue rectangle in
each map is the lamella plate settler and the blue shading that perpendicularly transects the sediment
basin is three rows of wire backed coir baffles attached to metal posts dividing the basin into four
sections. Lastly, reflectance values along with TSS and turbidity measurements from this study and
from Prior et al. [32] were compared (Figure 6). Table 3. Results of correlation analysis and linear regression. g
The initial analysis w
3.2. Regression Model Development TSS
Band
RE/G
NIR/G
NIR/R
RE/R
Intercept
Sample Size, n
r2
RMSE
RPD
MNB (%)
r value
0.950
0.932
0.898
0.929
–319.760
60
0.93
30.7
3.6
4.2
p value
<.0001
<0.0001
<0.0001
<0.0001
Coefficient
7935.402
–8115.633
15,933.000
–14,837.000
Turbidity
Band
RE/G
NIR/G
NIR/R
RE/R
Intercept
Sample Size, n
r2
RMSE
RPD
MNB (%)
r value
0.920
0.913
0.874
0.893
–328.016
60
0.85
44.6
2.5
2.9
p value
<0.0001
<0.0001
<0.0001
<0.0001
Coefficient
1656.912
–1352.279
2967.325
–2588.921
Averaged TSS
Band
RE/G
NIR/G
NIR/R
RE/R
Intercept
Sample Size, n
r2
RMSE
RPD
MNB (%)
r value
0.969
0.951
0.916
0.948
–319.775
20
0.97
21.8
5.0
1.5
p value
<0.0001
<0.0001
<0.0001
<0.0001
Coefficient
7932.678
–8112.650
15,927.000
–14,831.000
Averaged Turbidity
Band
RE/G
NIR/G
NIR/R
RE/R
Intercept
Sample Size, n
r2
RMSE
RPD
MNB (%)
r value
0.961
0.953
0.913
0.933
–328.013
20
0.93
30.9
3.5
1.2
p value
<0.0001
<0.0001
<0.0001
<0.0001
Coefficient
1657.235
–1352.618
2968.020
–2589.587
Figure 3. Scatter plots of measured TSS and turbidity compared to modeled results: (a) all TSS values
and modeled TSS values, (b) all turbidity values and modeled turbidity values, (c) averaged water
column TSS values and modeled averaged water column TSS values, (d) averaged water column
turbidity values and modeled averaged water column turbidity values. Table 3. Results of correlation analysis and linear regression. TSS
Band
RE/G
NIR/G
NIR/R
RE/R
Intercept
Sample Size, n
r2
RMSE
RPD
MNB (%)
r value
0.950
0.932
0.898
0.929
–319.760
60
0.93
30.7
3.6
4.2
p value
<.0001
<0.0001
<0.0001
<0.0001
Coefficient
7935.402
–8115.633
15,933.000
–14,837.000
Turbidity
Band
RE/G
NIR/G
NIR/R
RE/R
Intercept
Sample Size, n
r2
RMSE
RPD
MNB (%)
r value
0.920
0.913
0.874
0.893
–328.016
60
0.85
44.6
2.5
2.9
p value
<0.0001
<0.0001
<0.0001
<0.0001
Coefficient
1656.912
–1352.279
2967.325
–2588.921
Averaged TSS
Band
RE/G
NIR/G
NIR/R
RE/R
Intercept
Sample Size, n
r2
RMSE
RPD
MNB (%)
r value
0.969
0.951
0.916
0.948
–319.775
20
0.97
21.8
5.0
1.5
p value
<0.0001
<0.0001
<0.0001
<0.0001
Coefficient
7932.678
–8112.650
15,927.000
–14,831.000
Averaged Turbidity
Band
RE/G
NIR/G
NIR/R
RE/R
Intercept
Sample Size, n
r2
RMSE
RPD
MNB (%)
r value
0.961
0.953
0.913
0.933
–328.013
20
0.93
30.9
3.5
1.2
p value
<0.0001
<0.0001
<0.0001
<0.0001
Coefficient
1657.235
–1352.618
2968.020
–2589.587 Table 3. Results of correlation analysis and linear regression. g
The initial analysis w
3.2. Regression Model Development TSS
Band
RE/G
NIR/G
NIR/R
RE/R
Intercept
Sample Size, n
r2
RMSE
RPD
MNB (%)
r value
0.950
0.932
0.898
0.929
–319.760
60
0.93
30.7
3.6
4.2
p value
<.0001
<0.0001
<0.0001
<0.0001
Coefficient
7935.402
–8115.633
15,933.000
–14,837.000
Turbidity
Band
RE/G
NIR/G
NIR/R
RE/R
Intercept
Sample Size, n
r2
RMSE
RPD
MNB (%)
r value
0.920
0.913
0.874
0.893
–328.016
60
0.85
44.6
2.5
2.9
p value
<0.0001
<0.0001
<0.0001
<0.0001
Coefficient
1656.912
–1352.279
2967.325
–2588.921
Averaged TSS
Band
RE/G
NIR/G
NIR/R
RE/R
Intercept
Sample Size, n
r2
RMSE
RPD
MNB (%)
r value
0.969
0.951
0.916
0.948
–319.775
20
0.97
21.8
5.0
1.5
p value
<0.0001
<0.0001
<0.0001
<0.0001
Coefficient
7932.678
–8112.650
15,927.000
–14,831.000
Averaged Turbidity
Band
RE/G
NIR/G
NIR/R
RE/R
Intercept
Sample Size, n
r2
RMSE
RPD
MNB (%)
r value
0.961
0.953
0.913
0.933
–328.013
20
0.93
30.9
3.5
1.2
p value
<0.0001
<0.0001
<0.0001
<0.0001
Coefficient
1657.235
–1352.618
2968.020
–2589.587
Figure 3. Scatter plots of measured TSS and turbidity compared to modeled results: (a) all TSS values
and modeled TSS values, (b) all turbidity values and modeled turbidity values, (c) averaged water
column TSS values and modeled averaged water column TSS values, (d) averaged water column
turbidity values and modeled averaged water column turbidity values. Table 3. Results of correlation analysis and linear regression. Table 3. Results of correlation analysis and linear regression. Figure 3. Scatter plots of measured TSS and turbidity compared to modeled results: (a) all TSS values
and modeled TSS values, (b) all turbidity values and modeled turbidity values, (c) averaged water
column TSS values and modeled averaged water column TSS values, (d) averaged water column
turbidity values and modeled averaged water column turbidity values. 9 of 15
10 of 16 Drones 2020, 4, 54
Drones 2020, 4, x FO Generated sUAS average TSS and turbidity maps (blue rectangle is the lamella plate s
ge TSS map at time equals 0 h, (b) average turbidity map at time equals 0 h, (c) avera
me equals 3 h, (d) average turbidity map at time equals 3 h, (e) average TSS map
h, (f) average turbidity map at time equals 6 h. g
The initial analysis w
3.2. Regression Model Development Generated sUAS average TSS and turbidity maps (blue rectangle is the lamella plate
ge TSS map at time equals 0 h, (b) average turbidity map at time equals 0 h, (c) avera
me equals 3 h, (d) average turbidity map at time equals 3 h, (e) average TSS map at time
erage turbidity map at time equals 6 h. Figure 4. Generated sUAS average TSS and turbidity maps (blue rectangle is the lamella plate settler):
(a) average TSS map at time equals 0 h, (b) average turbidity map at time equals 0 h, (c) average TSS
map at time equals 3 h, (d) average turbidity map at time equals 3 h, (e) average TSS map at time
equals 6 h, (f) average turbidity map at time equals 6 h. Figure 4. Generated sUAS average TSS and turbidity maps (blue rectangle is the lamella plate settler):
(a) average TSS map at time equals 0 h, (b) average turbidity map at time equals 0 h, (c) average TSS
map at time equals 3 h, (d) average turbidity map at time equals 3 h, (e) average TSS map at time equals
6 h, (f) average turbidity map at time equals 6 h. 10 of 15
11 of 16 Drones 2020, 4, 54
Drones 2020, 4, x FO . enerated sUAS total TSS and turbidity maps (blue rectangle is the lamella plate se
map at time equals 0 h, (b) total turbidity map at time equals 0 h, (c) total TSS map
(d) total turbidity map at time equals 3 h, (e) total TSS map at time equals 6 h,
map at time equals 6 h. Generated sUAS total TSS and turbidity maps (blue rectangle is the lamella plate
S map at time equals 0 h, (b) total turbidity map at time equals 0 h, (c) total TSS
s 3 h, (d) total turbidity map at time equals 3 h, (e) total TSS map at time equals 6 h,
map at time equals 6 h. . Figure 5. Generated sUAS total TSS and turbidity maps (blue rectangle is the lamella plate settler): (a)
total TSS map at time equals 0 h, (b) total turbidity map at time equals 0 h, (c) total TSS map at time
equals 3 h, (d) total turbidity map at time equals 3 h, (e) total TSS map at time equals 6 h, (f) total
Figure 5. 4. Discussion
4. Discussion From Table 2, there was a general increase in spectral reflectance with increased TSS and
turbidity. The red band was consistently the highest while the green band was consistently the lowest
(Table 2 and Figure 6). Red and NIR bands should have the highest spectral response with high
suspended sediment concentrations partnered with dampened response from the other spectral
bands; these results agree with several other studies comparing sediment concentrations to spectral
data [43–47]. From Figure 2, it should be noted that the second sampling location reached equilibrium
within the time of sampling, thus allowing for the water column to be homogenous. Conversely, the
first sampling location, closest to the inlet, still had varying TSS and turbidity measurements within
the water column. Despite these discrepancies, the models were able to produce high r2 values,
suggesting that water column status does not affect reflectance, at least with high concentrations. This
finding is reinforced by comparison between the generated models for average values and for all
values. The bands and coefficients for all values versus averaged values models were very similar
(Table 3) and the generated maps from these models are almost identical (Figures 4 and 5). Water
column status is potentially accounted for more holistically in the averaged models thus resulting in
From Table 2, there was a general increase in spectral reflectance with increased TSS and turbidity. The red band was consistently the highest while the green band was consistently the lowest (Table 2 and
Figure 6). Red and NIR bands should have the highest spectral response with high suspended sediment
concentrations partnered with dampened response from the other spectral bands; these results agree
with several other studies comparing sediment concentrations to spectral data [43–47]. From Figure 2,
it should be noted that the second sampling location reached equilibrium within the time of sampling,
thus allowing for the water column to be homogenous. Conversely, the first sampling location, closest
to the inlet, still had varying TSS and turbidity measurements within the water column. Despite these
discrepancies, the models were able to produce high r2 values, suggesting that water column status
does not affect reflectance, at least with high concentrations. This finding is reinforced by comparison
between the generated models for average values and for all values. 4. Discussion
4. Discussion The bands and coefficients for all
values versus averaged values models were very similar (Table 3) and the generated maps from these
models are almost identical (Figures 4 and 5). Water column status is potentially accounted for more
holistically in the averaged models, thus resulting in better model statistics (Table 3). column status is potentially accounted for more holistically in the averaged models, thus resulting in
better model statistics (Table 3). The models in Table 3, show that the use of multiple band ratios produced higher r2 values
versus single bands; this was expected since multiple band ratios have been shown to reduce the
effect of sun glint from the water’s surface on recorded reflectance [30,47,48]. The models exhibited
more significance with red and NIR bands. Averaging water measurements only substantially
increases the turbidity r2 and r values for NIR/R and RE/R. Overall, r2, RMSE, and RPD agree closely
as to which models perform well. Generally, RPD > 2 indicates a reliable model [49]. MNB indicated
that the models tend to overpredict, with the TSS model being the highest. The generated sediment
maps (Figures 4 and 5) show the expected decrease in suspended sediments over time. The resulting
maps also looked identical since generated models have similar coefficients (Table 2). This can be
excepted since the high levels of suspended solids were consistent throughout the water column
The models in Table 3, show that the use of multiple band ratios produced higher r2 values versus
single bands; this was expected since multiple band ratios have been shown to reduce the effect of
sun glint from the water’s surface on recorded reflectance [30,47,48]. The models exhibited more
significance with red and NIR bands. Averaging water measurements only substantially increases the
turbidity r2 and r values for NIR/R and RE/R. Overall, r2, RMSE, and RPD agree closely as to which
models perform well. Generally, RPD > 2 indicates a reliable model [49]. MNB indicated that the
models tend to overpredict, with the TSS model being the highest. The generated sediment maps
(Figures 4 and 5) show the expected decrease in suspended sediments over time. The resulting maps
also looked identical since generated models have similar coefficients (Table 2). This can be excepted
since the high levels of suspended solids were consistent throughout the water column, thus resulting
in similar models. g
The initial analysis w
3.2. Regression Model Development Generated sUAS total TSS and turbidity maps (blue rectangle is the lamella plate settler):
(a) total TSS map at time equals 0 h, (b) total turbidity map at time equals 0 h, (c) total TSS map at
time equals 3 h, (d) total turbidity map at time equals 3 h, (e) total TSS map at time equals 6 h, (f) total
turbidity map at time equals 6 h. 11 of 15
f Drones 2020, 4, 54 Figure 6. Comparison of results to Prior et al. [32]: (a) box and whisker plots of reflectance values from
this study (AU-ESCTF) and from Prior et al. [32] sampling at Moores Creek, Lanett, AL, USA (MC),
(b) box and whisker plots of TSS and turbidity from this study (AUESCTF) and from Prior et al. [32]
sampling at Moores Creek, Lanett, AL, USA (MC). Figure 6. Comparison of results to Prior et al. [32]: (a) box and whisker plots of reflectance values from
this study (AU-ESCTF) and from Prior et al. [32] sampling at Moores Creek, Lanett, AL, USA (MC),
(b) box and whisker plots of TSS and turbidity from this study (AUESCTF) and from Prior et al. [32]
sampling at Moores Creek, Lanett, AL, USA (MC). Figure 6. Comparison of results to Prior et al. [32]: (a) box and whisker plots of reflectance values from
this study (AU-ESCTF) and from Prior et al. [32] sampling at Moores Creek, Lanett, AL, USA (MC),
(b) box and whisker plots of TSS and turbidity from this study (AUESCTF) and from Prior et al. [32]
sampling at Moores Creek, Lanett, AL, USA (MC). Figure 6. Comparison of results to Prior et al. [32]: (a) box and whisker plots of reflectance values from
this study (AU-ESCTF) and from Prior et al. [32] sampling at Moores Creek, Lanett, AL, USA (MC),
(b) box and whisker plots of TSS and turbidity from this study (AUESCTF) and from Prior et al. [32]
sampling at Moores Creek, Lanett, AL, USA (MC). 4. Discussion
4. Discussion excepted since the high levels of suspended solids were consistent throughout the water column,
thus resulting in similar models. In contrast, Prior et al. [32] showed that separate models were required to relate sUAS
multispectral imagery to TSS for cases with low and high sediment loads due to stream flow
variation. Even with elevated suspended sediment levels present during high flow events, the models
still struggled, likely due to influence from stream bed reflectance. The results from the current
sediment basin study suggest that the relationship between reflectance and suspended sediment will
reach linearity when the bed is not visible due to either excessive cloudiness and or if the depth
obscures the bed. The linear regression approach probably produced stronger models than in Prior
In contrast, Prior et al. [32] showed that separate models were required to relate sUAS multispectral
imagery to TSS for cases with low and high sediment loads due to stream flow variation. Even with
elevated suspended sediment levels present during high flow events, the models still struggled, likely
due to influence from stream bed reflectance. The results from the current sediment basin study suggest
that the relationship between reflectance and suspended sediment will reach linearity when the bed is
not visible due to either excessive cloudiness and or if the depth obscures the bed. The linear regression
approach probably produced stronger models than in Prior et al. [32] since a much wider range of
suspended sediment values were collected (Figure 6). This wide range of values was much easier Drones 2020, 4, 54 12 of 15 to achieve since the sediment basin allowed for continuous sampling over six hours of settlement,
whereas stream suspended sediment load is heavily dependent on rainfall and watershed size. One of the main challenges of this study was syncing of the automatic water samplers among
each other and with the sUAS flight. Another challenge was connecting the sUAS to the multispectral
sensor within the 15-min break between sampling. Since the plan was to conduct samplings every
15 min for six hours, there should have been 24 samplings. However, only 20 successful samplings
were collected due to difficulty in connecting the multispectral sensor and the overheating of the
sUAS. Moreover, it should be noted that the automatic samplers purged the intake tubes each time
before sampling, causing air bubbles to rise through the water column. 4. Discussion
4. Discussion This could have affected the
settlement of particles and may be the source of the small inflections toward the end of sampling seen
in Figure 2. Timing the launch of the sUAS before the purge of the intake tubes was also difficult,
thus sUAS imagery during purging could have skewed some results due to the presence of air bubbles
at the surface in some instances. Despite these uncertainties, settlement of solids followed an overall
expected trend, and stepwise linear regression was able to produce models that could be used for TSS
and turbidity predictions. Several challenges might be faced when implementing sUAS multispectral suspended sediment
monitoring for management applications. The most important aspect of sUAS utilization is maintaining
a safe environment. This could be difficult to achieve if a sUAS is being used on a construction site. Maintaining a safe launch and landing zone, ensuring line of site visibility as well as keeping a safe
distance from obstacles could prove to be difficult on a construction site. Sensor calibration would
need to occur prior to every flight as well as ensuring that no shadows are being cast on the sediment
basin. Flights are best conducted on overcast days, usually during midmorning, to reduce the potential
sun glint offthe water surfaces. Hazardous weather, including rain and high wind speeds, should be
avoided. Standards and best practices would need to be established and accepted by industry and
academia as well as federal and state governments in order to ensure legitimate compliance through
sUAS multispectral remote sensing. Results of this study show that sUAS multispectral imagery could be alternatively used for TSS
and turbidity monitoring instead of relying on grab samples. Future studies could improve these
results by conducting multiple sediment basin tests with the same soil type to ensure consistent results. Additional tests with different soil types could result in varying spectral reflectance responses, if soil
types and/or grain sizes reflect differently. Incorporating sampling depth in tests with lower suspended
sediment concentrations could also be beneficial to determine the influence of bed reflectance on sUAS
multispectral measurements. 5. Conclusions Funding:
This research was funded by the Auburn University Office of Undergraduate Research,
Auburn University 100+ Women Strong, and the Interdisciplinary Graduate Education Program in Remote
Sensing at Virginia Tech. Acknowledgments: We would like to thank Barry G. Dorman and Robert Icenogle at the USDA-ARS National
Soil Dynamics Laboratory, Auburn, AL, for their technical support and the use of their ISCO automatic water
samplers and laboratory vacuum filtration equipment. Conflicts of Interest: The authors declare no conflict of interest. 5. Conclusions This study indicates that sUAS multispectral imagery and linear regression modeling can determine
elevated levels of TSS and turbidity with high accuracy in a sediment basin. The measured spectral
reflectance showed an expected increase with higher levels of TSS and turbidity in addition to the red
and NIR bands increasing the most. Developed models were able to measure a wide range of TSS and
turbidity levels while also accounting for both homogeneous and heterogenous water column states. Only the turbidity model that considered all measurements had an r2 value below 0.9. The turbidity
model that averaged water column values had slightly increased individual band ratio r values and an
over increased r2 value above 0.9. This study showed that depth and bed reflectance did not need to be accounted for with elevated
TSS and turbidity levels. Future studies should include multiple sediment basin experiments for
validation and correlation assurance. Additionally, future studies could include determining when
depth and bed reflectance need to be considered and if various soil types and compositions reflect
differently when in suspension. This study exhibits the potential for using sUAS technology to
assess, measure and monitor sediment basins after high flow events. Further studies are needed to 13 of 15 13 of 15 Drones 2020, 4, 54 establish monitoring protocols and to investigate if sUAS technology can be used on streams receiving
construction and municipal discharges during high flow events. establish monitoring protocols and to investigate if sUAS technology can be used on streams receiving
construction and municipal discharges during high flow events. Author Contributions: Data curation, C.B., W.N.D. and S.L.S.; formal analysis, G.B.R.; funding acquisition,
E.M.P., F.C.O. and C.B.; investigation, E.M.P.; methodology, E.M.P., C.B. and W.N.D.; project administration,
F.C.O.; resources, W.N.D. and S.L.S.; software, G.B.R.; visualization, E.M.P.; writing—original draft, E.M.P.;
writing—review and editing, E.M.P., F.C.O., C.B., W.N.D., G.B.R. and S.L.S. All co-authors reviewed and edited
the manuscript. All authors have read and agreed to the published version of the manuscript. Author Contributions: Data curation, C.B., W.N.D. and S.L.S.; formal analysis, G.B.R.; funding acquisition,
E.M.P., F.C.O. and C.B.; investigation, E.M.P.; methodology, E.M.P., C.B. and W.N.D.; project administration,
F.C.O.; resources, W.N.D. and S.L.S.; software, G.B.R.; visualization, E.M.P.; writing—original draft, E.M.P.;
writing—review and editing, E.M.P., F.C.O., C.B., W.N.D., G.B.R. and S.L.S. All co-authors reviewed and edited
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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/). © 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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Qeios · Definition, February 2, 2020 Open Peer Review on Qeios PDS5A Gene National Cancer Institute National Cancer Institute Qeios ID: VXRAFA · https://doi.org/10.32388/VXRAFA Source National Cancer Institute. PDS5A Gene. NCI Thesaurus. Code C126608. This gene plays a role in the regulation of both mitosis and cell proliferation. Qeios ID: VXRAFA · https://doi.org/10.32388/VXRAFA 1/1
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Attitudes of translation agencies and professional translators in Saudi Arabia towards translation management systems
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Saudi journal of language studies
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The current issue and full text archive of this journal is available on Emerald Insight at:
https://www.emerald.com/insight/2634-243X.htm The current issue and full text archive of this journal is available on Emerald Insight at:
https://www.emerald.com/insight/2634-243X.htm 11 Received 13 September 2023
Revised 2 November 2023
18 December 2023
Accepted 19 December 2023 © Waleed Obaidallah Alsubhi. Published in Saudi Journal of Language Studies. Published by Emerald
Publishing Limited. This article is published under the Creative Commons Attribution (CC BY 4.0)
licence. Anyone may reproduce, distribute, translate and create derivative works of this article (for both
commercial and non-commercial purposes), subject to full attribution to the original publication and
authors. The full terms of this licence may be seen at http://creativecommons.org/licences/by/4.0/
legalcode Attitudes of translation agencies
and professional translators in
Saudi Arabia towards translation
management systems
Waleed Obaidallah Alsubhi
Department of English and Translation, College of Science and Arts,
Jeddah University, Jeddah, Saudi Arabia Attitudes of
translation
agencies Abstract Purpose – Effective translation has become essential for seamless cross-cultural communication in an era of
global interconnectedness. Translation management systems (TMS) have redefined the translation landscape,
revolutionizing project management and execution. This study examines the attitudes of translation agencies
and professional translators towards integrating and utilizing TMS, with a specific focus on Saudi Arabia. Design/methodology/approach – The study’s design was based on a thorough mixed-methods strategy
that purposefully combined quantitative and qualitative procedures to create an array of findings. Through a
survey involving 35 participants (both project managers and professional translators) and a series of
interviews, this research explores the adoption of TMS, perceived benefits, influencing factors and future
considerations. This integrated approach sought to investigate the nuanced perceptions of Saudi translation
companies and expert translators about TMS. By combining the strengths of quantitative data’s broad scopes
and qualitative insights’ depth, this mixed-methods approach sought to overcome the limitations of each
method, ultimately resulting in a holistic understanding of the multifaceted factors shaping attitudes within
Saudi Arabia’s unique translation landscape. q
p
Findings – Based on questionnaires and interviews, the study shows that 80% of participants were familiar
with TMS, and 57% had adopted it in their work. Benefits included enhanced project efficiency, collaboration
and quality assurance. Factors influencing adoption encompassed cost, compatibility and resistance to change. The study further delved into participants’ demographic profiles and years of experience, with a notable
concentration in the 6–10 years range. TMS adoption was linked to improved translation processes, and
participants expressed interest in AI integration and mobile compatibility. Deployment models favored cloud-
based solutions, and compliance with industry standards was deemed vital. The findings underscore the
evolving nature of TMS adoption in Saudi Arabia, with diverse attitudes shaped by cultural influences,
technological compatibility and awareness. Originality/value – This research provides a holistic and profound perspective on the integration of TMS,
fostering a more comprehensive understanding of the opportunities, obstacles and potential pathways to
success. As the translation landscape continues to evolve, the findings from this study will serve as a valuable
compass guiding practitioners and researchers towards effectively harnessing the power of technology for
enhanced translation outcomes. Keywords Translation management systems, Attitudes, Saudi Arabia, Translation agencies,
Professional translators Professional translators Paper type Research paper © Waleed Obaidallah Alsubhi. Published in Saudi Journal of Language Studies. Published by Emerald
Publishing Limited. Saudi Journal of Language Studies
Vol. 4 No. 1, 2024
pp. 11-27
Emerald Publishing Limited
e-ISSN: 2634-2448
p-ISSN: 2634-243X
DOI 10.1108/SJLS-09-2023-0040 Attitudes of
translation
agencies Attitudes of
translation
agencies 1. Introduction The translation landscape is deeply intertwined with translation technology, prompting
researchers (Man et al., 2019; Cronin, 2003; Millan and Bartrina, 2013) to underscore the
intricate association between translators and technological tools. Significantly, Gough (2017)
posits that the translation process cannot be studied in isolation from the translation
technology ecosystem. This perspective underscores the imperative of understanding the
interplay between translators and technology. A series of prior studies have emphasized
translators’ active engagement with various translation technologies, prompting a crucial
inquiry into the translators’ level of awareness concerning these tools. Gough (2017)
emphasizes that translators should possess a foundational grasp of the fundamental
concepts underpinning translation technologies to effectively leverage them. Furthermore,
Mahdy et al. (2020) noted that the reluctance to adopt Computer-Assisted Translation (CAT)
tools often stems from a lack of understanding and familiarity among translators, rather than
intrinsic limitations of these tools. Thus, the possession of a sound comprehension of the
conceptual framework of translation technologies is pivotal not only for their successful
adoption but also for enhancing the efficiency of the translation process itself. 12 Effective translation is a fundamental pillar of seamless communication in today’s era of
rapid globalization and cross-cultural interaction. Within this context, the emergence of
translation management systems (TMS) has significantly transformed the translation
landscape, revolutionizing project management and execution. TMS, which amalgamates
innovative software tools and methodologies, has introduced efficiency, enhanced
collaboration, and improved quality control (Katan, 2009; Gambier and van Doorslaer,
2010). Amidst this transformative wave, it becomes crucial to comprehend the perspectives of
translation agencies and professional translators regarding the integration and use of TMS. This study delves into an exploration of these perspectives, focusing specifically on the
unique context of Saudi Arabia. q
The trajectory of TMS adoption mirrors the swift evolution of translation technology. Often coupled with CAT tools, TMS plays a pivotal role in project management, effectively
coordinating tasks such as file handling and optimizing translation memory (Federico et al.,
2012). These systems address conventional workflow limitations and offer practical solutions
for managing multilingual content (Al-Rumaih, 2021). Furthermore, within the intricate realm
of translation, TMS has emerged as a catalyst for fostering team synergy across geographical
boundaries. It ensures consistency in terminology, facilitating seamless communication
within linguistically diverse environments (Verplaetse and Lambrechts, 2019;
Tennent, 2005). A comprehensive review of the existing literature concerning TMS reveals a diverse
landscape of advantages, limitations, and implementation challenges. Abstract This article is published under the Creative Commons Attribution (CC BY 4.0)
licence. Anyone may reproduce, distribute, translate and create derivative works of this article (for both
commercial and non-commercial purposes), subject to full attribution to the original publication and
authors. The full terms of this licence may be seen at http://creativecommons.org/licences/by/4.0/
legalcode
Funding: The researcher received funding from the Grant Program for Studies and Research in the
Field of Translation of The Arabic Observatory of Translation to complete this research study in the
field of translation for the year 2023. Saudi Journal of Language Studies
Vol. 4 No. 1, 2024
pp. 11-27
Emerald Publishing Limited
e-ISSN: 2634-2448
p-ISSN: 2634-243X
DOI 10.1108/SJLS-09-2023-0040 © Waleed Obaidallah Alsubhi. Published in Saudi Journal of Language Studies. Published by Emerald
Publishing Limited. This article is published under the Creative Commons Attribution (CC BY 4.0)
licence. Anyone may reproduce, distribute, translate and create derivative works of this article (for both
commercial and non-commercial purposes), subject to full attribution to the original publication and
authors. The full terms of this licence may be seen at http://creativecommons.org/licences/by/4.0/
legalcode g
Funding: The researcher received funding from the Grant Program for Studies and Research in the
Field of Translation of The Arabic Observatory of Translation to complete this research study in the
field of translation for the year 2023. 1. Introduction These systems hold
the potential to significantly enhance translation efficiency, streamline project coordination,
and strengthen quality assurance (Mahdy et al., 2020). However, reaping these benefits comes
with its share of challenges. Compatibility issues, resistance to change, and linguistic and
cultural adaptation concerns have emerged as prominent obstacles (Esselink, 2000). Despite the ongoing discussions, a significant research gap exists in exploring attitudes
and perceptions toward TMS among translation agencies and professional translators,
particularly in Saudi Arabia. Comparable studies across various contexts highlight that
cultural influences are vital in shaping technology adoption (Al-Gahtani, 2004). This
underscores the need for a closer examination of these factors within the unique Saudi
Arabian landscape. The multifaceted nature of Saudi Arabia’s linguistic diversity and
cultural intricacies raises pertinent questions about integrating TMS into translation
practices. Additionally, theoretical frameworks that elucidate technology adoption patterns
within the translation domain offer a promising avenue for understanding the dynamics
behind the acceptance or resistance to TMS (Rogers, 2010). The significance of investigating these attitudes extends beyond mere technology
adoption; it delves into the broader discourse on the role of technology in a field characterized
by linguistic finesse and cultural awareness. The Saudi Arabian context adds complexity to
these perspectives, shedding light on the translation’s interplay between tradition,
modernity, and cultural consciousness. Machine Translation (MT) is a dynamic field with a rich history and evolving methods. It has significant implications for the translation industry, including translation management
systems (TMS), and is relevant to the study of the attitudes of translation agencies and
professional translators in Saudi Arabia towards TMS. The history of MT provides context
for understanding the advancements in translation technology. Early rule-based MT systems
struggled with linguistic complexity, leading to critical evaluations, as seen in the ALPAC
report (Aziz Berkan Karadeli, 2023). The challenges within MT, such as handling low-
resource languages, idiomatic expressions, and cultural context, are pertinent to the study of
attitudes toward TMS. Understanding these challenges informs the contextual landscape of
translation technology (Kumar et al., 2023). Evaluation metrics in MT, including BLEU and
METEOR, are essential for assessing translation quality. Recognizing the importance of
evaluation methods is relevant to the study’s assessment of attitudes toward TMS quality
and performance. MT systems have applications in various domains, including the business
and professional translation sectors (Lee et al., 2023). Understanding these applications is
fundamental to comprehending the role of TMS in the translation industry. Attitudes of
translation
agencies 1. Introduction The future of MT
is relevant to the study as it shapes expectations and attitudes toward TMS. Advances in AI
and deep learning promise more accurate and context-aware translation solutions, which can
influence the acceptance and integration of TMS in the translation landscape (Tasmedir
et al., 2023). 13 ,
)
Considering these aspects of MT in the literature review, the study on the attitudes of
translation agencies and professional translators in Saudi Arabia towards TMS gains a
broader context. It is evident that the evolution of MT, its challenges, and its applications are
all intertwined with the adoption and perceptions of TMS, particularly in the unique cultural
and linguistic landscape of Saudi Arabia. This study contributes to the broader
understanding of technology in translation and the specific factors that shape its
acceptance in Saudi Arabia. A glimpse into the academic landscape reveals the work of Alotaibi (2014) in Saudi Arabia,
Mahfouz (2018) in Egypt, Mahdy et al. (2020) in Yemen, and Abu Dayyeh (2020) in Palestine,
who have contributed to our understanding of CAT tool dynamics. Yet, the comprehensive
investigation of translation technology in its entirety and an assessment of awareness levels
towards these technological tools are aspects that await deeper exploration within the
existing research landscape. g
p
Taking a closer look, the study by Abu Dayyeh (2020) meticulously evaluated the
adoption of CAT tools within the context of Palestine. The study’s outcomes unveiled a
hierarchy of utilization, with online electronic dictionaries taking precedence, followed by
online translation software and Internet search engines. In stark contrast, interviews
conducted as part of the research uncovered a widespread lack of familiarity among
Palestinians with translation memory systems, terminology management tools, and the array
of available corpora resources. A point of distinction to highlight is that the research conducted by Almutawa and Izwaini
(2015) primarily concentrated on in-house translators situated within organizations and
agencies. This specific focus potentially introduces a set of organizational barriers that might
have influenced the perceptions and attitudes towards TMS adoption at the time. As this
study endeavors to bridge this gap by encompassing a wider spectrum of Saudi translators, it
aims to offer a comprehensive perspective on the evolving landscape of translation
technology awareness. The study aims to address the following research questions: RQ1. What are the attitudes and perspectives of translation agencies and professional
translators in Saudi Arabia regarding the adoption of TMS? RQ1. 1. Introduction What are the attitudes and perspectives of translation agencies and professional
translators in Saudi Arabia regarding the adoption of TMS? RQ2. What are the levels of awareness and familiarity with TMS among the participants
in this study? RQ2. What are the levels of awareness and familiarity with TMS among the participants
in this study? RQ2. What are the levels of awareness and familiarity with TMS among the participants
in this study? RQ3. What are the perceived benefits and satisfaction levels associated with TMS
integration among the participants? RQ3. What are the perceived benefits and satisfaction levels associated with TMS
integration among the participants? RQ3. What are the perceived benefits and satisfaction levels associated with TMS
integration among the participants? 14 RQ4. What implementation challenges and training needs are identified in the context of
TMS adoption in Saudi Arabia? RQ4. What implementation challenges and training needs are identified in the context of
TMS adoption in Saudi Arabia? RQ5. What are the participants’ perspectives on the future trajectory of TMS in Saudi
Arabia, including preferences for deployment models, compliance with industry
standards, and recommendations for advancements in the TMS industry? RQ5. What are the participants’ perspectives on the future trajectory of TMS in Saudi
Arabia, including preferences for deployment models, compliance with industry
standards, and recommendations for advancements in the TMS industry? In light of this, the study’s significance lies in its contribution to the understanding of the
interplay between translation technology and translators, emphasizing the importance of a
solid grasp of technology for successful adoption. It also highlights the transformative
potential of TMS in improving translation processes. The unique cultural and linguistic
context of Saudi Arabia is recognized as a crucial factor influencing technology adoption. Moreover, this research offers insights that are valuable not only for TMS adoption but also
for the broader discussion on technology in the translation field, aiding practitioners and
researchers in navigating this evolving landscape. gy
2.1 Study design
Th
d ’
d
i The study’s design was based on a thorough mixed-methods strategy that purposefully
combined quantitative and qualitative procedures to create an array of findings. This
integrated approach sought to investigate the nuanced perceptions of Saudi translation
companies and expert translators about TMS. By combining the strengths of quantitative
data’s broad scopes and qualitative insights’ depth, this mixed-methods approach sought to
overcome the limitations of each method, ultimately resulting in a holistic understanding of the
multifaceted factors shaping attitudes within Saudi Arabia’s unique translation landscape. p g
q
p
Quantitative methods contributed to the ability to quantify trends and derive statistically
significant patterns from collected data. Through structured surveys and well-designed
questions, the quantitative aspect of the study provided a numerical perspective to
participants’ viewpoints. Qualitative approaches added an element that probed into the
complexities of participants’ experiences, complementing this quantitative base. This aspect
illuminated underlying motives, personal anecdotes, and contextual intricacies that
quantitative data frequently misses through open-ended inquiries and narrative responses. 2.2 Study participants
The sample participants were meticulously selected to ensure a comprehensive
representation of the Saudi Arabian translation landscape. A purposive sampling
technique was used to handpick 35 participants, each embodying a distinct perspective
within the translation community. This approach aimed to capture a spectrum of viewpoints,
ensuring that attitudes towards TMS adoption were not homogenized. 2.3 Interviews 15 A series of interviews were conducted to enhance the comprehensiveness of our insights. These interviews adopted an informal conversational tone rather than a conventional formal
structure. Participants were engaged in dialogues centered around their experiences with
TMS. These conversations explored participants’ preferences, reservations, and
observations concerning TMS. Furthermore, participants were encouraged to share the
transformative impact of TMS on their work routines and workflows while delving into any
encountered challenges. These interviews were conducted in person, providing a conducive environment for open
and candid discussions. The interactive nature of the in-person setting facilitated participants’
willingness to express their opinions freely. Each interview session lasted approximately
30 min, ensuring a balanced exploration of the topics without overwhelming participants. Participants’ privacy and confidentiality were paramount throughout this process. Personal identities and private information were meticulously safeguarded, ensuring
anonymity and compliance with ethical research standards. These interviews aimed to
extract profound insights from participants’ experiences and opinions rather than intruding
into their details. Participants were also handed out the survey questionnaire for completion during the
interviews. This allowed participants to express their thoughts verbally during the interview
and in writing through the questionnaire. The survey questionnaires were collected after the
interview, ensuring a smooth and coordinated data collection process encapsulating both
spoken and written perspectives. The following table shows interview questions that have been carefully crafted to elicit
detailed responses, contributing to the depth and richness of the data collected through
qualitative interviews (see Table 1). 2.2 Study participants The selection process involved calibrating experience, linguistic pairs, and organizational
affiliations. Participants with varying experience levels, from novices to seasoned Attitudes of
translation
agencies professionals, were included to account for the potential impact of familiarity on attitudes. Diverse linguistic pairs enriched the sample, recognizing the role language dynamics might
play in shaping TMS attitudes. Both translation agencies and independent practitioners
acknowledged the varying organizational contexts shaping TMS attitudes. professionals, were included to account for the potential impact of familiarity on attitudes. Diverse linguistic pairs enriched the sample, recognizing the role language dynamics might
play in shaping TMS attitudes. Both translation agencies and independent practitioners
acknowledged the varying organizational contexts shaping TMS attitudes. 2.3 Interviews 2.4 The questionnaire q
The survey questionnaire, meticulously crafted to align with the research’s objectives,
functioned as the principal instrument for data collection. Designed with a keen focus on
established research standards, the questionnaire encompassed several sections, each
thoughtfully tailored to elicit insights into distinct dimensions of attitudes, perceptions, and
experiences associated with the integration of TMS: (1)
Introduction and consent: The questionnaire initiation included a comprehensive
introduction that elucidated the study’s objectives and underscored the voluntary
nature of participation, confidentiality assurance, and ethical considerations. Participants were invited to express their consent to participate in the study by
appending their signatures, ensuring a well-informed and consensual research
engagement. (2)
Participant background: By delving into the participants’ affiliations with
translation agencies or independent practice, this section sought to grasp the
diverse organizational contexts within which these attitudes towards TMS are
formed. Furthermore, inquiries about years of experience within the translation
domain provided valuable context for understanding the interplay between
experience and attitudes. Theme
Interview questions
(1) Awareness and familiarity with
TMS
a. Could you elucidate your level of awareness and familiarity
with translation management systems (TMS)? b. Have you gained practical experience with TMS
implementation, and if so, could you provide a detailed account
of your experiences? (2) Perceived benefits and satisfaction
a. From your vantage point, what discernible benefits result from
the integration of TMS into the translation workflow? b. Can you share specific instances where TMS has positively
influenced project efficiency, collaboration, or overall project
management? (3) Implementation challenges and
training needs
a. What multifaceted challenges did you encounter during the
implementation of TMS, encompassing both technical
intricacies and adaptation to new workflows? b. How did your team respond to the introduction of TMS, and
were there instances of resistance to change? c. Could you expound on the training process for TMS within
your organization, detailing the invested resources and
methods employed to familiarize team members with the new
system? (4) Future trajectory of TMS: evolving
needs and aspirations
a. Anticipating the future, what deployment models for TMS do
you prefer in your specific context? b. In what ways do you envision AI integration contributing to
advanced automation in TMS, drawing on either your
experience or aspirations? c. How vital do you consider mobile compatibility in TMS,
particularly for translators frequently on the move? d. Table 1.
Interview questions for
exploring perspectives
on TMS adoption 2.4 The questionnaire Ranging from their overall satisfaction
levels to their perception of valuable TMS features, this section sought to provide a
granular understanding of the tangible outcomes of TMS adoption. Furthermore,
participants were invited to share challenges encountered during TMS
implementation, affording insights into potential pain points and obstacles. Attitudes of
translation
agencies (7)
Decision factors: This part of the questionnaire explored the decision-making
dynamics regarding TMS adoption. This section provided a comprehensive vantage
point into the intricate interplay of considerations in the TMS adoption journey by
inquiring about essential TMS features, criteria for TMS selection, and influential
factors in the decision-making process. (8)
Impact and training: An exploration into the broader ramifications of TMS adoption
formed the crux of this segment. Through inquiries about the impact of TMS on the
overall translation process, insights were gained into how this technology reshaped
workflows, collaborations, and quality assurance mechanisms. Additionally, the
training aspect was addressed, with participants sharing their experiences and
levels of satisfaction with the training and support received. (9)
Future considerations: Participants’ future perspectives and preferences were
highlighted in this segment. By inquiring about intentions to explore other TMS
options, envisioning improvements in the TMS landscape, and expressing
deployment preferences, the questionnaire aimed to encapsulate forward-looking
insights that could inform future developments in the TMS domain. (10) (10)
Additional comments: A qualitative dimension was introduced in this section,
providing participants the opportunity to share unstructured insights and
comments on TMS or related themes. This open-ended space acknowledged the
uniqueness of individual perspectives, allowing participants to voice nuanced
opinions that predefined questions might not capture. (10)
Additional comments: A qualitative dimension was introduced in this section,
providing participants the opportunity to share unstructured insights and
comments on TMS or related themes. This open-ended space acknowledged the
uniqueness of individual perspectives, allowing participants to voice nuanced
opinions that predefined questions might not capture. (6)
Experience with TMS: For participants who had interacted with TMS, this segment
delved into the nuances of their experiences. Ranging from their overall satisfaction
levels to their perception of valuable TMS features, this section sought to provide a
granular understanding of the tangible outcomes of TMS adoption. Furthermore,
participants were invited to share challenges encountered during TMS
implementation, affording insights into potential pain points and obstacles. 2.5 Data analysis The quantitative data collected from the survey underwent a rigorous analytical process to
derive meaningful insights. Descriptive statistical methods were employed to synthesize the
responses into frequencies and percentages, offering a comprehensive overview of
participants’ perspectives and attitudes. This numerical representation provided a
foundation for understanding prevailing trends and patterns within the dataset. Furthermore, the qualitative responses from open-ended questions were subjected to
thematic analysis, a methodological approach to uncover recurrent themes and patterns
within the qualitative data. By coding and categorizing participants’ narrative responses, this
analysis sought to uncover nuanced insights, revealing more profound layers of attitudes,
experiences, and perceptions. 3. Results and discussion
3.1 Perspectives on TMS adoption: a multifaceted analysis
h
i
i
f
i
d
di
S
d
i 3. Results and discussion
3.1 Perspectives on TMS adoption: a multifaceted analysis
Th
i
i
f
i
d
di
TMS
d
i 2.4 The questionnaire Are there language-specific features or data security measures
that you deem indispensable for TMS to align with industry
standards? Source(s): Table by the author
Table 1. Interview questions for
exploring perspectives
on TMS adoption
SJLS
4,1
16 16 c. How vital do you consider mobile compatibility in TMS,
particularly for translators frequently on the move? d. Are there language-specific features or data security measures
that you deem indispensable for TMS to align with industry
standards? Table 1. Interview questions for
exploring perspectives
on TMS adoption (3)
Awareness and familiarity with TMS: To establish a foundational understanding,
participants were prompted to indicate their prior awareness of the TMS concept,
gauging the extent to which this technology was known within their professional
sphere. Additionally, respondents were asked to gauge their familiarity with TMS,
offering insights into the existing knowledge landscape within the Saudi Arabian
translation industry. (4)
Benefits and perceptions: This section unveiled participants’ perceptions of TMS
benefits in the translation workflow. Through multiple-choice queries, the
questionnaire aimed to uncover the facets of TMS that resonated most with the
participants: enhanced project efficiency, improved collaboration, elevated quality
assurance, streamlined management, or other facets. (4)
Benefits and perceptions: This section unveiled participants’ perceptions of TMS
benefits in the translation workflow. Through multiple-choice queries, the
questionnaire aimed to uncover the facets of TMS that resonated most with the
participants: enhanced project efficiency, improved collaboration, elevated quality
assurance, streamlined management, or other facets. (5)
Challenges and implementation factors: This section’s exploration of barriers to
TMS adoption was a central focus. By soliciting participants’ insights into factors
impeding TMS integration, such as cost considerations, compatibility concerns, and
resistance to change, the questionnaire aimed to unravel the complexities involved
in TMS implementation. Participants’ perceptions of the financial implications of
TMS deployment were probed, adding another layer to the exploration of
challenges. (6)
Experience with TMS: For participants who had interacted with TMS, this segment
delved into the nuances of their experiences. Ranging from their overall satisfaction
levels to their perception of valuable TMS features, this section sought to provide a
granular understanding of the tangible outcomes of TMS adoption. Furthermore,
participants were invited to share challenges encountered during TMS
implementation, affording insights into potential pain points and obstacles. (6)
Experience with TMS: For participants who had interacted with TMS, this segment
delved into the nuances of their experiences. 3. Results and discussion
3.1 Perspectives on TMS adoption: a multifaceted analysis The examination of attitudes regarding TMS adoption among translation agencies and
professional translators reveals a multifaceted interplay of factors and perspectives. The insights highlight the complex interplay between cost, compatibility, awareness, and
technical challenges that influence the decision to adopt TMS. The perceived benefits of TMS
adoption, including streamlined project management, enhanced collaboration, and improved SJLS
4,1 translation quality, underscore its potential to revolutionize the translation workflow. However, challenges related to change resistance, implementation complexities, and training
requirements must be effectively addressed to harness TMS’s benefits fully. The interviews
also shed light on the evolving nature of TMS, with participants expressing interest in
advanced features like AI integration and mobile compatibility, indicating a continuous quest
for innovation within the translation industry. translation quality, underscore its potential to revolutionize the translation workflow. However, challenges related to change resistance, implementation complexities, and training
requirements must be effectively addressed to harness TMS’s benefits fully. The interviews
also shed light on the evolving nature of TMS, with participants expressing interest in
advanced features like AI integration and mobile compatibility, indicating a continuous quest
for innovation within the translation industry. y
3.1.1 Awareness and familiarity with TMS. The interviews revealed a spectrum of
awareness levels and prior experiences with TMS. Most participants were well-versed in the
concept of TMS and had firsthand experience with its implementation, while others exhibited
limited or no familiarity. This variation aligns with the findings of previous studies (Carrillo,
2007; Shuttleworth and Cowie, 2014; Alkan, 2016), suggesting that adopting TMS is still in
varying stages of maturity across different industry segments. 3.1.1 Awareness and familiarity with TMS. The interviews revealed a spectrum of
awareness levels and prior experiences with TMS. Most participants were well-versed in the
concept of TMS and had firsthand experience with its implementation, while others exhibited
limited or no familiarity. This variation aligns with the findings of previous studies (Carrillo,
2007; Shuttleworth and Cowie, 2014; Alkan, 2016), suggesting that adopting TMS is still in
varying stages of maturity across different industry segments. 18 y g
g
y
y
g
3.1.2 Perceived benefits and satisfaction. Participants with direct experience highlighted
tangible benefits of TMS integration, including heightened project efficiency, improved
collaboration among team members, and enhanced project management. y g
g
y
y
g
3.1.2 Perceived benefits and satisfaction. SJLS
4,1 3. Results and discussion
3.1 Perspectives on TMS adoption: a multifaceted analysis Participants with direct experience highlighted
tangible benefits of TMS integration, including heightened project efficiency, improved
collaboration among team members, and enhanced project management. TMS streamlined project management, reducing manual tasks. It also improved collaboration,
leading to higher translation quality, remarked one participant. Another interviewee noted, “Centralizing translation assets ensured consistency and saved time. Real-time updates helped track project progress effectively.” The utilization of TMS was particularly noted for its contribution to managing translation
assets, leading to increased consistency in translated content and quality assurance. 3.1.3 Implementation challenges and training needs. Challenges encountered during TMS
implementation were multifaceted, ranging from technical hurdles to the adaptation to new
workflows. Resistance to change was a barrier. Some team members were hesitant about using new technology,
shared an interviewee. Another participant added, “Setting up and customizing the TMS to fit our needs took time and
effort. Technical issues also cropped up.” The need for training was emphasized as well. Training was necessary but time-consuming. We had to invest resources to familiarize everyone
with the new system, another respondent explained. These findings resonate with earlier studies that emphasized cost, compatibility, and change
resistance as impediments to TMS adoption (Gambier et al., 2004; Folaron, 2012), underlining
the importance of addressing user concerns to foster successful integration. 3.1.4 Future trajectory of TMS: evolving needs and aspirations. Participants offered
insights into the future trajectory of TMS in the Saudi Arabian context. Preferences for
deployment models, the importance of complying with industry standards, and
recommendations for advancements in the TMS industry were discussed. “We’re looking for AI integration in TMS for advanced automation. This would revolutionize how
translations are managed,” remarked one interviewee. Another participant stated, “Mobile compatibility would be great. Translators on the go could
benefit from managing tasks and tracking progress.” Moreover, participants stressed the need
for language-specific features and better data security to align TMS with industry standards. These forward-looking perspectives emphasize the requirement for continuous research
and development in TMS technologies to address the evolving needs and aspirations of
translation agencies and professional translators in Saudi Arabia. The themes and excerpts from the interviews provide a rich understanding of attitudes, challenges, and opportunities
related to adopting and utilizing TMS. from the interviews provide a rich understanding of attitudes, challenges, and opportunities
related to adopting and utilizing TMS. 3. Results and discussion
3.1 Perspectives on TMS adoption: a multifaceted analysis Attitud
transl
age from the interviews provide a rich understanding of attitudes, challenges, and opportunities
related to adopting and utilizing TMS. 3.2 Quantitative insights 3.2 Quantitative insights
The survey participants comprised 35 individuals, including translation agency
representatives and independent professional translators. Their years of experience in the
translation industry varied, with 8 participants (23%) having 0–2 years of experience, 9
participants (26%) having 3–5 years, 8 participants (23%) having 6–10 years, and 7
participants (20%) having 11þ years of experience. 19 The survey unveiled a substantial awareness level within the participants’ domain. An impressive 28 respondents (80%) demonstrated prior familiarity with TMS, signifying a
commendable industry cognizance. Conversely, a minority of 7 participants (20%) indicated
an absence of knowledge concerning TMS. An intriguing observation emerged regarding TMS adoption rates (Figure 1). Among the
surveyed individuals, 20 participants (57%) reported practical usage of TMS in their
translation undertakings, emphasizing a noteworthy uptake of TMS within the community. Delving into the perceived advantages of TMS incorporation, 18 participants (90%)
acknowledged TMS as a catalyst for augmenting project efficiency. Acknowledging the
significance of collaborative synergy, 17 participants (85%) attributed improved
collaboration among team members to integrating TMS. 14 participants (70%)
underscored TMS’s impact on enhancing quality assurance mechanisms, thereby fostering
consistency across translations. In comparison, 16 participants (80%) highlighted TMS’s role
in streamlining project management and tracking, contributing to efficient resource
allocation. The survey delved into the nuanced factors influencing the adoption of TMS (Figure 2),
shedding light on the decision-making dynamics among participants. A significant majority
of 25 participants (71%) underscored the financial aspect, deeming the cost of implementation
as a pivotal determinant of TMS adoption. 21 participants (60%) accentuated the importance
of compatibility with existing tools and workflows in shaping their inclination towards TMS. The human element came into play, as 14 participants (40%) cited resistance from team Figure 1. Perceived benefits
of TMS Figure 2. Factors influencing
TMS adoption
SJLS
4,1
20 20 Figure 2. Factors influencing
TMS adoption members as a tangible challenge in adopting TMS. At the same time, 8 participants (23%)
highlighted the role of awareness deficiency as a deterrent to TMS adoption. Delving into participants’ financial perceptions of TMS implementation, the survey
identified distinct patterns in participants’ outlooks. A discerning 10 participants (29%)
considered TMS implementation moderately expensive. 15 participants (43%) perceived
TMS implementation to fall within an affordable range, implying a balanced cost-benefit
proposition. Notably, 7 participants (20%) indicated optimism in the long-term cost-
effectiveness of TMS adoption. p
Participants’ future intentions regarding exploring other TMS options were diverse. 3.2 Quantitative insights While 17 participants (49%) expressed openness to considering alternative options, 18 (51%)
indicated their intention to remain with their current TMS. Participants’ perspectives on the future of TMS indicate a forward-looking orientation: (1)
AI Integration: 16 participants (46%) expressed keenness for integrating artificial
intelligence (AI) within TMS, recognizing its potential to drive advanced automation
and revolutionize the translation landscape. (2)
Mobile Compatibility: An interest in enhanced mobile compatibility was voiced by 11
participants (31%), reflecting a growing need for flexibility and accessibility in
translation management. (3)
Language-Specific Features: The significance of catering to linguistic nuances
resonated with 7 participants (20%), suggesting that tailoring TMS features to
language-specific requirements would enhance its utility. In considering deployment models, the survey revealed a preference for cloud-based
solutions (51%) over on-premises (31%) or hybrid (17%) alternatives. A noteworthy 21
participants (60%) underscored the imperative nature of complying with industry standards
in developing and deploying TMS. In considering deployment models, the survey revealed a preference for cloud-based
solutions (51%) over on-premises (31%) or hybrid (17%) alternatives. A noteworthy 21
participants (60%) underscored the imperative nature of complying with industry standards
in developing and deploying TMS. In considering deployment models, the survey revealed a preference for cloud-based
solutions (51%) over on-premises (31%) or hybrid (17%) alternatives. A noteworthy 21
participants (60%) underscored the imperative nature of complying with industry standards
in developing and deploying TMS. Turning to training and integration, 19 participants (54%) received formal training from
TMS providers, with 9 participants (26%) benefiting from external training programs. Satisfaction levels with the training provided varied, with 7 participants (37%) being “very
satisfied,” 10 participants (53%) “satisfied,” and 2 participants (10%) remaining “neutral.” Attitudes of
translation
agencies Regarding integration with Computer-Assisted Translation (CAT) tools, 23 participants
(66%) experienced seamless integration, 10 participants (29%) encountered some
compatibility issues, and 2 participants (5%) reported no integration capabilities. When asked about any additional comments, participants shared their perspectives,
offering valuable insights into various aspects related to TMS: Participants emphasized the impact of TMS on improving translation efficiency and
acknowledged opportunities for further enhancement. They emphasized customizable
workflows, integration with machine translation, and real-time collaboration as areas with
potential for enhancement. Participants believed that these features could substantially
enhance overall productivity and streamline translation workflows. Attitudes of
translation
agencies 3.2 Quantitative insights In addition, they
emphasized the importance of robust data security measures, harnessing the potential of
neural machine translation, and staying updated on evolving industry trends for the
continued advancement of TMS technology. 21 Several participants emphasized the need to raise awareness about TMS software,
particularly among certain sectors and managers. They pointed out that some managers
lacked familiarity with TMS and sometimes confused it with basic machine translation tools
like Google Translate. This lack of awareness was particularly notable among specific
international companies operating in Saudi Arabia. Addressing this gap in understanding
was deemed crucial to promoting wider adoption and effective utilization of TMS within
these sectors. Participants expressed a need for expanded educational initiatives, including workshops
and lectures with a focus on TMS. Their request for more structured learning opportunities
reflected their recognition of the importance of comprehensive understanding and effective
use of TMS tools. The call for enhanced education underscores participants’ enthusiasm to
develop their skills and knowledge related to TMS technologies. Another observation pertains to the readiness of the local translation market for TMS
adoption. Participants noted that cost-related factors pose a significant challenge to the
integration of TMS within the industry. Furthermore, the fragmented nature of the
translation market was identified as another obstacle. This fragmentation appears to
contribute to the absence of a unified approach toward TMS adoption, impacting its seamless
implementation across various segments of the market. These results offer a comprehensive insight into the diverse attitudes, considerations, and
perceived benefits surrounding the adoption of TMS. This study contributes to the broader
understanding of TMS integration within the Saudi Arabian translation landscape by
delving into the nuanced factors and individual perceptions. 3.3 Interpretation and significance Cost emerged as a prominent concern, echoing earlier studies that
underscore the significance of financial implications in adopting new technologies (Gambier
et al., 2004; Folaron, 2012). Compatibility with existing tools and workflows was another key
determinant, aligning with the broader technology adoption literature emphasizing seamless
integration with established systems (Rogers, 2010). 3.3.2 Factors influencing adoption. The research identifies several factors that impact the
decision to adopt TMS. Cost emerged as a prominent concern, echoing earlier studies that
underscore the significance of financial implications in adopting new technologies (Gambier
et al., 2004; Folaron, 2012). Compatibility with existing tools and workflows was another key
determinant, aligning with the broader technology adoption literature emphasizing seamless
integration with established systems (Rogers, 2010). Resistance to change and lack of awareness were challenges in TMS adoption. Resistance
from team members points to the involvement of human factors in technology adoption,
indicating the importance of effective change management strategies (Orlikowski, 2000). The need for increased awareness emphasizes the importance of educational and
communication efforts to promote the benefits of TMS among potential users
(Shuttleworth and Cowie, 2014). (
,
)
3.3.3 Future considerations and innovation. Participants’ forward-looking perspectives
provide insights into the future trajectory of TMS within the Saudi Arabian translation
landscape. The expressed interest in AI integration reflects the ongoing shift towards
automation and the potential role of AI in revolutionizing translation processes (Jiang and Lu,
2020). The need for mobile compatibility aligns with the evolving work patterns, emphasizing
the importance of flexibility and accessibility in managing translation tasks (Hampshire and
Porta Salvia, 2010). Furthermore, the suggestion of language-specific features emphasizes the
diversity of linguistic requirements in translation and localization processes (Esselink, 2000). 3.3.4 Deployment models and standards. The preference for cloud-based deployment
models resonates with the broader trend of organizations adopting cloud technologies for
enhanced scalability and accessibility (Armbrust et al., 2010). The importance placed on
complying with industry standards underscores the role of standards in ensuring
interoperability and quality within the translation domain (ISO17100, 2015). 3.3.5 Training and integration. The findings suggest that formal training plays a
significant role in ensuring effective TMS implementation. The varying levels of satisfaction
with training underline the need for tailored and comprehensive training programs to
address diverse user needs (O’Brien, 2011). The smooth integration of TMS with CAT tools
highlights the technical compatibility required for seamless workflows and enhanced
productivity (Somers, 2003). 3.3.5 Training and integration. 3.3 Interpretation and significance 3.3 Interpretation and significance The combined insights from the interviews and survey questionnaire have shed light on the
complex landscape of TMS adoption and utilization in Saudi Arabia. These findings lay the
groundwork for a comprehensive discussion of the key themes, implications, and potential
avenues for future research. The implications of these findings extend to multiple dimensions
within the translation ecosystem. Practitioners and agencies can benefit from the insights
into the benefits and challenges associated with TMS integration. Decision-makers can
consider the highlighted factors influencing adoption, such as cost and compatibility, while
strategizing TMS implementation plans. Moreover, the identification of future considerations
like AI integration and mobile compatibility provides a forward-looking lens, guiding
potential enhancements in TMS technology. In terms of scholarly contributions, this research bridges a gap in the existing literature by
offering a focused exploration of TMS adoption within the Saudi Arabian context. Previous
studies have explored technology adoption in translation in a general context (Gambier et al., 2004; Al-Gahtani, 2004). This study, however, focuses on the specific landscape of TMS
adoption. By doing so, it enhances the body of knowledge around translation technology’s
practical realities within a distinct cultural and linguistic milieu. 2004; Al-Gahtani, 2004). This study, however, focuses on the specific landscape of TMS
adoption. By doing so, it enhances the body of knowledge around translation technology’s
practical realities within a distinct cultural and linguistic milieu. 3.3.1 Adoption and perceived benefits. The research findings indicate significant attention
and adoption of TMS within the Saudi Arabian translation industry. The interviews and
survey responses point to a prevalent awareness of TMS, with most participants
demonstrating prior familiarity. This heightened awareness reflects the global trend of
growing recognition of the potential benefits offered by TMS in enhancing translation
workflows (Christensen and Schjoldager, 2017; Wallis, 2006). 22 (
j
g
)
Participants generally hold positive perceptions of TMS, which are substantiated by
tangible benefits resulting from its integration. The reported benefits encompass enhanced
project efficiency, improved collaboration, heightened quality assurance, and more efficient
project management. The research has revealed that TMS have garnered substantial
attention and adoption within the Saudi Arabian translation industry. These findings
resonate with previous research highlighting TMS’s potential to significantly improve
translation processes (Bowker, 2002; Gough, 2021). 3.3.2 Factors influencing adoption. The research identifies several factors that impact the
decision to adopt TMS. SJLS
4,1 3.3 Interpretation and significance The findings suggest that formal training plays a
significant role in ensuring effective TMS implementation. The varying levels of satisfaction
with training underline the need for tailored and comprehensive training programs to
address diverse user needs (O’Brien, 2011). The smooth integration of TMS with CAT tools
highlights the technical compatibility required for seamless workflows and enhanced
productivity (Somers, 2003). 3.3.6 Understanding awareness and usage patterns of TMS. In relation to TMS, one
plausible rationale for the observed limited awareness of CAT tools could stem from their
relatively recent introduction to the Arab context. Al-Ajmi (2011) highlighted that the dearth
of such corpora in the Arabic domain might be attributed to the absence of requisite programs 3.3.6 Understanding awareness and usage patterns of TMS. In relation to TMS, one
plausible rationale for the observed limited awareness of CAT tools could stem from their
relatively recent introduction to the Arab context. Al-Ajmi (2011) highlighted that the dearth
of such corpora in the Arabic domain might be attributed to the absence of requisite programs to compile such resources. Although Al-Ajmi’s (2011) study was conducted some years ago, a
recent investigation by Man et al. (2019) aligns with this notion, suggesting that translators
are inclined to favor technologies that offer swift solutions. The intricate functionalities of
certain translation technologies might pose a hurdle to their acquisition, as they frequently
necessitate time and customization to fully comprehend and utilize. to compile such resources. Although Al-Ajmi’s (2011) study was conducted some years ago, a
recent investigation by Man et al. (2019) aligns with this notion, suggesting that translators
are inclined to favor technologies that offer swift solutions. The intricate functionalities of
certain translation technologies might pose a hurdle to their acquisition, as they frequently
necessitate time and customization to fully comprehend and utilize. The outcomes of the study demonstrate a prevailing recognition among the respondents
regarding the necessity of acquiring proficiency in translation technologies. Their consensus
revolves around the advantages of having a practical guide that elucidates the array of
translation technologies available. Furthermore, there is a consensus on the indispensability
of incorporating instruction on translation technologies within undergraduate degree
programs. Notably, close to half of the participants admit to never having participated in a
dedicated course on utilizing translation technology. Despite this, their expressed intent to
leverage technology underscores a noticeable knowledge gap in this domain. 3.4 Limitations
Whil
hi While this research strives to provide valuable insights into the attitudes and perspectives of
translation agencies and professional translators towards the integration and utilization of
TMS in the Saudi Arabian context, several limitations warrant acknowledgment. Firstly, the
study’s scope is confined to a specific geographic area, namely Saudi Arabia. This limitation
potentially restricts the generalizability of findings to other cultural and linguistic contexts,
necessitating caution when extrapolating the results to different regions. Secondly, the research’s reliance on a mixed-methods approach, combining survey
questionnaires and semi-structured interviews, might inadvertently introduce biases or
discrepancies. Participants’ self-reporting of their attitudes and practices, while valuable,
could be influenced by social desirability bias or the tendency to present oneself in a favorable
light. Thirdly, due to time and resource constraints, the sample size of the study might not
fully encompass the diversity and nuances within the entire population of translation
agencies and professional translators in Saudi Arabia. This could potentially affect the
representation of minority perspectives or underrepresented groups. Attitudes of
translation
agencies 3.3 Interpretation and significance These findings
resonate with Han’s (2020) study that the conventional teaching approach is becoming
obsolete. This alignment is further underscored by Man et al.’s (2019) conclusion,
emphasizing the fundamental role of systematic training in nurturing technological
competence. In light of these insights, this study underscores the urgency for more robust
training initiatives to effectively heighten awareness and proficiency in translation
management systems. 23 3.5 Recommendations Moving forward, this study lays the groundwork for future research endeavors to explore the
intricate landscape of TMS adoption and utilization. In light of the insights gleaned, several
valuable avenues for further investigation emerge. Firstly, there is significant potential in
conducting cross-cultural comparative studies that transcend geographical boundaries. These studies could encompass diverse countries and regions, unraveling variations in
attitudes, practices, and challenges related to TMS adoption. Such a global perspective could
enrich the understanding of the universal and context-specific factors influencing the
integration of TMS. Secondly, the implementation of longitudinal studies, carried out over an extended period,
holds promise in tracking the evolution of attitudes and practices towards TMS. These
longitudinal observations could unveil dynamic trends and shifts in adoption patterns as
translation technology continues to evolve. This longitudinal lens could also shed light on
emerging challenges and adaptations over time. Delving deeper into the factors that contribute to resistance against TMS adoption stands as another avenue for future research. By conducting in-depth analyses of the specific barriers faced by translation agencies and
professional translators, researchers can pinpoint the precise challenges that need to be
addressed. This knowledge could pave the way for more tailored interventions and strategies
to overcome reluctance towards TMS integration. contribute to resistance against TMS adoption stands as another avenue for future research. By conducting in-depth analyses of the specific barriers faced by translation agencies and
professional translators, researchers can pinpoint the precise challenges that need to be
addressed. This knowledge could pave the way for more tailored interventions and strategies
to overcome reluctance towards TMS integration. A promising research focus lies in investigating the efficacy of various training and
educational initiatives geared towards TMS adoption. Understanding which training
methods are most effective in equipping translation professionals with the necessary skills
and knowledge can facilitate smoother integration and maximize the benefits of TMS
technology. Furthermore, the exploration of hybrid models, which seamlessly combine
human expertise with TMS capabilities, presents an innovative direction for future
investigation. The potential of striking a balance between automation and human creativity
could revolutionize translation practices and enhance the efficiency of workflows. 24 Considering the ethical dimensions of TMS integration is essential in this digital age. Future research could delve into the realms of data privacy, security, and potential biases
introduced by TMS. This exploration ensures that the integration of TMS remains aligned
with ethical standards and societal considerations. 3.5 Recommendations Lastly, as emerging technologies like
artificial intelligence and natural language processing gain traction, probing their
intersections with TMS could be a productive avenue. Investigating how these
technologies can synergize with TMS to create more advanced and efficient translation
workflows could shape the future of the translation industry. SJLS
4,1 References References
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study has unveiled the intricate web of factors influencing TMS adoption, encompassing
financial considerations, technical intricacies, human dynamics, and the imperative of
heightened awareness. The elucidation of these dimensions enriches our comprehension of
the multifaceted nature of TMS integration. The significance of TMS’s integration lies not only in its immediate benefits but also in its
potential to revolutionize and elevate translation workflows in the long run. The recognition
of its potential advantages and the participants’ contemplation of future possibilities
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prevailing challenges. Notably, the cost implications, the intricate web of compatibility
requirements, the resistance posed by entrenched practices, and the essential need for
comprehensive training stand out as formidable challenges that must be addressed with
strategic measures. In a broader sense, the insights garnered from this research extend beyond the immediate
scope of TMS integration. They contribute to the ongoing discourse surrounding the broader
domain of translation technology adoption and adaptation. By shedding light on the nuances,
intricacies, and considerations surrounding TMS, this study offers a roadmap for translation
agencies, professionals, and researchers who strive to navigate the swiftly evolving
technological landscape within the translation domain. In essence, this research provides a holistic and profound perspective on the integration of
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John Benjamins, Amsterdam and Philadelphia. Attitudes of
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agencies Verplaetse, H. and Lambrechts, A. (2019), “Surveying the use of CAT tools, terminology management
systems and corpora among professional translators: general state of the art and adoption of
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systems”, (Master’s thesis), University of Ottawa, Ottawa. 27 Verplaetse, H. and Lambrechts, A. (2019), “Surveying the use of CAT tools, terminology management
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02.01. About the author Dr Waleed Obaidallah Alsubhi is Assistant Professor of Translation at the University of Jeddah. He
received his Master’s Degree in Translation from Sheffield University, and his Ph.D. in Translation
Studies from Birmingham University, UK. He worked as a translation project manager for several
translation and localization projects. He also works as Consultant for several institutions. His research
interests are translation and cognition, translation technology, audiovisual translation and localization. Waleed Obaidallah Alsubhi can be contacted at: woalsubhi@uj.edu.sa For instructions on how to order reprints of this article, please visit our website:
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The reformed confessions: embarrassment or blessing to a missionary church?
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The reformed confessions: embarrassment
or blessing to a missionary church? J.J. Fritz Krüger
School of Ecclesiastical Sciences (Missiology)
Potchefstroom Campus
North-West University
POTCHEFSTROOM
E-mail: fritzkruger@telkomsa.net The reformed confessions: embarrassment or blessing to a
missionary church? The historic reformed confessions are sometimes blamed for
the apparent lack of missionary zeal among churches of the
RCSA, and called an embarrassment to the church. This article
investigates these allegations and attempts a missionary read-
ing of the confessions. Because of the specific focus of the con-
fessions, they should not be expected to give guidance on the
whole life of the church. However, careful reading of the creeds
does offer a surprisingly abundant missionary harvest. They
offer both the foundations and the limits for mission, and in
defining the identity of Christians, also define the missionary
identity of the church. As the community of fellowship with
Christ, the church is both a unique instrument in the “missio
Dei” and the end objective of mission (mission as church plant-
ing). Creeds offer beautiful expositions of the loving, fellowship-
seeking heart of God who has not given up on fallen humanity,
but who continues to call people to fellowship with Him in his
self-revelation in creation, Scripture and the incarnation of the
Son. Surprisingly, it is the Canons of Dordt (2.5) which offers
the only explicit call to mission in the three creeds! 2
Since 2005, annual mission conferences, organised by congregations of the
RCSA and the Faculty of Theology of the North-West University, present a
forum for discussion of these matters. The viewpoints referred to here were ex-
pressed during these conferences. 1
The abbreviation RCSA (Reformed Churches in South Africa) is the English
equivalent of the Afrikaans GKSA (Gereformeerde Kerke in Suid Afrika). Die gereformeerde belydenisse: verleentheid of seën vir ’n
missionêre kerk? Die histories gereformeerde belydenisskrifte kry soms die skuld
vir die klaarblyklike gebrek aan sendingywer by kerke van die
GKSA, en word selfs ’n verleentheid vir die kerke genoem. Hier-
die artikel ondersoek die uitsprake en onderneem ’n missionêre In die Skriflig 41(4) 2007:549-570 549 The reformed confessions: embarrassment or blessing to a missionary church? lees van die belydenisskrifte. As gevolg van die spesifieke
fokus van die belydenisskrifte, moet daar nie van hulle verwag
word om leiding te gee op alle terreine van die kerklike lewe
nie. Nogtans lewer ’n versigtige lees van die belydenisskrifte ’n
verbasend groot missionêre oes op. Hulle bied sowel die fon-
damente as die grense van sending, en deur die identiteit van
Christene te definieer, definieer hulle ook die missionêre identi-
teit van die kerk. As gemeenskap van mense wat in gemeen-
skap met Christus lewe, is die kerk sowel ’n unieke instrument
in die “missio Dei” asook die doel van sending (sending as kerk-
planting). Belydenisskrifte bied wonderlike uiteensettings van
die liefdevolle, gemeenskapsoekende hart van God wat nie op-
gegee het met die gevalle mensdom nie, maar wat voortgaan
om mense tot gemeenskap met Hom te roep deur sy self-
openbaring in die skepping, in die Skrif en in die inkarnasie van
die Seun. Verbasend genoeg is dit juis die Dordtse Leerreëls
(2.5) wat die enigste eksplisiete oproep tot sending in al drie
belydenisskrifte laat hoor! 1. Introduction During the past few years, we have seen a growing discussion
within the RCSA1 on the missionary calling of our churches – or
rather: the perceived lack of missionary consciousness and activity.2
In discussions, some people have tried to blame this on the doctrinal
teaching of the church, more specifically on the reformed con-
fessions, and specifically the doctrine of divine predestination (cf. Van der Merwe, 1960:2). In their opinion, the confessions contain
nothing that would inspire members or congregations to participate
in mission, neither do they contribute to the formation of a mission-
ary RCSA. In a sense, the confessions have therefore become an
embarrassment to the church, rather than a blessing. Haak (1999:61-62), speaking with obvious piety about the reformers
and the reformed confessions, defends the confessions against
these claims by conceding that the confessions do not present a
missionary and inviting ecclesiology, but rather a defensive and
conservative one, reflecting a church concerned only with herself In die Skriflig 41(4) 2007:549-570 550 J.J. Fritz Krüger and her own survival. However, this is the result of the context within
which the confessions originated, and of the fact that no single
confession can be absolutely comprehensive in its following of
Scripture. The limitations of the reformed confessions are therefore
understandable. and her own survival. However, this is the result of the context within
which the confessions originated, and of the fact that no single
confession can be absolutely comprehensive in its following of
Scripture. The limitations of the reformed confessions are therefore
understandable. In speaking of the missionary calling of reformed churches, the
question is whether the confessions really deserve to be the em-
barrassment they are to some. Do they deserve to take the blame
for the perceived lack of missionary zeal of our churches? The
following remarks will attempt an answer to this question, by offering
a missionary reading of the confessions. Due to constraints of
space, only a relatively small selection out of the total wealth of the
confessions can be offered. 2. The function of confessions According to Schaff (1877:4), a creed is a confession for public use
– which in itself renders the document one of expression and
outward communication of the doctrine of the church (cf. Schulze,
1991:2, 3). The very possession of confessions should already
stimulate missionary activity: they are not intended to be treasured
possessions carefully locked away in the vaults of the church, only
put on view at certain solemn occasions and then polished before
they are once again consigned to the underground vaults of the
treasury. They are intended to be printed on banners and hung
above the doors of our churches – on the outside, inviting in; they
are intended to be printed and handed out as summaries of our
faith, inviting careful thought and consideration; they are intended to
be hung out like flags, commanding attention; they are intended to
be raised like banners in front of a marching crowd, urging them on
to enter the world in the name of Jesus with his message of
reconciliation and peace. In this sense, then, there is little difference
between an apologetic and a missionary function of the confessions. In its Greek form, the word suvmbolon means a mark, badge or
watchword. In other words, it functions as an indication of identity,
especially in the military sense (cf. the church as ecclesia militans):
Which army is this? The answer: the army of Christ! Such a mark of
identity or military banner always has two functions: for those who
belong to the identified group, it indicates belonging, safety, security. Confessional statements indicate the boundaries of the faith
community, both delineating and protecting the community (cf. confessions as symbols by which the church is distinguished from
the world – Schulze, 1991:4-5; Belgic Confession Art. 29). To those In die Skriflig 41(4) 2007:549-570 551 The reformed confessions: embarrassment or blessing to a missionary church? who do not belong, it is a social boundary marker that excludes
them, but also an indirect kind of invitation: this is who we are, join
us! Important for our topic is that creeds were usually formulated in a
context of controversy concerning the true meaning of Scripture, so
that the teaching of the church could be regulated and so that the
church could be guarded against error. 2. The function of confessions This fact gives every one of
the creeds a very specific thrust, so that we should guard against
over-asking the creeds in our expectation of their missionary role in
our churches of today. Whatever missionary prompting they may
present, will probably be indirect, coming from the consequences of
doctrinal statements not primarily concerned either with mission as
such or with the missionary calling of the church (cf. Haak, 1999:61-
64, 67). Some digging will therefore be required. The remarks below
represent such digging, from a missionary point of view. Hopefully,
the results will inspire some discussion, if not a clear statement of
the mission of the church according to the confessions. 3. The Heidelberg Catechism In its origins, the Heidelberg Catechism was only slightly polemic. Its
intention was never dispute with opponents (with the possible
exception of the contentious Question and Answer 80), but rather
religious instruction for the young (cf. Schaff, 1877:531-532). It was
written as a summary of the Christian faith from the Word of God. Indeed, in the reformed world, it also functions as a standard of
public doctrine, explaining as it does so very clearly the Calvinistic
system (Schaff, 1877:540). 3.1 The foundations and limits of mission Although it cannot be said that the famous introduction to the
Heidelberg Catechism in Answer 1 had mission in any form in mind,
it certainly sets out the constitution for all mission. In its un-
ashamedly exclusive stress on our only comfort in life and death
being our belonging to and serving of Jesus Christ, it defines both
the foundations and the limits of all mission. The scope of this first
answer is breathtaking in its profundity. It speaks of comfort, thereby recognising the universal human con-
dition of misery, of alienation from all comfort. It recognises the
human heart in its pain and in its cry for comfort. At the root of man’s
misery is the fact that there is nobody he can call father. He is like
an abandoned child, alone in a terrifying world. He has to fend for In die Skriflig 41(4) 2007:549-570 552 J.J. Fritz Krüger himself in a world where our highest effort usually delivers no more
than thorns and thistles (Gen. 3:18). He has nobody to defend him in
adversity – only his own futile efforts which are so easily over-
whelmed by this sad world (cf. 9:26; 103). This is therefore no sterile doctrinal document: it is a pastoral hand
reaching out to the lost and the lonely, the suffering and the crying,
those lost in despair and hidden in oppression. If there were a fault,
it is not in the Catechism but in the heart that reads it. How can I
read and speak of comfort, even confess it, as the only comfort
available, but treasure it only for myself? How can I rejoice in libera-
tion from sin and the tyranny of Satan, revelling in the freedom of
serving Christ, and yet be blind and deaf to the suffering of the world
around me? The Catechism speaks of belonging to a Saviour both body and
soul, thus including total human existence in all its diversity in the
desperate need for one Saviour: not one for the body and another
for the soul. No, the very same Saviour will be responsible for the re-
demption of both body and soul (cf. Bavinck, 1954:63-64). 3
The numbering used in heading 3 and its subparagraphs refers to the relevant
Lord’s Day: Question and Answer of the Heidelberg Catechism, so that 9:26
refers to Lord’s Day 9, Question and Answer 26, following common practice in
this regard. 4
Cf. in this regard also the explanation of the first commandment in 34:94-95.
This commandment can be called the foundation of all mission: there is only one
true God, and He must be worshiped with heart and soul by one and all! This
emphasis is echoed in 45:117, where the nature of prayer is discussed. 3.1 The foundations and limits of mission Con-
sequently, in whatever way we get involved in the lives of people,
we cannot say that we want to save only their souls, while ignoring
their plight in terms of poverty, hunger, disease, injustice and
oppression, claiming that it is none of our business. Also, inversely,
we cannot focus only on the material and bodily life of people,
declaring the soul to be respected neutral territory where we dare
not influence the sovereign right of the individual to choose for
himself. The whole life of man cries out to be comforted, and this
first answer points the way. Our cry for comfort, whatever its origins
may be and whatever the circumstances causing us to cry out, can
only be answered by one Person: Jesus Christ. The content of our missionary preaching is also pointed out clearly:
How is the cry of the human heart for comfort answered?: by the
gospel of payment for our sins; by the blood of Jesus; and by his
delivering us from the tyranny of the devil (cf. 5:12-8:25). Individual
and collective sin, and the tyranny of Satan are the roots of misery
and the robbers of God’s comforting gifts of grace (4:9). In die Skriflig 41(4) 2007:549-570 553 The reformed confessions: embarrassment or blessing to a missionary church? In this way the Catechism penetrates right through all liberal ecu-
menical and humanistic probing of the human condition. What is at
the root of all human misery? Some people say political injustice, or
oppression and violence. Others point to poverty and economic
imbalances between North and South. Still others speak of disease
and lack of education. However, the Catechism is clear that sin and
Satan are at the root of human misery (2:3-4:11). This emphasis of
the Heidelberg Catechism makes mission, as gospel teaching, both
possible and necessary! All those other things might be the visible
manifestations of sin and Satan, but they are not the root cause. Only once this has been accepted, can we go back and, in the name
of Jesus, fight for liberation, side by side with the victims of injustice,
oppression, poverty, disease and bad education. In this way the Catechism penetrates right through all liberal ecu-
menical and humanistic probing of the human condition. What is at
the root of all human misery? Some people say political injustice, or
oppression and violence. 3.1 The foundations and limits of mission He has declared: ‘Cursed is everyone who
does not continue to do everything written in the Book of the
Law’. now and in eternity. He has declared: ‘Cursed is everyone who
does not continue to do everything written in the Book of the
Law’. This fact makes our mission of reconciliation so much more des-
perate in its urgency as we cry out to people: “we implore you on
Christ’s behalf: be reconciled to God!”. “We know what it is to fear
God, [therefore] we try to persuade men. […] Christ’s love compels
us” (2 Cor. 5:20, 11, 14). 3.1 The foundations and limits of mission Others point to poverty and economic
imbalances between North and South. Still others speak of disease
and lack of education. However, the Catechism is clear that sin and
Satan are at the root of human misery (2:3-4:11). This emphasis of
the Heidelberg Catechism makes mission, as gospel teaching, both
possible and necessary! All those other things might be the visible
manifestations of sin and Satan, but they are not the root cause. Only once this has been accepted, can we go back and, in the name
of Jesus, fight for liberation, side by side with the victims of injustice,
oppression, poverty, disease and bad education. Although the Catechism never uses the word conversion at this
point, it is clear that it speaks of a total reorientation of human life if
its seeks to be comforted. The Catechism is no modern-day re-
specter of the integrity of the sovereign individual: I am not my own,
but I belong body and soul to Jesus Christ, living for him and not for
myself, for sin or for Satan. Christ is the magnetic north that realigns
all human life according to its creationally intended directions (3:6),
thereby bringing it to its created potential and liberating it from all
constraints and chafing bonds. The chaos of human life is brought to
order in one way only: by the magnetic field of Jesus Christ in his
sovereign, loving, saving Lordship (cf. also the exclusive statement
of 11:29-30, echoing the words of Acts 4:12). To be truly free, is to
be a slave of Jesus Christ only.4 The emphasis in 1:1 on the grace of God in Jesus Christ does not in
any way detract from the sobriety of the Catechism in terms of the
anger of God about human sin and injustice. This, of course, is al-
ways the other side of the coin of our missionary preaching; the
warning note that tells people that, even if we proclaim the mercy
and the love of a saving God, He is not a God to be toyed with: God
is not only merciful, but also just. In the words of 4:10 (cf. 4:11;
19:52; 32:87): He is terribly angry about the sin we are born with as well as the
sins we personally commit. As a just judge He punishes them In die Skriflig 41(4) 2007:549-570 554 J.J. Fritz Krüger now and in eternity. 3.2 Our Christian identity Perhaps the answer that speaks most directly to the heart of
Christians about our task in the world, is to be found in 12:32, where
our identity as Christians is defined: Why are you called a Christian? Why are you called a Christian? At this mighty event of Pentecost, the
prophecy mentioned by quoting Joel 2:28-32 clearly also included
witnessing to, warning and pleading with unbelievers: “Save
yourselves from this corrupt generation!” (Acts 2:40). Note also
that the young church’s mission to the world starts with the
anointing of the Spirit, which transforms ordinary men and women
into prophets of Jesus Christ and sends them to unbelievers,
urging them to repent and turn to God! Confessing the name of
Christ (12:32) thereby becomes a prophetic speaking about
God’s will of salvation: addressing sin directly, calling to repen-
tance, speaking about Christ in a way that will attract people to
Him and to the same union with Him that I enjoy. • Prophet: According to Friedrich (1968:853), prophecy is also
proclamation, just like evangelism. Prophecy in the Early Church
served to admonish, console, encourage, reveal sin and call to
repentance (Friedrich, 1968:848). Although evangelism is as a
rule directed to unbelievers, and prophecy to the congregation of
believers, Acts 2:17 indicates that even prophecy is sometimes
evangelistic in its intent. At this mighty event of Pentecost, the
prophecy mentioned by quoting Joel 2:28-32 clearly also included
witnessing to, warning and pleading with unbelievers: “Save
yourselves from this corrupt generation!” (Acts 2:40). Note also
that the young church’s mission to the world starts with the
anointing of the Spirit, which transforms ordinary men and women
into prophets of Jesus Christ and sends them to unbelievers,
urging them to repent and turn to God! Confessing the name of
Christ (12:32) thereby becomes a prophetic speaking about
God’s will of salvation: addressing sin directly, calling to repen-
tance, speaking about Christ in a way that will attract people to
Him and to the same union with Him that I enjoy. • Priest: the classic texts about the priesthood of believers are
1 Peter 2:5 and 9. If we compare these verses, we see that
offering spiritual sacrifices to God and declaring (“broadcasting” –
cf. Schniewind, 1964:67) the praises of him who called us out of
darkness into his wonderful light are parallel actions, if not one
and the same thing. In other words, our liturgical service in the
holy presence of God is at the same time directed to the world. Why are you called a Christian? Because by faith I am a member of Christ and so I share in his
anointing. I am anointed to confess his name, to present myself
to Him as a living sacrifice of thanks, to strive with a good
conscience against sin and the devil in this life, and afterward to
reign with Christ over all creation for all eternity. There is an element of wonderful piety here, which is all too often
understood by well-meaning Christians in a pietistic way as being
relevant only to the inner life of my individual soul. However,
commentaries on the Heidelberg Catechism identify this answer as
one of the most missionary in the whole confession (cf. Veldkamp,
s.a.:168-172; Spykman, 1984:87-88). First of all, it is essential to recognise the all important emphasis of
this answer. There can be no discussion about my identity or calling
as a Christian apart from that very personal, intimate (mystic!) union
with Christ. It is this union alone that makes me what I am. It is from
the transforming power of this union alone that I speak and confess
and witness (cf. John 15:1-5). It is the three-fold office of Christ
alone which defines and motivates and makes possible my work in
this world, which in this sense remains the work of Christ Himself,
done through me. In this sense, we should say that no mission is
possible without intimate union with Christ, and also that true union
with Christ can have no other effect than missionary activation of
believers and churches, for the simple reason that the Christ with
whom we are joined in mystic union never stops reaching out to the
world. In die Skriflig 41(4) 2007:549-570 555 The reformed confessions: embarrassment or blessing to a missionary church? From this union with Christ, his anointing as Prophet, Priest and
King becomes also my anointing to these same offices. Each of
these three offices can be shown to have missionary elements to
them: • Prophet: According to Friedrich (1968:853), prophecy is also
proclamation, just like evangelism. Prophecy in the Early Church
served to admonish, console, encourage, reveal sin and call to
repentance (Friedrich, 1968:848). Although evangelism is as a
rule directed to unbelievers, and prophecy to the congregation of
believers, Acts 2:17 indicates that even prophecy is sometimes
evangelistic in its intent. Why are you called a Christian? It
is not for nothing that in the very next verses (1 Peter 2:11-12)
Peter continues by calling Christians aliens and strangers who
are called to live among pagans in such a way that even these
pagans will one day glorify God (cf. Bavinck, 1954:55-56). We
find the same idea in 32:86, the introduction to the third main
section of the Catechism, concerning our gratitude for salvation:
the good works we do serve to win our neighbours for Christ
(1 Peter 3:1-2). We are to live missionary lives by the simple act
of obedience to the Word of God – this is our spiritual sacrifice to
God. So we see that the offering of our spiritual sacrifices to God,
offering our bodies as living sacrifices in spiritual worship (Rom. 12:1), and praising God for our salvation are not acts intended to In die Skriflig 41(4) 2007:549-570 556 J.J. Fritz Krüger be done in private, hidden away in dark, mysterious, incense-
filled sacred spaces, but out in the open, in the world, among the
pagans … with the express intention of bringing them to Christ
and to the moment of glorifying God on the day of judgement (cf. Kistemaker, 1987:91-97). This is mission in its essence! be done in private, hidden away in dark, mysterious, incense-
filled sacred spaces, but out in the open, in the world, among the
pagans … with the express intention of bringing them to Christ
and to the moment of glorifying God on the day of judgement (cf. Kistemaker, 1987:91-97). This is mission in its essence! • King: to share in the anointing of Christ means, according to
12:32, that I will strive with a good conscience against sin and the
devil in this life. Returning again to the Scripture references given
by the Catechism, we find the same text of 1 Peter 2 mentioned,
but now verse 11: “Dear friends, I urge you, as aliens and
strangers in the world, to abstain from sinful desires, which war
against the soul.” This might seem a very private war, waged in
the hidden depths of the heart, but verse 12 shows again that
there is a missionary intention even in my private struggle against
secret sin: I have to live such a good life among unbelievers that
they too will end up glorifying God! 5
Contrary to this interpretation, cf. Floor (1995:206) and Grosheide (1960:96).
Neither of these commentators, however, make mention of the obvious parallel
of Isaiah 52:7, nor do they consider the semantic implications of readiness
(eJtoimasiva), which is readiness for action – action defined as such by the
dynamic word eujanggevlion. 6
With reference to Isaiah 52:7. Why are you called a Christian? Another reference here is to Ephesians 6:11, where Paul urges
believers to put on the full armour of God. Again, this is not merely
for a private battle, but for a full-scale war against rulers and
authorities, the powers of this dark world and even against the
spiritual forces of evil in the heavenly realms (Eph. 6:12). The battle
is not merely defensive, but aims to conquer new territory: we are to
go out with feat fitted with readiness to proclaim the gospel of peace
(Eph. 6:15 – most English translations follow the notion that
“readiness” indicates readiness to proclaim or witness5 – cf. Grundman, 1964:704-706; MacDonald, 2000:345-346;6 Louw &
Nida, 1989:6837 and Oepke, 1967:312), armed with the sword of the
Spirit, the Word of God. In the wide definition of the “enemy” in this
battle, it is clear that this war can never be fought only in the privacy
or our own hearts, but will be waged in the midst of the world, in the
lives of people, and on the public stages of our society. In die Skriflig 41(4) 2007:549-570 557 The reformed confessions: embarrassment or blessing to a missionary church? 3.3 The church: product of Christ’s fellowship-seeking and
saving love Fritz Krüger (2 Cor. 5:18-20; cf. Bavinck, 1954:53), which we should be ready to
proclaim. This is the Christ whose fellowship-seeking heart is turned
towards the world, seeking and calling the lost, and taking the
church with Him in this great saving work. This is why Bavinck
(1954:70) can say so unequivocally that mission is the work of the
church, yet at the same time it is the work of Christ Himself through
his church (cf. also Bosch, 1991:372, 378-381). Thirdly, it is not without reason that we confess that Christ gathers
his church through his Word and his Spirit. This is the sovereign
work of the Lord of all things. Never once is either the initiative or the
power in the hands of people, however holy or powerful or rich they
might be, for the Word of Christ alone is the saving power of God in
the lives of people (Rom. 1:16). There is no one who dares to
preach without having been sent by Christ and speaking the words
given by Christ (Rom. 10:14-17), and there is no one who can
believe without the Spirit of Christ granting faith and changing hearts
(1 Cor. 1:10-16). Always we have to say with Paul: “I …, yet not I but
the grace of God …” (1 Cor. 15:10).8 Fourthly, this is a profound comfort to all involved in the beautiful,
yet seemingly impossible work of mission. Many missionaries will
attest that they often find themselves heading for seemingly im-
penetrable walls, not knowing what to do or how to do it. They
sometimes see their work dashed to pieces on the rocks of sin and
hardened hearts. They see unfortunate circumstances making
months of hard work meaningless in one day. They see people in
whom they trusted turn away from Christ and following their old
ways. Many are the times they ask in despair: What is the meaning
of all this? Why do we do this? The answer is: because Christ
gathers for Himself a community chosen for eternal life! Again, we
have to repeat, always in great humility: “I … and yet not I …”. Fifthly, mission is universal. We cannot say that we should focus our
missionary work only on the people of the Third World, or only on
those who are under oppression, or those who are poor or sick. 3.3 The church: product of Christ’s fellowship-seeking and
saving love Now, when we turn to the church, we find that the identity of
individual believers is also the identity of the church, and the
anointing of individuals to prophets, priests and kings, also implies
that the church is a prophetic, priestly, and royal fellowship – doing
the same work corporately in its fellowship as its members do
individually in their personal spheres of influence. Especially important here, though, is the quality of the fellowship of
the church. As Bavinck says (1954:56-57; translation – JJFK), with
reference to Philippians 2:14-15: All work, regardless of how much we paid for it or how im-
pressive it is in its design, that does not rest in a congregation
that has found the secret of mutual love, will prove to be futile. Both mission and evangelism simply cannot do without that one
moment of creating jealousy (among onlookers standing
outside the church and looking in). Our confession about the church (21:54-55) is once again a
poignant expression of trust in Jesus Christ: I believe that the Son of God through his Spirit and Word, out of
the entire human race, from the beginning of the world to its
end, gathers, protects, and preserves for Himself a community
chosen for eternal life and united in true faith. And of this
community I am and always will be a living member. This simple statement contains a wealth of missionary persepctives. The following points highlight a few of them. Firstly, we should honour the emphasis of the Heidelberg Catechism
by starting with Christ. He works through all ages of history to gather
a community of people as his personal possession, for personal
fellowship with Him, in order to share his gifts and treasures with
those He gathers. Here we see the loving, fellowship-seeking and
saving heart of Christ at work as He is present in the world of
comfortless humanity! This is the missio Dei in the Person of Jesus
Christ (cf. Bosch, 1991:389-393). One cannot help but hear his cry
in Matthew 11:28 – “Come to Me, all you who are weary and bur-
dened, and I will give you rest”! Secondly, this is the Christ who is present in this world through his
chosen people, speaking to the tormented hearts of lost people with
the gospel of peace and the words of the ministry of reconciliation In die Skriflig 41(4) 2007:549-570 558 J.J. 8
A good description of the growth of the church through the Word of Christ is
given in Noordegraaf (1983). ‘Your kingdom come’ means, […] Keep your church strong, and
add to it ... ‘Your kingdom come’ means, […] Keep your church strong, and
add to it ... ‘Your kingdom come’ means, […] Keep your church strong, and
add to it ... Another perspective is offered by 26:69-27:74, which speaks of the
sacrament of baptism as the mark of being a member of Christ
(26:70) and of being received into the church of Christ (27:74). The
two promises concerning baptism, quoted from Scripture in the
Catechism, are both taken from a context of mission (Matt. 28:19
and Mark 16:16). Therefore, mission, the sacrament of baptism and
the church as the body of and fellowship with Christ are all intimately
connected. 3.3 The church: product of Christ’s fellowship-seeking and
saving love If
we confess with the first few Lord’s Days of the Heidelberg Cate-
chism that indeed all people are lost and without comfort, then
mission should likewise extend to all people. That is why the
inclusion of this phrase is so important: “The Son of God … out of
the entire human race … gathers … for Himself a community”! It is 8 In die Skriflig 41(4) 2007:549-570 559 The reformed confessions: embarrassment or blessing to a missionary church? high time that the old missionary direction from North to South and
from West to East come to an end: in the universal church of Christ,
He moves from all to all, excluding no single church. Sixthly, the end objective of mission is often seen to be the salvation
of the individual. Once (a promise of) conversion is achieved, the
goal has been reached. However much emphasis the Catechism
places on the individual, even addressing the individual throughout,
this part of our confession is very clear as to Christ’s objective: He is
gathering a church, a fellowship of saved people, a community. This
fellowship character of the church is seen at its clearest in the
sacrament of the Lord’s Supper, which shows that we are “flesh of
his flesh and bone of his bone” (28:70). Regarding the communion
of the saints, 21:55 is very exclusive in its language: believers share
in Christ and in all his treasures and gifts “as members of this
community”! This is the well-known metaphor of the church as the
body of Christ (cf. 1 Cor. 12:12-13), which leads us to conclude that
the objective of mission work is the planting of churches (cf. Bavinck, 1954:160-161). Significantly, at the heart of the prayer life
of the church is the Lord’s prayer, where we ask in the second
request: “Let your Kingdom come!” (48:123), which is then explained
as follows: 9
The numbering in heading 4 and subparagraphs refers to the chapter and
paragraph numbers of the Canons of Dordt, so that 1:1-3 refers to chapter 1,
paragraphs 1-3, following common practice in this regard. 4. The Canons of Dordt The Canons of Dordt are the most polemic of the three reformed
confessions. As a result of the contra-Arminian objectives of the
Canons, we cannot expect a broad and comprehensive approach to
all matters doctrinal. Unlike the Heidelberg Catechism, the Canons
of Dordt never pretended to present a complete overview of
Christian faith as such, but instead narrowed its focus to specific
soteriological matters. However, despite this, we find in the Canons In die Skriflig 41(4) 2007:549-570 560 J.J. Fritz Krüger of Dordt a wonderful view of the loving heart of a God who searches
out all who belong to Him and restores them to life as He intended. 4.1 The loving heart of God seeking salvation for man from
eternity to eternity Right at the beginning of the Canons of Dordt, under the first main
point of doctrine regarding divine election and reprobation, we find a
beautiful introduction with a truly evangelical heart (1:1-39): yes, it is
true that all people have sinned and have come under the curse of
death, thus destroying all comfort for mankind, but the love of God is
not satisfied with this. In his mercy, He sends preachers of the joyful
gospel of his love in Jesus Christ, so that lost people may be
brought to repentance and faith in Christ. God, says 1:7, calls and
draws the chosen ones into Christ’s fellowship through his Word and
Spirit – He finds us, we do not have to search for Him. With infinite
patience, but with unstoppable resolve, God continues to fulfil his
promise and achieve his objectives, searching out those who belong
to Him, cleansing and washing them to perfection through the blood
of Christ and the transforming grace of his Word and Spirit (cf. 3/4:11 and 16). Once again, a short but powerful indication of the
missio Dei (cf. Bosch, 1991:389-393): God is at work in this world
through his Word and his ministers to turn the darkness of the curse
into the light of blessing (cf. also Heyns, 1978:93-94, with reference
to Barth). In 1:6-7 we also find the expression of the mysterious link between
the visible result of the preaching of the gospel, namely repentance
in the hearts of some, and the invisible decision of God to choose
some for eternal life and others not (cf. Eph. 1:4, 11). However
unpopular this doctrine of divine predestination is, it does provide us
with solid bedrock under our feet as we go out into the world to
preach the gospel: there will always be fruit; there will always and
inevitably be those who turn to God (just as there will always be
those who do not – cf. 2:6), regardless of faults in the preaching or
the preachers, regardless of circumstances and regardless of initial
resistance to the gospel. Even though many years may pass without
one single, visible convert, we may still fall back on the certainty that
those who have been chosen (in the sense of Eph. 1) will 9 In die Skriflig 41(4) 2007:549-570 561 The reformed confessions: embarrassment or blessing to a missionary church? 4.1 The loving heart of God seeking salvation for man from
eternity to eternity irrevocably come to repentance and faith through the irresistible
grace of God (cf. 3/4:12), despite the gates of hell seeking vainly to
prevail (2:9). As Van der Merwe has rightly remarked, missionaries
cannot make gold, they can only discover with the tools of the Word
and their witness the gold that has already been given by God (Van
der Merwe, 1960:91; cf. also John 17:6, 9). From the Heidelberg
Catechism, we already know that Christ is gathering his church
without fail. From the Canons of Dordt, we now know that the end is
also guaranteed: all those who have been intended for eternal life,
will inherit it. Between these two solid rocks, we are allowed to do
our work in peace and assurance as the anointed ones of Christ:
prophets, priests and kings in this world in the Name of Christ. How can we not be inspired by the sight of God moving into this
world, seeking out his beloved chosen ones, softening their
hardened hearts, breaking down their resistance, with infinite
gentleness cleansing and freeing them from the dark mire of sin and
death? In fact, this confession is the only one to order us out into the
world in no uncertain terms: This promise, together with the command to repent and believe,
ought to be announced and declared without differentiation or
discrimination to all nations and people, to whom God in his
good pleasure sends the gospel (2:5). 4.2 The radical corruption of man The Canons of Dordt, in reaction to the teachings of the Arminians
and Pelagians, are the most outspoken of all three our confessions
regarding the total and radical corruption of man. One aspect of this
doctrine which has direct bearing on missiological thinking in our
time, is to be found in 3/4:4-5. The Canons clearly teach that there is
no possibility of finding, knowing or turning to God simply by means
of the light of nature, without the Word and Spirit of God. This is not
even possible for those who have the Law of Moses. We read in
3/4:6: What, therefore, neither the light of nature nor the law can do,
God accomplishes by the power of the Holy Spirit, through the
Word or the ministry of reconciliation. This is the gospel about
the Messiah, through which it has pleased God to save
believers, in both the Old and the New Testament. This accent of the Canons of Dordt cuts off at the root all discussion
about the saving potential of other religions and anonymous Chris-
tianity (cf. Bosch, 1991:486), and severely limits the possibility of In die Skriflig 41(4) 2007:549-570 562 J.J. Fritz Krüger serious dialogue with other religions as called for by some in the
ecumenical movement. True ecumenicity is only possible within and
on the basis of overt fellowship in Jesus Christ (cf. the critical
discussion of this topic in Zuidema, 1965:123-158). serious dialogue with other religions as called for by some in the
ecumenical movement. True ecumenicity is only possible within and
on the basis of overt fellowship in Jesus Christ (cf. the critical
discussion of this topic in Zuidema, 1965:123-158). This, of course, does not mean that careful, listening conversation
with those of other faiths is impossible, or that one should not seek
to understand the hearts and minds of people of other faiths (cf. Bosch, 1991:483) from a position of solidarity with the world of
sinners (Heyns, 1978:370-372). However, if listening means uncriti-
cal acceptance, or the impossibility of appealing to those of other
faiths to be reconciled to God through Jesus Christ because He is
the only Way and Truth, we have to draw a line. 4.2 The radical corruption of man However deep our
solidarity may go with those of other faiths, based on our common
humanity, it will always have to be a critical solidarity, based on the
exclusivity of the gospel of Jesus Christ (Heyns, 1978:372-374). On the basis of our confession, we therefore have to deny the pos-
sibility that people can approach God along the paths of their own
religions, without the revealing and gracious work of God Himself
through his Word and Spirit. Perhaps in our time, just as in the time
of the Reformation, there should be a renewed emphasis on the
unbreakable link between Word and Spirit (cf. Jonker, 1981:50-58)
and a strong rejection of natural theology (cf. Van Genderen &
Velema, 1992:126-131), which is often at the root of religious univer-
salism. The Spirit of God works where the Word of Christ is
preached, heard and confessed (Van Genderen & Velema,
1992:112-114). The universality of mission, which is not restricted in movement from
West to non-West and from North to South, seems to be indirectly
indicated in 3/4:7, as discussed under 3.3 above. 10
The references in 5 and subparagraphs are to the Belgic Confession, e.g.
Article/(Art.) 1 refers to Article 1 of the Confession, following common practice. 5. The Belgic Confession The Belgic Confession was written at a time of fierce religious per-
secution in the Low Countries, especially under Philip II of Spain,
whose reign extended also over the Low Countries. Written by
Guido de Brès in 1561, with the help of a few others, it was intended
to give an explanation of the reformed faith from the Word of God,
especially in order to distinguish reformed Christians from the radical
Anabaptists (Schulze, 1991:32). Just like the Heidelberg Catechism,
the Belgic Confession is therefore broader in scope than the Canons
of Dordt. In die Skriflig 41(4) 2007:549-570 563 The reformed confessions: embarrassment or blessing to a missionary church? תעד11
Cf. the analysis of the noun
in Psalm 19:2 by Swanson (2001, par. 1981). 564
In die Skriflig 41(4) 2007:549-570 12
This does not mean that Art. 2 teaches the possibility of natural theology (cf. 4.2
above). It states clearly that what is needed for our salvation and for the glory of
God can only be found in Scripture. Article 7 states this even more clearly. 5.1 The God who seeks fellowship with man in his self-
revelation In a world where many are seeking God and even more claim to
have found Him, while some say that He cannot be known or found
at all, the Belgic Confession is characteristically clear in its con-
fessional language: God is there, He is the only God (Art. 1)10 and
He makes Himself known to man (Art. 2). God’s existence as the
only God and his self-revelation is one of the strongest missionary
motifs in Scripture and in our confessions. This is the foundation of
the missio Dei: God comes to man and reveals Himself to us, calling
us into fellowship with Him. God’s dwelling place and heavenly glory
is inaccessible to man, so that the only possibility for knowing God is
if He should come to us, in a way that would be accessible to us as
sinful, broken humans. It is proof of his grace that God does not
come to us in dazzling glory and heavenly splendour that frightens,
overwhelms and threatens to destroy us, but that He comes to us in
the creation, preservation and government of the universe, in the
human language of Scripture and in the humanity of his Son (cf. Heyns, 1992:66). God’s revelation is a fellowship-seeking revelation (cf. Heyns, 1978:
6-7; Van Genderen & Velema, 1992:39). He never reveals Himself
simply for the purpose of factual knowledge, but in order to be
known by man (“to know” being used here in the Hebrew sense of
the verb ידע).11 We can therefore, say that He reveals Himself to
us in order to draw us into a relationship with Him – even more, into
a union with Him in which we can delight in the richness of his love,
grace and goodness (cf. Berkhof, 1986:126). This is also the per-
spective of Article 2, which mentions that God reveals Himself more
fully in Scripture for the salvation of his own, meaning for those who
belong to Him, who live in fellowship with Him and who therefore
have been given life in Him. In a world where many absolutely refuse to know God (Rom. 1:18-
20, 23-25), God does not withdraw in indignant silence or hide from In die Skriflig 41(4) 2007:549-570 564 J.J. Fritz Krüger man. 14
Of course, this is only vulnerability when viewed from the perspective of man.
From God’s point of view, the qualification of 1 Corinthians 1:17-25 applies,
namely what is weak and powerless in the eyes of man, is strong and victorious
in the eyes of God. 5.1 The God who seeks fellowship with man in his self-
revelation No, He continues to reveal Himself in nature12 and Scripture
(breathed by the Spirit), and ultimately in his Son,13 to present
Himself to man as a gracious, fellowship-seeking God who is willing
to lay bare his heart to us in loving vulnerability (cf. Berkhof,
1986:52, 142 – who prefers to speak of “defencelessness”) – open
to the brutal rejection of those who hate Him. 13
All self-revelation of God is therefore always trinitarian (cf. Art. 8-9). 5.2 The God who seeks fellowship with man in the incarnation
of his Son God’s vulnerability in seeking out lost mankind is most graphically
illustrated in the Person of Jesus Christ, of whom we confess in
Article 10 that He is the exact image of the Father, the true eternal
God, the Almighty, whom we invoke, worship and serve. God Him-
self is so serious about seeking those who belong to Him, that He
was willing to make Himself nothing and take on the nature of a
servant, “being made in human likeness” (Phil. 2:6-7; cf. Art.18),
becoming a mere man in the person of Jesus. Jesus demonstrated
his defenceless vulnerability especially in the ridicule and rejection
He was willing to accept and ultimately in his willingness to die a
cursed death on the cross, mocked by the people of Jerusalem and
the Romans.14 The incarnation of the Son of God, in which God
comes to us in Person as Immanuel (God with us, and also like us in
knowing personally our temptations and weakness; Heb. 4:15) and
demonstrates to us his caring and saving solidarity with us, therefore
becomes the highest revelation of his love for us (Art. 26) and
perhaps one of the great cornerstones of mission. We can now say that God’s self-revelation, together with all the
means He uses for this purpose (including the incarnation of his
Son), constitutes his own mission to the world. God seeks out lost
mankind, and calls his scattered children to Himself, as when He
came into the garden of Eden and called to Adam and Eve, saying: In die Skriflig 41(4) 2007:549-570 565 The reformed confessions: embarrassment or blessing to a missionary church? “Where are you?” (Gen. 3:9). We confess this in Article 17, where
we say: “Where are you?” (Gen. 3:9). We confess this in Article 17, where
we say: We believe that our good God, by his marvellous wisdom and
goodness, seeing that man had plunged himself in this manner
into both physical and spiritual death and made himself com-
pletely miserable, set out to find him, though man, trembling all
over, was fleeing from Him. This missio Dei is the only basis of the missio ecclesiae – the
mission of the church: we serve God in his fellowship-seeking self-
revelation to the world that does not know Him, but rejects Him as
God. 5.2 The God who seeks fellowship with man in the incarnation
of his Son It is a humbling task to reflect, as a church and as a Christian,
this loving, seeking heart of God in a brutal world where the
presence of God is often not only denied but actively resisted. 5.3 The God who seeks fellowship with man in creation God’s fellowship-seeking revelation to man is a continuation and
reaffirmation of his intentions with the creation of man: man was
created for fellowship with God, and creation as such was already
an act of fellowship. As Berkhof (1986:157-158) says: The creative act bears the same stamp as we discovered in the
revelation encounter as originating in the very being of God. To
create means that God stoops down, that He limits Himself, that
He provides living and breathing space for the other … This is confessed first in Article 12, where we say that everything
was created for its own unique way of serving God. The whole
created order was put in place and is now sustained by God through
Jesus Christ (cf. Art. 13), for the sake of man, who needs this larger
order to be able to serve God himself. Creation and the providence
of God (and therefore also all history) are aimed at man as God’s
servant, who lives in fellowship with and obedience to God, and
witness to God’s grace and fatherly care for his children. In fact, the
very idea that man was created in the image of God already speaks
of relationship and fellowship. Berkhof (1986:186) says: “man is
apparently a being who is made to encounter God, to respond to his
Word. Man is a responding creature”. It is precisely for the sake of this response from the side of man that
God returns to fallen man to seek Him out and restore Him in love
and grace. In his providential reign over man and all of creation, God
does not have to accept the results of the fall as irrevocable, but
immediately sets out to redeem his tragically broken and lost crea- In die Skriflig 41(4) 2007:549-570 566 J.J. Fritz Krüger tion. This story of the missio Dei becomes in the end the story of the
missio ecclesiae! 5.4 The God who seeks fellowship with man in the church The Belgic Confession also speaks about the church as the holy
congregation or gathering of those who are saved in Christ and who
are his subjects. Although the motif of fellowship between Christ and
those who belong to Him is not immediately evident in Article 27, the
motif of Christ as Lord and believers as his subjects provide
adequate material to speak of the church as the sphere of Christ’s
fellowship with those who confess his name. This fellowship is made
evident in the washing by the blood of Christ, in the sanctification
and sealing by the Holy Spirit and in the preservation by the Father. The church can therefore be called the sacred environment of the
triune God’s life-giving and life-preserving fellowship with those who
have been saved (cf. Berkhof, 1968:343-349, who speaks of the
church as a covenant community of fellowship). It is in this sense that the beginning words of Article 28 should also
be understood – however contentious they might appear to some to-
day: We believe that since this holy assembly and congregation is
the gathering of those who are saved and there is no salvation
apart from it, no one ought to withdraw from it, content to be by
himself, regardless of his status or condition (emphasis – JJFK). The church is where Christ exercises his discipline and instruction,
where believers are built-up by the gifts of fellow believers. It is the
life-giving body of which individuals are both dependent and con-
tributing members. The church, in its separation from the un-
believing world (cf. Art. 28), is also the sign to the world of the
kingdom of Christ as the alternative and inevitably approaching
reality of the end-time (cf. Heyns, 1977:23-29; Bosch, 1991:374-
377). However, this last emphasis is not found in the Belgic
Confession, which does not indicate the task of the church towards
the unbelieving world. Still, the necessity of the church and church
planting as the result of mission must be emphasised (cf. 3.3
above): Where there is faith, there will be the fellowship of faith – the
church. Bosch (1991:372) quotes Braaten (1977:55) when saying: “a
church without mission or a mission without the church are both
contradictions. Such things do exist, but only as pseudo-structures”. This brings us to Article 29, which deals with the marks of the true
church, Here a slightly critical question should be asked. 5.4 The God who seeks fellowship with man in the church Reading In die Skriflig 41(4) 2007:549-570 567 The reformed confessions: embarrassment or blessing to a missionary church? this article in the light of the circumstances and events giving rise to
the Belgic Confession (especially the polemic against the Roman
Catholic Church and the Anabaptists), it is understandable that the
formulation is as it is. However, it could be asked whether this is all
that can be said concerning the marks of a true church. What seems
to be missing is that vital aspect of the missionary heart of the
church, which belongs to the very being of the church (based on
1 Peter 2:9, already discussed above; cf. also Bosch, 1991:372): as
God is a missionary God, his church is a missionary church. Of
course, it then becomes a point of discussion whether a church
which is not active in mission can be called a false church! There
are a number of possible solutions to this problem: this article in the light of the circumstances and events giving rise to
the Belgic Confession (especially the polemic against the Roman
Catholic Church and the Anabaptists), it is understandable that the
formulation is as it is. However, it could be asked whether this is all
that can be said concerning the marks of a true church. What seems
to be missing is that vital aspect of the missionary heart of the
church, which belongs to the very being of the church (based on
1 Peter 2:9, already discussed above; cf. also Bosch, 1991:372): as
God is a missionary God, his church is a missionary church. Of
course, it then becomes a point of discussion whether a church
which is not active in mission can be called a false church! There
are a number of possible solutions to this problem: • It could be argued that mission is implied in holding to the pure
Word of God in all respects. True preaching of the Word will
always include calling people to repentance, on the basis of
God’s revealed love in Jesus Christ. Baptism as sacrament in
itself also constitutes a boundary of the true church, bringing
people into the fellowship of the church and Christ. Holy
Communion as sacrament is at its heart a celebration of the
fellowship-seeking heart of God, who not only seeks but also
provides that fellowship through the body and blood of his Son. 5.4 The God who seeks fellowship with man in the church Finally, discipline is also a means to safeguard the identity of the
church, preventing her from becoming so much like the world that
she loses her own, distinctive and witnessing identity in the world. • Heyns (1977:110-111) attempts to extend the first mark of the
true church, the pure preaching of the gospel, to include mis-
sionary preaching to the world, and remarks that the church
which does not do this thereby denies its own calling and right to
exist (thus effectively calling mission the raison d’être of the
church). • Bosch (1991:373), however, is more convincing in his solution
that a missionary church is not always and overtly involved in
missionary projects. Its missionary dimension can also be
revealed in the way it welcomes strangers to its regular services,
in the way its members participate in the ministry of their church
and are equipped for their service in society. However, a
missionary church will also be involved with society and move
beyond its own walls. In die Skriflig 41(4) 2007:549-570 568 J.J. Fritz Krüger 6. Conclusion From the above, it is clear that the reformed confessions do not
deserve to be called either an embarrassment to the church, or a
stumbling block in the way of a church seeking to find and activate
its missionary calling. However, it is also clear that the missionary
implications of our confessions are mostly hidden gold, which will
not be found through superficial and half-hearted digging. It is there
to be discovered, but it will require some serious effort. Maybe what
is needed now is a document offering a clear exposition of the
missionary calling of the church on the basis of the confessions. Perhaps the greater embarrassment for the church should be the
fact that the historic confessions have become largely forgotten and
that the church itself is no longer a confessing church. Without the
urge to declare publicly and intentionally the praises of Him who
called us out of darkness into his wonderful light, neither the con-
fessions we have nor the ones that might still be written will change
anything in our missionary inactivity. It is not what we have (or don’t
have) on paper that should be of concern, but what we have written
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BELGIC CONFESSION. http://www.gksa.co.za Date of access: 18 Feb. 2006. BERKHOF, H. 1986. Christian faith: an introduction to the study of the faith. Grand Rapids: Eerdmans. BELGIC CONFESSION. http://www.gksa.co.za Date of access: 18 Feb. 2006. BERKHOF, H. 1986. Christian faith: an introduction to the study of the faith. Grand Rapids: Eerdmans. BOSCH, D.J. 1991. Transforming mission: paradigm shifts in theology of
mission. Maryknoll: Orbis. BOSCH, D.J. 1991. Transforming mission: paradigm shifts in theology of
mission. Maryknoll: Orbis. y
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VELDKAMP, H. s.a. Zondagskinderen: kanttekeningen bij de Heidelbergse
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Catechismus. Deel 1. Franeker: Wever. ZUIDEMA, S.U. 1965. De Christus der Schriften en oecumenische theologie. Amsterdam: Buijten & Schipperheijn. ZUIDEMA, S.U. 1965. De Christus der Schriften en oecumenische theologie. Amsterdam: Buijten & Schipperheijn. Key concepts: Christian identity
church planting: goal of mission
mission
missionary church
reformed confessions
Kernbegrippe:
Christelike identiteit
gereformeerde belydenisskrifte
kerkplanting: doel van sending
missionêre kerk
sending Christian identity
church planting: goal of mission
mission
missionary church
reformed confessions In die Skriflig 41(4) 2007:549-570 Kernbegrippe: In die Skriflig 41(4) 2007:549-570 570
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Cytomegalovirus reactivation enhances the virulence of
Staphylococcus aureus pneumonia in a mouse model
S. Hraiech, J. Bordes, Jean-Louis Mege, X. De Lamballerie, S. Lehingue, R. Charrel, C. Guervilly, J. M. Forel, Didier Raoult, L. Papazian Cytomegalovirus reactivation enhances the virulence of
Staphylococcus aureus pneumonia in a mouse model
S. Hraiech, J. Bordes, Jean-Louis Mege, X. De Lamballerie, S. Lehingue, R. Charrel, C. Guervilly, J. M. Forel, Didier Raoult, L. Papazian To cite this version: S. Hraiech, J. Bordes, Jean-Louis Mege, X. De Lamballerie, S. Lehingue, et al.. Cytomegalovirus
reactivation enhances the virulence of Staphylococcus aureus pneumonia in a mouse model. Clinical
Microbiology and Infection, 2017, 23 (1), pp.38-45. 10.1016/j.cmi.2016.09.025. hal-01521473 Cytomegalovirus reactivation enhances the
virulence of a staphylococcus aureus pneumonia
in a mouse model S Hraiech1,2, J Bordes3, JL Mège2*, X De Lamballerie4*, S Lehingue1, C Guervilly1, J-M Forel1, D Raoult2,
L Papazian1,2 From ESICM LIVES 2015
Berlin, Germany. 3-7 October 2015 From ESICM LIVES 2015
Berlin, Germany. 3-7 October 2015 Daily weight, signs of sepsis and spontaneous mortality
were noted. After 15 days, surviving mice were eutha-
nized. Blood and lung were collected for bacterial cul-
ture and histological examination. MCMV reactivation
was assessed by RT-PCR in lungs and salivary glands. A
second cohort of 20 mice were treated according to the
same protocol (10 MCMV positive and 10 MCMV
negative mice) but were sacrificed at day 2 and day 5
after pneumonia. Results The aim of this study was to assess the virulence of a
staphylococcal pneumonia developed during CMV reac-
tivation in a mouse model. No mortality from staphylococcal pneumonia was
observed in the control group whereas the mortality
rate was of 10 % in the MCMV group (p = 0.15). Mean
weight loss at day 1 was higher in MCMV mice than in
control (1.5 g versus 0.9 g respectively). Macroscopic
observation and bacteriological analysis of lungs showed
staphylococcal abscesses in 4/20 (20%) mice in MCMV
group as compared to 0/20 in control group at day 15. At day 5, 3/5 mice had lung abscesses in MCMV group
as compared to 0/5 in control group. No lung abscesses
were present at day 2 after pneumonia. Overall, 7/30
(23%) mice had lung staphylococcal abscesses in MCMV
group as compared to 0% in control group (p = 0,005). Mean lung bacterial count was significantly higher in
MCMV mice as compared to control at day 2 (2.4 × 105
vs. 2.4 × 102 CFU/ lung, p = 0.009), day 5 (2.1 ×105 vs. 5.5 × 102 CFU/lung, p = 0.02) and day 15 (5.5 × 101 vs. 0 CFU/lung, p = 0.04). HAL Id: hal-01521473 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. Hraiech et al. Intensive Care Medicine Experimental 2015, 3(Suppl 1):A831
http://www.icm-experimental.com/content/3/S1/A831 Hraiech et al. Intensive Care Medicine Experimental 2015, 3(Suppl 1):A831
http://www.icm-experimental.com/content/3/S1/A831 Introduction Cytomegalovirus (CMV) reactivation is common in
immunocompetent mechanically ventilated patients. Lungs are a frequent site of reactivation. CMV reactiva-
tion may be associated with higher mortality among
these patients [1]. Some studies have suggested that
CMV reactivation may be associated with higher inci-
dence of nosocomial pneumonia [2,3]. © 2015 Hraiech et al.; 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 use, distribution, and reproduction in any medium, provided the
original work is properly cited. Methods The study was approved by our local ethic committee. Female BALB/c mice were used in all experiments. CMV primo-infection was obtained by intra-peritoneal
inoculation of 2 x 104 PFU of murine CMV (MCMV)
Smith strain. Seropositivity was confirmed by immuno-
fluorescence in serum. MCMV was considered to be
latent 4 months later. Reactivation was triggered by
cecal ligature and puncture. Mice were considered to
have a CMV reactivation 2 weeks later [4]. After this, 20
MCMV positive mice underwent an intra-nasal inocula-
tion with 5 × 108 CFU of Staphylococcus aureus to
induce pneumonia. Twenty MCMV negative BALB/c
mice were treated according to the same protocol,
including cecal ligature and puncture (control group). 2IHU Méditerranée Infection, URMITE CNRS IRD INSERM UMR 7278, Marseille,
France
4Unite des Virus Emergents UMR 190 ‘Emergence des Pathologies Virales’,
Marseille, France
Full list of author information is available at the end of the article Conclusions In a mouse model, CMV reactivation leads to the switch
from a non lethal to a lethal staphylococcal pneumonia, Page 2 of 2 Page 2 of 2 Hraiech et al. Intensive Care Medicine Experimental 2015, 3(Suppl 1):A831
http://www.icm-experimental.com/content/3/S1/A831 Published: 1 October 2015 Published: 1 October 2015 References
1. Kalil, et al: Crit Care 2011. 2. Jaber, et al: Chest 2005. 3. Limaye, et al: JAMA 2008. 4. Cook, et al: J Infect Dis 2002. doi:10.1186/2197-425X-3-S1-A831
Cite this article as: Hraiech et al.: Cytomegalovirus reactivation enhances
the virulence of a staphylococcus aureus pneumonia in a mouse model. Intensive Care Medicine Experimental 2015 3(Suppl 1):A831. References
1. Kalil, et al: Crit Care 2011. 2. Jaber, et al: Chest 2005. 3. Limaye, et al: JAMA 2008. 4. Cook, et al: J Infect Dis 2002. doi:10.1186/2197-425X-3-S1-A831
Cite this article as: Hraiech et al.: Cytomegalovirus reactivation enhances
the virulence of a staphylococcus aureus pneumonia in a mouse model. Intensive Care Medicine Experimental 2015 3(Suppl 1):A831. Authors’ details
1 1Réanimation - Détresses Respiratoires et Infections Sévères, APHM, CHU
Nord, Marseille, France. 2IHU Méditerranée Infection, URMITE CNRS IRD
INSERM UMR 7278, Marseille, France. 3Fédération d’Anesthésie-Réanimation-
Urgences-Surveillance Continue-Centre de Dialyse BCRM Toulon Hôpital
d’Instruction des Armées Sainte-Anne Toulon, Toulon, France. 4Unite des
Virus Emergents UMR 190 ‘Emergence des Pathologies Virales’, Marseille,
France. References 3. Limaye, et al: JAMA 2008. 4. Cook, et al: J Infect Dis 2002. doi:10.1186/2197-425X-3-S1-A831
Cite this article as: Hraiech et al.: Cytomegalovirus reactivation enhances
the virulence of a staphylococcus aureus pneumonia in a mouse model. Intensive Care Medicine Experimental 2015 3(Suppl 1):A831. Submit your manuscript to a
journal and benefi t from:
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es
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Sistema de desarrollo ministerial para los líderes de una organización cristiana
| null | 2,020
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cc-by-sa
| 48,983
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Sistema de desarrollo ministerial para
los líderes de una organización cristiana
Item Type
info:eu-repo/semantics/bachelorThesis
Authors
Guevara Ramírez, Pablo Cesar; Benites Cuenca, Erick Saúl
Citation
[1] P. C. Guevara Ramírez and E. S. Benites Cuenca, “Sistema
de desarrollo ministerial para los líderes de una organización
cristiana,” Universidad Peruana de Ciencias Aplicadas (UPC),
Lima, Perú, 2019. http://hdl.handle.net/10757/648664
DOI
http://doi.org/10.19083/tesis/648664
Publisher
Universidad Peruana de Ciencias Aplicadas (UPC)
Rights
info:eu-repo/semantics/openAccess; Attribution-ShareAlike 4.0
International
Download date
25/06/2024 12:07:45
Item License
http://creativecommons.org/licenses/by-sa/4.0/
Link to Item
http://hdl.handle.net/10757/648664
UNIVERSIDAD PERUANA DE CIENCIAS APLICADAS
FACULTAD DE INGENIERÍA
PROGRAMA ACADÉMICO DE INGENIERÍA DE SISTEMAS
Sistema de desarrollo ministerial para los líderes de una organización cristiana
TESIS
Para optar el título profesional de Ingeniero de Sistemas
AUTORES
Guevara Ramírez, Pablo Cesar (0000-0003-4561-6872)
Benites Cuenca, Erick Saúl (0000-0002-6341-7634)
ASESORES
Contreras Caja, Luis Lenin (0000-0002-0097-1291)
Perona Galluccio, Sandra Guisella (0000-0003-3483-8867)
Lizardo Silva, Ubaldo (0000-0001-7208-0044)
Lima, 14 de octubre de 2019
DEDICATORIA
Con mucho cariño este trabajo va dedicado en primer lugar a Dios por darnos la
perseverancia para poder terminar este proyecto, a nuestras esposas por su apoyo
incondicional en todo momento, a nuestra familia que estuvo allí siempre.
I
AGRADECIMIENTOS
Agradecemos a Raúl Llamoza por abrirnos las puertas de tan prestigiosa institución y poder
realizar este proyecto. Al pastor José León por su apoyo constante y a nuestro querido pastor
Pedro Hornung por darnos siempre toda la cobertura y ayuda necesaria para ingresar a la
institución y poder culminar con esfuerzo y dedicación nuestra tesis.
II
RESUMEN
El presente trabajo de tesis tiene por objetivo plantear los lineamientos para el desarrollo de
un sistema basado en CRM (Gestión de la relación con los clientes) para la gestión de la
información de las personas que asisten a una organización cristiana.
Para ello se realiza el análisis de la situación actual de la organización y del proceso de
desarrollo ministerial de los líderes como objeto de estudio, con el fin de comprender y
definir los casos de uso del negocio, sus respectivos flujos de actividades y reglas de
negocios los cuales contribuyen a plantear una propuesta de solución basado en un software
a medida coherente con los objetivos establecidos. Dentro de este desarrollo, se hace uso de
la notación UML para el modelado de los artefactos que intervienen en el presente análisis.
Asimismo, se describe los requerimientos funcionales y no funcionales, modelo de caso de
uso de sistema, atributos de caso de uso de sistema, modelo conceptual, diccionario de datos
del proyecto, arquitectura propuesta de la solución y el desarrollo de los casos de uso del
núcleo central con las pruebas correspondientes para el pase a producción. Por otro lado, se
describe la política, los objetivos, y las normas de calidad definidas por la organización que
aplican al desarrollo de software y que las satisface.
Finalmente se incluye la gestión del proyecto donde se muestra la planificación con un
cronograma de actividades para las diferentes etapas del sistema.
Palabras clave: iglesia; líder; ministerio; software; fidelización.
III
Ministerial development system for the leaders of a Christian organization
ABSTRACT
The aim of this thesis work is to propose the guidelines for the development of a CRM
(Customer Relationship Management) based-system for the information management of the
people who attend a Christian organization.
For this, the analysis of the current situation of the organization and the ministerial
development process of the leaders as an object of study is carried out, in order to understand
and define the cases of business use, their respective activity flows and business rules which
contribute to propose a solution based on a customized software consistent with the
established objectives. Within this development, the UML notation is used for the modeling
of the artifacts involved in the present analysis.
Also, it describes the functional and non-functional requirements, system use case model,
system use case attributes, conceptual model, project data dictionary, proposed solution
architecture and the development of use cases of the central core with the corresponding tests
for the production stage. On the other hand, it describes the policy, objectives, and quality
standards defined by the organization that applies to software development and that satisfies
them.
Finally, project management is included where planning is shown with a schedule of
activities for the different stages of the system.
Keywords: church; leader; ministry; software; loyalty.
IV
TABLA DE CONTENIDOS
1.
CAPITULO 1: FUNDAMENTOS TEÓRICOS............................................................ 2
1.1
INTRODUCCIÓN ................................................................................................. 2
1.2
MARCO TEÓRICO .............................................................................................. 2
1.2.1
FUNDAMENTOS TEÓRICOS SOBRE EL NEGOCIO .................................. 2
1.2.2
FUNDAMENTOS TEÓRICOS SOBRE LAS TENDENCIAS Y
TECNOLOGÍAS ACTUALES ..................................................................................... 6
1.3
OBJETO DE ESTUDIO ...................................................................................... 17
1.3.1
ORGANIZACIÓN OBJETIVO ...................................................................... 17
1.3.2
DESCRIPCIÓN DE ÁREAS (Fuente: CCAV) .............................................. 18
1.3.3
MISIÓN ........................................................................................................... 19
1.3.4
VISIÓN ............................................................................................................ 20
1.3.5
OBJETIVOS ESTRATÉGICOS (Fuente: CCAV).......................................... 20
1.3.6
ORGANIGRAMA ........................................................................................... 21
1.4
CAMPO DE ACCIÓN ......................................................................................... 22
1.4.1
BREVE DESCRIPCIÓN ................................................................................. 22
1.4.2
PROCESOS DEL NEGOCIO ......................................................................... 23
1.4.3
SISTEMAS AUTOMATIZADOS VINCULADOS CON EL CAMPO DE
ACCIÓN ...................................................................................................................... 24
1.5
ANÁLISIS CRÍTICO DE LOS PROBLEMAS DE INFORMACIÓN ................ 25
1.5.1
SITUACIÓN PROBLEMÁTICA Y PROBLEMAS A RESOLVER ............. 25
1.6
CONCLUSIONES ............................................................................................... 26
2
CAPITULO 2: PROPUESTA DE LA SOLUCIÓN ................................................. 27
2.1
INTRODUCCIÓN ............................................................................................... 27
2.2
OBJETIVOS DEL PROYECTO ......................................................................... 27
2.2.1
OBJETIVO GENERAL .................................................................................. 27
2.2.2
OBJETIVOS ESPECÍFICOS .......................................................................... 27
2.2.3
FUNDAMENTACIÓN DE LOS OBJETIVOS .............................................. 28
2.2.4
INDICADORES DE LOGRO DE OBJETIVOS ............................................ 29
2.3
BENEFICIOS DEL PROYECTO ........................................................................ 30
2.3.1
BENEFICIOS TANGIBLES ........................................................................... 30
2.3.2
BENEFICIOS INTANGIBLES ....................................................................... 30
V
2.4
ANTECEDENTES .............................................................................................. 30
2.4.1
SOLUCIONES ENCONTRADAS.................................................................. 31
2.4.2
ANÁLISIS COMPARATIVO ......................................................................... 31
2.4.3
EVALUACIÓN DE LA MEJOR SOLUCIÓN ............................................... 33
2.4.4
FUNCIONES PREVISTAS ............................................................................ 34
2.5
TENDENCIAS Y TECNOLOGÍAS PROPUESTAS .......................................... 36
2.6
CONCLUSIONES ............................................................................................... 41
3
CAPITULO 3: MODELADO DEL NEGOCIO....................................................... 42
3.1
INTRODUCCIÓN ............................................................................................... 42
3.2
REGLAS DEL NEGOCIO .................................................................................. 42
3.3
MODELO DE CASOS DE USO DEL NEGOCIO .............................................. 44
3.3.1
ACTORES DEL NEGOCIO ........................................................................... 44
3.3.2
CASOS DE USO DEL NEGOCIO ................................................................. 45
3.3.3
DIAGRAMA DE CASOS DE USO DEL NEGOCIO .................................... 46
3.4
MODELO DE ANÁLISIS DEL NEGOCIO........................................................ 47
3.4.1
TRABAJADORES DEL NEGOCIO .............................................................. 47
3.4.2
ENTIDADES DEL NEGOCIO ....................................................................... 49
3.4.3
DIAGRAMA DE CLASES DEL NEGOCIO ................................................. 56
3.5
REALIZACIÓN DE LOS CASOS DE USO DEL NEGOCIO ............................ 59
3.5.1
AV_CUN01_GESTIONAR INICIACIÓN ..................................................... 59
3.5.2
AV_CUN02_CONSOLIDAR CREYENTES ................................................. 63
3.5.3
AV_CUN03_ORGANIZAR ENCUENTROS ................................................ 67
3.5.4
AV_CUN04_REALIZAR ESCUELA DE LÍDERES .................................... 71
3.5.5
AV_CUN05_ORGANIZAR GRUPOS FAMILIARES .................................. 74
3.6
CONCLUSIONES ............................................................................................... 76
4
CAPITULO 4: REQUERIMIENTOS ...................................................................... 77
4.1
INTRODUCCIÓN ............................................................................................... 77
4.2
ESPECIFICACIÓN DE REQUERIMIENTOS DE SOFTWARE....................... 77
4.2.1
REQUERIMIENTOS FUNCIONALES ......................................................... 77
4.2.2
REQUERIMIENTOS NO FUNCIONALES................................................... 81
4.3
MODELO DE CASOS DE USO DEL SISTEMA ............................................... 86
4.3.1
ESPECIFICACIÓN DE LOS ACTORES DEL SISTEMA ............................ 86
4.3.2
DIAGRAMA DE ACTORES DEL SISTEMA ............................................... 88
VI
4.3.3
DIAGRAMA DE PAQUETES ....................................................................... 89
4.3.4
DIAGRAMA DE CASOS DE USO DEL SISTEMA POR PAQUETE ......... 90
4.3.5
ATRIBUTOS DE LOS CASOS DE USO DEL SISTEMA ............................ 97
4.3.6
ESPECIFICACIONES ALTO NIVEL DE LOS CASOS DE USO DEL
SISTEMA .................................................................................................................... 99
4.3.7
ESPECIFICACIONES DETALLADAS DE LOS CASOS DE USO DEL
NÚCLEO CENTRAL................................................................................................ 112
4.4
MODELO CONCEPTUAL ............................................................................... 162
4.4.1
DIAGRAMA DEL MODELO CONCEPTUAL ........................................... 162
4.4.2
DICCIONARIO DEL MODELO CONCEPTUAL ...................................... 163
4.5
CONCLUSIONES ............................................................................................. 168
5
CAPITULO 5: ARQUITECTURA DE SOFTWARE ........................................... 169
5.1
INTRODUCCIÓN ............................................................................................. 169
5.2
DIAGRAMA DE CASOS DE USO MAS SIGNIFICATIVOS PARA LA
ARQUITECTURA ........................................................................................................ 170
5.3
METAS DE LA ARQUITECTURA DE SOFTWARE ..................................... 171
5.4
RESTRICCIONES DE LA ARQUITECTURA DE SOFTWARE .................... 172
5.5
MECANISMOS ARQUITECTURALES.......................................................... 173
5.6
VISTA LÓGICA DE LA ARQUITECTURA DE SOFTWARE ....................... 174
5.7
VISTA DE IMPLEMENTACIÓN DE LA ARQUITECTURA DE SOFTWARE
175
5.8
VISTA DE DESPLIEGUE DE LA ARQUITECTURA DE SOFTWARE ........ 176
5.9
CONCLUSIONES ............................................................................................. 177
6
CAPÍTULO 6: CALIDAD Y PRUEBAS DE SOFTWARE .................................. 178
6.1
INTRODUCCIÓN ............................................................................................. 178
6.2
PLAN DE CALIDAD DE SOFTWARE ........................................................... 178
6.2.1
Políticas De Calidad ...................................................................................... 178
6.2.2
Objetivos De Calidad..................................................................................... 178
6.2.3
Normatividad Aplicable ................................................................................ 179
6.2.4
MÉTRICAS DE CALIDAD DEL SOFTWARE .......................................... 184
6.2.5
ANÁLISIS DE RESULTADOS DE LA MEDICIÓN .................................. 189
6.3
6.3.1
PRUEBAS DEL SOFTWARE .......................................................................... 194
PLAN DE PRUEBAS ................................................................................... 194
VII
6.3.2
OBJETIVO .................................................................................................... 194
6.3.3
ROLES Y RESPONSABILIDADES DEL EQUIPO DE PRUEBAS .......... 194
6.3.4
PRERREQUISITOS ...................................................................................... 195
6.3.5
ALCANCE .................................................................................................... 195
6.3.6
ESTRATEGIA DE PRUEBAS ..................................................................... 195
6.3.7
DOCUMENTACIÓN DE RESPALDO ........................................................ 195
6.3.8
FUNCIONALIDAD DEL SISTEMA ........................................................... 195
6.3.9
CASOS DE PRUEBA ................................................................................... 196
6.3.10
AMBIENTE DE PRUEBAS ..................................................................... 197
6.3.11
PRUEBAS ESPECIALES ......................................................................... 198
6.3.12
PRUEBAS DE ACEPTACIÓN................................................................. 198
6.3.13
TIEMPOS Y RECURSOS REQUERIDOS .............................................. 198
6.4
EJECUCIÓN DE LOS CASOS DE PRUEBAS ................................................ 199
6.4.1
CASO DE PRUEBA AV_CP040_NOTIFICACIÓN DE SEGUIMIENTO AL
LÍDER 199
6.4.2
CASO DE PRUEBA AV_CP024_IMPORTAR INFORMACIÓN DE
CURSOS LLEVADOS Y DISPONIBLES ............................................................... 199
6.5
CONCLUSIONES ............................................................................................. 200
7
CAPITULO 7: CONSTRUCCIÓN ........................................................................ 201
7.1
INTRODUCCIÓN ............................................................................................. 201
7.2
PATRONES DE LA SOLUCIÓN PROPUESTA.............................................. 201
7.2.1
PATRÓN MVC ............................................................................................. 201
7.2.2
PATRÓN REPOSITORY.............................................................................. 207
7.2.3
PATRÓN ADAPTER .................................................................................... 209
7.3
MODELO DE DATOS ...................................................................................... 211
7.3.1
MODELO DE DATOS FÍSICO DEL SISTEMA ......................................... 212
7.3.2
DICCIONARIO DE DATOS ........................................................................ 213
7.4
CONCLUSIONES ............................................................................................. 221
8
CAPITULO 8: GESTIÓN DEL PROYECTO ....................................................... 222
8.1
INTRODUCCIÓN ............................................................................................. 222
8.2
REGISTRO DE INTERESADOS ..................................................................... 222
8.3
EDT ................................................................................................................... 223
8.4
CRONOGRAMA DE EJECUCIÓN .................................................................. 224
VIII
8.5
ACTAS DE ACEPTACIÓN .............................................................................. 226
8.6
CONCLUSIONES ............................................................................................. 229
9
CONCLUSIONES GENERALES ......................................................................... 230
10
GLOSARIO DE TÉRMINOS................................................................................ 232
11
SIGLARIO............................................................................................................. 234
12
REFERENCIAS .................................................................................................... 235
13
ANEXOS ............................................................................................................... 238
IX
ÍNDICE DE TABLAS
Tabla 1: Cuadro de funcionalidades CRM (Fuente: propia) .............................................. 12
Tabla 2: Análisis de FODA de la CCAV (Fuente: Propia) ................................................ 17
Tabla 3: Situación problemática vs Problemas a resolver (Fuente: propia) ....................... 26
Tabla 4: Indicadores de logro de objetivos (Fuente propia) ............................................... 29
Tabla 5: Solución encontrada Softlife (Fuente: http://misoftlife.com) ................................ 31
Tabla 6: Solución encontrada Administrador Fiel (Fuente: administradorfiel.com) .......... 31
Tabla 7: Análisis comparativo de otras soluciones (Fuente propia) ................................... 32
Tabla 8: Apriori – Ejemplo de conjunto de datos (Fuente: propia) .................................... 39
Tabla 9: Apriori – Ejemplo de conjunto de resultados (Fuente: propia) ............................ 40
Tabla 10: Apriori – Ejemplo de conjunto de resultados de pares (Fuente: propia) ............ 40
Tabla 11: Apriori – Ejemplo de conjunto de resultados de triplos (Fuente: propia) .......... 41
Tabla 12: Reglas del Negocio (Fuente: propia) .................................................................. 43
Tabla 13: Actores del Negocio (Fuente: propia) ................................................................ 44
Tabla 14: Casos de Uso del Negocio (Fuente: Propia)....................................................... 45
Tabla 15: Trabajadores del Negocio (Fuente: propia) ........................................................ 49
Tabla 16: Entidades del Negocio (Fuente: propia) ............................................................. 55
Tabla 17: Atributos de los Casos del Sistema (Fuente: propia).......................................... 99
Tabla 18: AV_CUS001_Registro de Hoja de Datos (Fuente: propia) .............................. 99
Tabla 19: AV_CUS002_Registrar pedido de oración (Fuente: propia) ............................. 99
Tabla 20: AV_CUS003_Consultar contenidos y noticias (Fuente: propia) ....................... 99
Tabla 21: AV_CUS004_Registrar comentarios (Fuente: propia) .................................... 100
Tabla 22: AV_CUS005_Registrar Ganado (Fuente: propia)............................................ 100
Tabla 23: AV_CUS006_Consultar Hoja de Datos (Fuente: propia) ................................ 100
Tabla 24: AV_CUS007_Actualizar Hoja de Datos (Fuente: propia) ............................... 100
Tabla 25: AV_CUS008_Publicar contenidos y noticias (Fuente: propia)........................ 101
Tabla 26: AV_CUS009_Asignar Líder (Fuente: propia) ................................................. 101
Tabla 27: AV_CUS010_Consultar Creyentes Asignados (Fuente: propia) ..................... 101
Tabla 28: AV_CUS011_Programar fecha de contacto (Fuente: propia) .......................... 102
Tabla 29: AV_CUS012_Generar Reporte de Contacto (Fuente: propia) ......................... 102
Tabla 30: AV_CUS013_Consultar Reporte de Contacto (Fuente: propia) ...................... 102
Tabla 31: AV_CUS014_Consultar Reporte General de Consolidación (Fuente: propia) 102
Tabla 32: AV_CUS015_Programar encuentros (Fuente: propia) .................................... 103
X
Tabla 33: AV_CUS016_Generar reporte del encuentro (Fuente: propia) ........................ 103
Tabla 34: AV_CUS017_Consultar programación de encuentros (Fuente: propia) .......... 103
Tabla 35: AV_CUS018_Registrar reserva de participación a encuentro (Fuente: propia)
........................................................................................................................................... 103
Tabla 36: AV_CUS019_Consultar reservas de participación a encuentros (Fuente: propia)
........................................................................................................................................... 104
Tabla 37: AV_CUS020_Aprobar participación a encuentro (Fuente: propia) ................. 104
Tabla 38: AV_CUS021_Actualizar asistencia a encuentro (Fuente: propia) ................... 104
Tabla 39: AV_CUS022_Publicar contenido del encuentro (Fuente: propia) ................... 105
Tabla 40: AV_CUS023_Acceder a contenido del encuentro (Fuente: propia) ................ 105
Tabla 41: AV_CUS024_Importar información de cursos llevados y disponibles (Fuente:
propia) ............................................................................................................................... 105
Tabla 42: AV_CUS025_Promocionar cursos sugeridos (Fuente: propia) ....................... 105
Tabla 43: AV_CUS026_Consultar cursos sugeridos (Fuente: propia)............................. 106
Tabla 44: AV_CUS027_Registrar ficha de interés a cursos (Fuente: propia).................. 106
Tabla 45: AV_CUS028_Consultar fichas de interés (Fuente: propia) ............................. 106
Tabla 46: AV_CUS029_Registrar nuevo grupo familiar (Fuente: propia) ...................... 106
Tabla 47: AV_CUS030_Actualizar información de grupo familiar (Fuente: propia) ...... 107
Tabla 48: AV_CUS031_Consultar reuniones de grupo familiar (Fuente: propia) ........... 107
Tabla 49: AV_CUS032_Aceptar invitación a grupo familiar (Fuente: propia) ............... 107
Tabla 50: AV_CUS033_Confirmar asistentes (Fuente: propia)....................................... 108
Tabla 51: AV_CUS034_Publicar contenido de grupo familiar (Fuente: propia) ............. 108
Tabla 52: AV_CUS035_Acceder a contenido de grupo familiar (Fuente: propia) .......... 108
Tabla 53: AV_CUS036_Generar_Reporte de crecimiento anual (Fuente: propia) .......... 108
Tabla 54: AV_CUS037_Generar Reporte de eficiencia del líder (Fuente: propia).......... 109
Tabla 55: AV_CUS038_Generar Reporte de desarrollo ministerial del creyente (Fuente:
propia) ............................................................................................................................... 109
Tabla 56: AV_CUS039_Generar Reporte general de satisfacción de servicios (Fuente:
propia) ............................................................................................................................... 109
Tabla 57: AV_CUS040_Notificación de seguimiento al líder (Fuente: propia) .............. 109
Tabla 58: AV_CUS041_Notificación de eventos al creyente (Fuente: propia) ............... 110
Tabla 59: AV_CUS042_Actualizar encuesta de satisfacción (Fuente: propia)................ 110
Tabla 60: AV_CUS043_Ejecutar encuesta de satisfacción (Fuente: propia) ................... 110
XI
Tabla 61: AV_CUS044_Responder encuesta de satisfacción (Fuente: propia) ............... 111
Tabla 62: AV_CUS045_Consultar reporte de encuestas de satisfacción (Fuente: propia)
........................................................................................................................................... 111
Tabla 63: Diccionario del Modelo Conceptual – Creyente (Fuente: propia) ................... 163
Tabla 64: Diccionario del Modelo Conceptual – Líder (Fuente: propia) ......................... 163
Tabla 65: Diccionario del Modelo Conceptual – Consolidación (Fuente: propia)........... 164
Tabla 66: Diccionario del Modelo Conceptual – Programación Contacto (Fuente: propia)
........................................................................................................................................... 164
Tabla 67: Diccionario del Modelo Conceptual – Reporte Contacto (Fuente: propia)...... 164
Tabla 68: Diccionario del Modelo Conceptual – Reporte Consolidación (Fuente: propia)
........................................................................................................................................... 165
Tabla 69: Diccionario del Modelo Conceptual – Curso Bíblico (Fuente: propia) ........... 165
Tabla 70: Diccionario del Modelo Conceptual – Curso programado (Fuente: propia) .... 165
Tabla 71: Diccionario del Modelo Conceptual – Inscripción (Fuente: propia) ................ 166
Tabla 72: Diccionario del Modelo Conceptual – Curso Asociación (Fuente: propia) ..... 166
Tabla 73: Diccionario del Modelo Conceptual – Grupo Familiar (Fuente: propia) ......... 166
Tabla 74: Diccionario del Modelo Conceptual – Encuentro (Fuente: propia) ................. 167
Tabla 75: Diccionario del Modelo Conceptual – Asistente (Fuente: propia) ................... 167
Tabla 76: Diccionario del Modelo Conceptual – Encuentrista (Fuente: propia) .............. 167
Tabla 77: Metas de la Arquitectura de Software (Fuente: propia) ................................... 171
Tabla 78: Restricciones de la Arquitectura de Software (Fuente: propia)........................ 172
Tabla 79: Mecanismos de la Arquitectura de Software (Fuente: propia) ......................... 173
Tabla 80: Capas de la Vista Lógica (Fuente: propia) ....................................................... 174
Tabla 81: Métrica de Uso (Fuente: propia)....................................................................... 184
Tabla 82: Métrica Interna 01 (Fuente: propia) ................................................................. 185
Tabla 83: Métrica Interna 02 (Fuente: propia) ................................................................. 186
Tabla 84: Métrica Externa 01 (Fuente: propia) ................................................................ 187
Tabla 85: Métrica Externa 02 (Fuente: propia) ................................................................ 188
Tabla 86: Resultados de Métrica de Uso (Fuente: propia) ............................................... 189
Tabla 87: Resultados de Métrica Interna 01 (Fuente: propia) .......................................... 190
Tabla 88: Resultados de Métrica Interna 02 (Fuente: propia) .......................................... 191
Tabla 89: Resultados de Métrica Externa 01 (Fuente: propia) ......................................... 192
Tabla 90: Resultados de Métrica Externa 02 (Fuente: propia) ......................................... 193
XII
Tabla 91: Roles y responsabilidades del equipos de Pruebas (Fuente: propia) ................ 195
Tabla 92: Casos de Prueba (Fuente: propia) ..................................................................... 196
Tabla 93: Matriz caso de uso AV_CUS040 (Fuente: propia)........................................... 196
Tabla 94: Matriz caso de uso AV_CUS024 (Fuente: propia)........................................... 197
Tabla 95: Pruebas y control de calidad (Fuente: propia) .................................................. 198
Tabla 96: Patrón MVC (Fuente: propia)........................................................................... 202
Tabla 97: Patrón Repository (Fuente: propia) .................................................................. 207
Tabla 98: Patrón Adapter (Fuente: propia) ....................................................................... 209
Tabla 99: Creyentes (Fuente: propia) ............................................................................... 213
Tabla 100: Departamentos (Fuente: propia) ..................................................................... 214
Tabla 101: Provincias (Fuente: propia) ............................................................................ 214
Tabla 102: Distritos (Fuente: propia) ............................................................................... 214
Tabla 103: Líderes (Fuente: propia) ................................................................................. 214
Tabla 104: Cursos programados (Fuente: propia) ............................................................ 215
Tabla 105: Consolidaciones (Fuente: propia) ................................................................... 215
Tabla 106: Programaciones (Fuente: propia) ................................................................... 216
Tabla 107: Reportes (Fuente: propia) ............................................................................... 216
Tabla 108: Cursos bíblicos (Fuente: propia) .................................................................... 216
Tabla 109: Areas (Fuente: propia) .................................................................................... 216
Tabla 110: Creyentes áreas (Fuente: propia) .................................................................... 217
Tabla 111: Inscripciones (Fuente: propia) ........................................................................ 217
Tabla 112: Cursos relaciones (Fuente: propia) ................................................................. 217
Tabla 113: Migraciones (Fuente: propia) ......................................................................... 218
Tabla 114: Grupos (Fuente: propia) ................................................................................. 218
Tabla 115: Instancias (Fuente: propia) ............................................................................. 219
Tabla 116: Roles (Fuente: propia) .................................................................................... 219
Tabla 117: Creyentes_roles (Fuente: propia) ................................................................... 219
Tabla 118: Peticiones (Fuente: propia) ............................................................................. 219
Tabla 119: Tipopeticiones (Fuente: propia) ..................................................................... 220
Tabla 120: Reportes_estados (Fuente: propia) ................................................................. 220
Tabla 121: Cursos_adquiridos (Fuente: propia) ............................................................... 220
Tabla 122: Cursos_sugeridos (Fuente: propia) ................................................................. 220
Tabla 123: Registro de Interesados del Proyecto (Fuente: propia)................................... 222
XIII
Tabla 124: Cronograma de Ejecución del Proyecto (Fuente: Propia) .............................. 225
XIV
ÍNDICE DE FIGURAS
Figura 1: Relación entre tipos y componentes CRM (Fuente: Croxatto (2005)).................. 9
Figura 2: Procesos y Arquitectura CRM (Fuente: ON Soluciones 2013) ........................... 12
Figura 3: Contenido de un Sistema CRM Clientes vs contactos (Fuente: propia) ............. 13
Figura 4: Contenido de un Sistema CRM Motivo vs sub motivos (Fuente: propia) ......... 14
Figura 5: Contenido de un Sistema CRM Referidos (Fuente propia)................................. 14
Figura 6: Contenido de un Sistema CRM Interacción con todas las partes (Fuente: propia)
............................................................................................................................................. 16
Figura 7: Mapa Estratégico General (Fuente: propia) ........................................................ 20
Figura 8: Organigrama de la Organización CCAV (Fuente: Propia) ................................. 21
Figura 9: Proceso del Desarrollo Ministerial (Fuente: Propia) .......................................... 22
Figura 10: Mapa de procesos de Comunidad Cristiana Agua Viva (Fuente: Propia) ........ 23
Figura 11: Área de Consolidación (Fuente: propia) ........................................................... 24
Figura 12: Arquitectura del Android (Fuente: Xataka Android)......................................... 37
Figura 13: Algoritmo Apriori (Fuente: Agrawal & Srikant, 1994)..................................... 39
Figura 14: Diagrama de Casos de Usos del Negocio (Fuente propia) ................................ 46
Figura 15: Diagrama de Clases – Gestionar Iniciación (Fuente propia) ............................. 56
Figura 16: Diagrama de Clases – Consolidar Creyentes (Fuente propia)........................... 56
Figura 17: Diagrama de Clases – Organizar Encuentros (Fuente propia) .......................... 57
Figura 18: Diagrama de Clases – Realizar Escuela de Líderes (Fuente propia)................. 57
Figura 19: Diagrama de Clases – Organizar Grupos Familiares (Fuente propia)............... 58
Figura 20: Diagrama de Actividades – Gestionar Iniciación (Fuente propia) .................... 61
Figura 21: Diagrama de Actividades – Consolidar Creyentes (Fuente propia) .................. 65
Figura 22: Diagrama de Actividades – Organizar Encuentros (Fuente propia).................. 69
Figura 23: Diagrama de Actividades – Realizar Escuela de Líderes (Fuente propia) ........ 72
Figura 24: Diagrama de Actividades – Organizar Grupos Familiares (Fuente propia) ...... 75
Figura 25: Diagrama de Actores del Sistema (Fuente propia)............................................ 88
Figura 26: Diagrama de Paquetes del Sistema (Fuente propia) .......................................... 89
Figura 27: Diagrama de CUS por paquetes – Admisión a la Comunidad (Fuente propia) 90
Figura 28: Diagrama de CUS por paquetes – Consolidar Creyentes (Fuente propia) ........ 91
Figura 29: Diagrama de CUS por paquetes – Organización de Encuentros (Fuente propia)
............................................................................................................................................. 92
XV
Figura 30: Diagrama de CUS por paquetes – Promoción de Cursos y Talleres (Fuente
propia) ................................................................................................................................. 93
Figura 31: Diagrama de CUS por paquetes – Organización de Grupos Familiares (Fuente
propia) ................................................................................................................................. 94
Figura 32: Diagrama de CUS por paquetes – Control de Crecimiento (Fuente propia) ..... 95
Figura 33: Diagrama de CUS por paquetes – Seguridad (Fuente propia) .......................... 96
Figura 34: AV_CUS024 - Pantalla 01 de Mockup (Elaborado con Justinmind).............. 114
Figura 35: AV_CUS024 - Pantalla 02 de Mockup (Elaborado con Justinmind).............. 115
Figura 36: AV_CUS040 - Pantalla 01 de Mockup (Elaborado con Justinmind).............. 118
Figura 37: AV_CUS001 - Pantalla 01 de Mockup (Elaborado con Justinmind).............. 125
Figura 38: AV_CUS001 - Pantalla 02 de Mockup (Elaborado con Justinmind).............. 125
Figura 39: AV_CUS001 - Pantalla 03 de Mockup (Elaborado con Justinmind).............. 126
Figura 40: AV_CUS009 - Pantalla 01 de Mockup (Elaborado con Justinmind).............. 129
Figura 41: AV_CUS009 - Pantalla 02 de Mockup (Elaborado con Justinmind).............. 129
Figura 42: AV_CUS009 - Pantalla 03 de Mockup (Elaborado con Justinmind).............. 130
Figura 42: AV_CUS009 - Pantalla 04 de Mockup (Elaborado con Justinmind).............. 130
Figura 43: AV_CUS011 - Pantalla 01 de Mockup (Elaborado con Justinmind).............. 134
Figura 44: AV_CUS011 - Pantalla 02 de Mockup (Elaborado con Justinmind).............. 134
Figura 45: AV_CUS011 - Pantalla 03 de Mockup (Elaborado con Justinmind).............. 135
Figura 46: AV_CUS011 - Pantalla 04 de Mockup (Elaborado con Justinmind).............. 135
Figura 48: AV_CUS012 - Pantalla 01 de Mockup (Elaborado con Justinmind).............. 140
Figura 49: AV_CUS012 - Pantalla 02 de Mockup (Elaborado con Justinmind).............. 140
Figura 50: AV_CUS029 - Pantalla 01 de Mockup (Elaborado con Justinmind).............. 146
Figura 51: AV_CUS029 - Pantalla 02 de Mockup (Elaborado con Justinmind).............. 147
Figura 52: AV_CUS029 - Pantalla 03 de Mockup (Elaborado con Justinmind).............. 147
Figura 53: AV_CUS029 - Pantalla 04 de Mockup (Elaborado con Justinmind).............. 148
Figura 54: AV_CUS029 - Pantalla 05 de Mockup (Elaborado con Justinmind).............. 148
Figura 55: AV_CUS029 - Pantalla 06 de Mockup (Elaborado con Justinmind).............. 149
Figura 56: AV_CUS030 - Pantalla 01 de Mockup (Elaborado con Justinmind).............. 154
Figura 57: AV_CUS030 - Pantalla 02 de Mockup (Elaborado con Justinmind).............. 155
Figura 58: AV_CUS030 - Pantalla 03 de Mockup (Elaborado con Justinmind).............. 155
Figura 59: AV_CUS030 - Pantalla 04 de Mockup (Elaborado con Justinmind).............. 156
Figura 60: AV_CUS030 - Pantalla 05 de Mockup (Elaborado con Justinmind).............. 156
XVI
Figura 61: AV_CUS037 - Pantalla 01 de Mockup (Elaborado con Justinmind).............. 161
Figura 62: AV_CUS037 - Pantalla 02 de Mockup (Elaborado con Justinmind).............. 161
Figura 63: Diagrama del Modelo Conceptual ................................................................... 162
Figura 64: Diagrama de CUS más significativos para la Arquitectura ............................. 170
Figura 65: Vista Lógica (Fuente: propia) ......................................................................... 174
Figura 66: Vista de Implementación (Fuente: propia)...................................................... 175
Figura 67: Vista de Despliegue (Fuente: propia) .............................................................. 176
Figura 68: Modelo de calidad del Producto de Software (Fuente http://iso25000.com) .. 181
Figura 69: Modelo de calidad de Uso (Fuente ISO/IEC 25010) ....................................... 182
Figura 70: Relación de los tipos de métricas de calidad (Fuente ISO/IEC 25023) ........... 183
Figura 71: Patrón MVC (Fuente propia)........................................................................... 202
Figura 72: Estructura MVC (Fuente: propia) ................................................................... 203
Figura 73: Modelo Grupo.php (Fuente: propia) ............................................................... 204
Figura 74: Vista grupos/view.blade.php (Fuente: propia) ................................................ 205
Figura 75: Controlador GruposController.php (Fuente: propia) ...................................... 206
Figura 76: Patrón Repositorio (Fuente https://bosnadev.com/2015/03/07/using-repositorypattern-in-laravel-5/) ......................................................................................................... 208
Figura 77: Evidencia del uso del Patrón Repositorio (Fuente: propia) ............................ 208
Figura 78: Patrón Adapter (Fuente propia) ....................................................................... 210
Figura 79: Cliente (Fuente: propia) .................................................................................. 210
Figura 80: Adaptador (Fuente: propia) ............................................................................. 211
Figura 81: Modelo de Datos Físico del Sistema (Fuente: propia) .................................... 212
Figura 82: Estructura de Desglose del Trabajo del Proyecto (Fuente: propia)................. 223
XVII
INTRODUCCIÓN
El presente proyecto tiene como objetivo principal diseñar, desarrollar e implementar un
sistema para la buena gestión de la información de la Iglesia Comunidad Cristiana Agua
Viva (CCAV), en el proceso de desarrollo ministerial, mediante herramientas web y móviles,
para poder tener un rápido acceso y manejo de la información de manera adecuada basado
en un sistema CRM (Gestión de la relación con el cliente) que después de analizar y entender
los procesos de la organización se hace uso de la tecnología con el sistema que se propone
para así poder retener a las personas que llegan a las sedes de la organización y se desarrollen
como líderes potenciales.
El análisis de los procesos de la organización pudo llevar a identificar la problemática que
presenta y que a través de los años la organización ha tenido un crecimiento de personas,
pero la gran debilidad ha sido que no se han podido retener y en las diferentes áreas del
proceso ministerial ha ido disminuyendo la cantidad de personas que se puedan quedar en la
organización, sobre todo en las áreas de consolidación, encuentros, escuela de líderes y en
los grupos familiares. Además, el manejo de la información que tiene la organización
actualmente es solo con ingreso de datos por medio de formularios y muestra reportes de
listados de personas y lo que están necesitando es sacar nueva información como son los
indicadores de resultados del crecimiento y logros de los lideres para la toma de decisiones,
y otros aspectos más para ver en qué áreas hay debilidades y se pueda reforzar para poder
retener a las personas que llegan a la organización.
Por lo expuesto anteriormente se propone dar una solución tecnológica que ayude a la
organización a tener un buen manejo de la información, que esté actualizada, y sea fácil de
usar de una forma rápida en plataformas web y móvil basados en un sistema CRM.
En el contenido de este documento se puntualiza la problemática que motivó a desarrollar el
presente proyecto y los argumentos que lo justifican. Este documento también expresa
detalladamente la metodología que se implementa, así como la planificación del proyecto.
Al finalizar el documento se hace mención de los resultados que se obtienen y conclusiones
del proyecto, así como la bibliografía consultada para la realización del mismo.
1
1. CAPITULO 1: FUNDAMENTOS TEÓRICOS
1.1
INTRODUCCIÓN
El presente capítulo trata sobre la mejora en el proceso ministerial que tiene que ver con la
gestión eficiente de la información de las personas que llegan a la organización y se puedan
quedar en ella, consiguiendo la fidelización. Además, se abarcan los fundamentos teóricos,
en donde se detallan la situación actual del objeto de estudio, campo de acción y el análisis
crítico de los problemas de información.
1.2
MARCO TEÓRICO
1.2.1
FUNDAMENTOS TEÓRICOS SOBRE EL NEGOCIO
ANTECEDENTES
Historia de la Iglesia Evangélica en el Perú
Las iglesias llamadas protestantes y evangélicas llegaron al Perú con los emigrantes de
Norteamérica con la determinación de difundir la Biblia. Podemos destacar a Diego
Thomson, un escocés que llegó al Perú el 28 de junio de 1822 quien recibió una invitación
de Don José de San Martín, el libertador del Perú. El plan de San Martín era que Thomson
desarrollara el sistema de formación de los profesores de escuela, a fin de hacer popular la
educación, que en esa época era solo para los criollos y a los adinerados coloniales. Luego
se destaca en la difusión de la fe cristiana el misionero italiano Reverendo Francisco Penzotti
quien llegó al Perú en julio del año 1888. Los esfuerzos de difundir las Sagradas Escrituras
impulsados por Francisco Penzotti lograron luego la fundación de la Iglesia Metodista, que
fue la primera congregación llamada protestante en el Perú (INCHE, Iglesias Evangélicas o
protestantes, 2011).
Las iglesias evangélicas a través de la historia en otros países como el Presbiterianismo,
Anglicanismo, Metodismo o Luteranismo también basados en la fe cristiana, tienen poca
presencia y se destacan por su aporte social y político, reconociendo la importancia del amor
al prójimo y la solidaridad acompañando a la fe. La iglesia evangélica se hace presente
fundando centros médicos, colegios, comedores populares, etc. Iglesias como las Asambleas
de Dios del Perú y la Alianza Cristiana Misionera nacen y crecen, extendiendo su influencia
por todo el territorio peruano, en una intensa labor misionera de ir a muchos lugares del Perú
difundiendo la palabra de Dios y la misión apostólica, que es de abrir nuevos locales donde
se comparta el evangelio (INCHE, Iglesias Evangélicas o protestantes, 2011).
2
En los años de los 70 y 80 se inician nuevas iglesias evangélicas, tales como: Agua Viva,
Camino de Vida, Iglesia Bíblica Emmanuel, Movimiento Misionero Mundial, Movimiento
Evangelístico Misionero, son los nombres de algunas de las muchas iglesias que cada día
nacen, fundamentando su trabajo en la predicación del mensaje del evangelio y el
discipulado de los nuevos creyentes para luego formar líderes, formando también redes o
células(grupos familiares) dentro de hogares cristianos, distribuidos y organizados en los
distritos de cada departamento y ciudad del Perú.
La cantidad de personas que conforman las iglesias protestantes y evangélicas se estima en
alrededor de 4 millones de miembros. En su mayoría estas personas se congregan en las
iglesias evangélicas como la iglesia Comunidad Cristiana Agua Viva. Es importante destacar
que en el Perú el crecimiento de la iglesia evangélica comenzó en los años 70, desde el 1%
y ha llegado a pasar el 12% el año 2006 (INCHE, 2011).
La Iglesia Católica y la Iglesia Evangélica ven a Jesucristo como cabeza de la iglesia, pero
la diferencia es que las evangélicas no reconocen al Papa del Vaticano como una autoridad
espiritual en la Tierra. Es por esto que no hay un líder único dentro de la Comunidad
Cristiana ni una entidad colegiada que tome decisiones por encima de toda la congregación
nacional, tampoco hay una unidad dogmática, con rituales ni de otro tipo. Aunque, existen
asociaciones que agrupan a algunas congregaciones y a pastores del Perú que son líderes de
congregaciones que guían a los miembros que asisten a sus iglesias. Entre ellos están el
Concilio Nacional Evangélico del Perú (CONEP), la Unión Nacional de Iglesias Cristianas
del Perú (UNICEP), y la Fraternidad Internacional de Pastores Cristianos (FIPAC) (INCHE,
2011).
Comunidad Cristiana Agua Viva
La iglesia Comunidad Cristiana Agua Viva (CCAV) nace el año 1980 con 6 parejas
matrimoniales, con el transcurso de los años la congregación ha ido creciendo y hoy en día
cuenta con más de 50 mil miembros de diferentes lugares de la ciudad, debido a este
crecimiento se ha empezado a presentar problemas en cuanto al manejo de información por
falta de un correcto control y seguimiento de la misma por parte de los líderes de la
organización que realizan diversas funciones en la organización. (CCAV, 2017).
CCAV es una organización sin fines de lucro que busca ayudar a las familias de la sociedad
con el fin de ser una buena influencia enseñando principios y valores en cada una de las
3
reuniones los fines de semana y en los eventos que pueda realizar. El conjunto de familias
compone la sociedad y la nación, si las familias son fuertes la nación lo será. (Fuente: propia)
La Iglesia Comunidad Cristiana agua Viva(CCAV) se encuentra inscrita voluntariamente en
el Registro de Entidades Religiosas creado en el Ministerio de Justicia por el Decreto
Supremo 003-2003-JUS y este registro tiene como finalidad primordial el reconocimiento
de la personería jurídica civil de las entidades religiosas, y facilitar sus relaciones con el
Estado. Las organizaciones religiosas inscritas tienen personería jurídica de derecho privado
sin fines de lucro. Su organización y funciones se rigen por la Ley de Libertad Religiosa N°
29635 y su reglamento, también por sus propias normas y estatutos (Justicia, 2003).
Estrategia de la organización
Los pastores [13], Sergio y Carla Hornung como líderes principales de CCAV motivan a los
miembros de la organización con el fin de cumplir las estrategias bíblicas para poder alcanzar
a más personas con la predicación de los mensajes bíblicos y el buen ejemplo, y uno de los
objetivos es la de hacer de cada miembro de la iglesia un líder, desarrollando en él su máximo
potencial.
Es así que cada hombre, mujer, joven o adolescente tiene la oportunidad de prepararse como
líder, invirtiendo tiempo en el estudio bíblico y teniendo una mejor vida que puede
experimentar en la búsqueda de Dios y sus principios. Este proceso comienza con la
asistencia al Pre Encuentro [20], el cual es requisito básico para el siguiente paso, que es el
Encuentro.
“El Encuentro es el tiempo que uno separa para estar con Dios quien traerá sanidad sobre las
vidas. Una vez que se ha experimentado un Encuentro con Jesús, se sigue el curso de
Discipulado. Allí, estudiamos los principios básicos de la doctrina cristiana con el fin de
alcanzar el siguiente paso de la preparación: el Liderazgo” (AguaViva, 2015).
Los estudios de Liderazgo preparan a los creyentes en el servicio a Dios como líderes de un
Grupo y aquí es donde se aprende a poder trabajar en equipo.
Fundamentos de fe (AguaViva, 2015).
La Organización tiene fundamentos de fe que se resume en las siguientes:
•
Creer en un sólo Dios, creador de todas las cosas, que es perfecto y Todopoderoso.
4
•
Creer en Jesucristo que es el único intermediario entre Dios y los hombres. Fue
afligido y murió reemplazándonos en una cruz para ofrecer su sangre por nuestros
pecados, y resucitó.
•
Creer en la necesidad de una verdadera y profunda evangelización en el Perú
(Individuo-familia-sociedad-nación), la cual traerá como consecuencia la
recuperación de los valores morales, pondrá freno al odio, a la violencia y a la
corrupción y traerá paz, desarrollo, respeto a la vida y bienestar.
Necesidades de la Organización
Una gran parte de iglesias tradicionales como por ejemplo “Las Asambleas de Dios del Perú”
(INCHE, 2015) y otras más en la actualidad manejan la información en papeles o en sistemas
antiguos, lo cual ha venido acarreando un constante trabajo en revisión de la información y
de manera anticuada.
El correcto seguimiento y control es uno de los requerimientos básicos de CCAV, motivo
por el cual se ha visto en la necesidad de recurrir a la realización e implementación de un
sistema para poder manejar su información de una manera adecuada usando plataformas web
y móvil, evitando así la pérdida de información y de tiempo. Para poder identificar los
problemas y planteamientos de solución previamente se realizó un análisis del proceso
ministerial [9] de la organización. (Fuente: propia)
5
1.2.2
FUNDAMENTOS TEÓRICOS SOBRE LAS TENDENCIAS Y
TECNOLOGÍAS ACTUALES
En lo correspondiente a este punto, sobre las tendencias y tecnologías actuales, se hará un
estudio del CRM (Gestión de la Relación con el cliente).
Investigación de trabajos anteriores
En investigaciones a nivel nacional en el trabajo titulado “Impacto de un modelo de sistema
CRM en la fidelización de los clientes”, concluyeron que, aunque una empresa empiece una
estrategia CRM, esto no garantiza que de inmediato o a largo plazo empiece a ganar la
fidelización de los clientes, para lograr esto, el CRM debe ser parte de la cultura de la
organización y también se necesita que los clientes involucrados en el proceso puedan
colaborar. Mendoza Quijada y Vilela Goicochea (2014)
Los autores llegaron a la conclusión que, para hacer una buena implementación, el CRM
debe ser aceptado por todos los involucrados en el proceso.
Al realizar la implementación del sistema CRM que se propone, se mantuvo una buena
comunicación con los usuarios involucrados en el proceso de desarrollo ministerial.
En investigaciones a nivel internacional en el proyecto de grado “El CRM (Customer
Relationship Management) para tener una mejor calidad en el servicio al cliente” recomienda
que la estrategia CRM debe ser adoptado por toda la institución y no solo por algunas áreas
o departamentos, para que haya una mejora en la relación con los clientes en su filosofía
empresarial, también recomienda que la institución debe capacitar constantemente al
personal involucrado, sobre todo a los usuarios que usarán la aplicación. Noboa (2016)
En el trabajo titulado “Sistema de información web para la gestión de clientes (CRM)”
implementaron un sistema CRM y concluyeron que, para cumplir con las demandas de los
clientes, las organizaciones necesitan velocidad en el desarrollo del software, y que este
pueda tener nuevas actualizaciones, y es necesario emplear técnicas de adaptabilidad. Díaz
Alba y Pinzón Salgado (2014)
El CRM en la historia
Antes de definir los conceptos del CRM es necesario ver la historia de esta estrategia. Los
negocios a inicios del siglo XIX se realizaban de acuerdo a métodos artesanales de
producción, los clientes y los fabricantes tenían una relación estrecha, porque estaban
geográficamente cerca. Era un periodo donde las empresas y los clientes tenían un
6
conocimiento personal, de esta manera las empresas daban a sus clientes productos
personalizados, por lo que la fidelidad de este se mantenía siempre. Después de la
Revolución Industrial, a inicios de siglo XX, empezó una era de mecanización, que buscaba
el aumento de los niveles de productividad, el cliente y el trabajador tenían líneas
departamentales intermediarias, el contacto con el cliente cada vez estaba más lejos. García
(2001)
En los años 50 y 60, las empresas querían satisfacer la demanda de bienes y servicios que
estaba en crecimiento. En los años 80, los productos comenzaron a incorporar la tecnología,
las empresas adoptaron sistemas de gestión. El avance de las tecnologías de información y
las telecomunicaciones hizo que surgieran cambios en la economía y en los procesos de
negocio. Los productos se empiezan a estandarizar, y las empresas para que puedan vender
y marcar la diferencia buscan cercanía al cliente y poder influenciar en su comportamiento
ya que ahora este ahora tiene el poder. García (2001)
El concepto CRM es una filosofía de negocio que integra las diferentes áreas de una
organización y que tiene contacto con el cliente, lo que busca es darle un valor agregado, y
que sea de beneficio para ambas partes. García (2001)
La evolución de la tecnología ha cambiado significativamente la forma de relación de la
organización con sus clientes, así como la de sus empleados entre sí. (Croxatto, 2005)
Definición del CRM
Las definiciones que se puede encontrar sobre el CRM son de dos tipos. Primero la definición
del CRM como una estrategia de negocio, y la segunda como una aplicación que lleva una
estrategia de negocio que involucre la relación con los clientes y la organización. (Croxatto,
2005, pág. 37)
A continuación, se detallan definiciones de algunos autores:
CRM es una estrategia de negocios que optimiza los ingresos y la rentabilidad, buscando la
satisfacción y la lealtad de sus clientes. Las tecnologías de CRM identifican y gestionan las
estrategias con los clientes, y puede ser en persona o de manera virtual. El software de CRM
provee funcionalidad a las empresas en cuatro aspectos: ventas, marketing, servicio al cliente
y comercio digital. (Gartner, 2017)
CRM es una estrategia de negocios que las compañías están usando para tener mayores
ingresos, optimizar sus ganancias y la satisfacción de sus clientes. Lo que busca es obtener
7
un balance entre el valor de mercado de la empresa y el valor del cliente. La tecnología
integra la organización a todos los canales que ese cliente puede usar: vendedores, call
center, socios de negocios, canales de venta, comerciantes minoristas y otros. Las
tecnologías ayudan a tener un mayor conocimiento del cliente, un acceso mayor, una
integración de los canales y una interacción más eficiente. (Brunetta, 2014, pág. 82)
Un concepto en común de los autores, es que se necesita de una base tecnológica para que
sea procesada la información de los consumidores e interacciones con los mismos. De
acuerdo a estos conceptos se considera a esta plataforma tecnológica como sistema CRM, y
en este proyecto, se está implementando un sistema CRM con el fin de mejorar la gestión de
la información de los creyentes.
Las relaciones con el cliente son algo que puede ser administrado. ¿Por qué las personas que
vengan a la organización se quedarían en esta? La respuesta es porque tenemos un modelo
de administración de los clientes que marca la diferencia ante los demás. Por ejemplo,
conocer muy bien a cada persona que llega a la iglesia, saber las necesidades que tienen ya
sea a nivel espiritual, emocional o material. Saber de sus gustos e intereses y poder hacer
una buena gestión de sus datos para beneficio de ellos. (Fuente: propia)
Poder usar este conocimiento para relacionarnos con las personas que llegan a la
organización y decidir a quienes ofrecer algún beneficio de acuerdo a su necesidad, quienes
necesitan ir a un encuentro o necesiten alguna consejería porque este pasando por algún
problema. (Fuente: propia).
Es por eso que la empresa debe escuchar y responder activamente a través de diferentes
canales para mantener la competitividad del negocio.
8
Figura 1: Relación entre tipos y componentes CRM (Fuente: Croxatto (2005))
Componentes de un CRM
La ilustración #1 nos muestra como los componentes CRM forman parte de la integración
de los diversos tipos de CRM. Los componentes CRM son fundamentales para los procesos
y ciclos de información del sistema (Croxatto, 2005), y los usuarios del CRM no son
únicamente los clientes, sino también los empleados de la empresa, operadores de contacto,
vendedores, jefes de productos, responsables del marketing, ejecutivos, proveedores, etc.
Portal
El portal es la principal puerta de acceso a las facilidades del CRM, es el medio por el cual
diferentes grupos o usuarios tendrán acceso a una visión totalmente diferente del conjunto
de información, bienes y servicios que estén disponibles (Greenberg, 2003).
Funcionalidad específica para cada negocio
Como se ve en la ilustración #1, la funcionalidad específica para cada área de negocio, es la
que soporta los procesos que componen el núcleo del CRM operacional. Estas permiten
automatizar los procesos de las áreas “Front Office” y “Back Office” (incluidas las
relaciones con ventas, marketing y servicio al cliente) (Croxatto, 2005).
9
Proceso analítico
Funcionalidad que permite el análisis de la información del sistema soportado por el uso de
la tecnología del almacén de datos (Chen y Popovich, 2003). Esta consiste en un servidor
que se encuentra entre el almacén de datos y el usuario y constituye lo que es el CRM
analítico y el negocio inteligente.
Repositorio de Datos (Motor del CRM)
El repositorio de datos forma parte del CRM operacional y es el lugar donde se captura y
almacena toda la información de los clientes, productos, posibles contactos, campañas, etc.
(Todman, 2000).
Facilidad de integración
Esta permite la unión de información de los sistemas CRM con otras aplicaciones, también
permite la integración con otros sistemas heredados de la empresa y la comunicación entre
distintos sistemas CRM. (Croxatto, 2005).
Módulos de un CRM
Los sistemas CRM pueden estar compuestos por varios módulos según las necesidades de
la organización. Los módulos más comunes son ventas, marketing, y servicios. A
continuación, se describirá las funciones que cumple cada módulo en los sistemas CRM.
Módulo de Marketing
Este módulo permite a la organización conocer quiénes son sus clientes, que desean y como
y cuando desean realizar negocios con la organización, ofreciendo experiencias
personalizadas a través de diversos canales para fidelizar clientes. Este tiene funcionalidades
como análisis de mercado y clientes, planificación de marketing, campañas de marketing
para clientes con características parecidas, creación de listas, identificación de oportunidades
de ventas cruzadas, análisis de retención de clientes, personalizar y monitorear eventos,
calendarios y actividades (Cabello, 2007).
Módulo de ventas (soporte a la fuerza de ventas)
Este módulo está centrado en el equipo de ventas de la organización e impulsa las iniciativas
CRM. Su objetivo es fortalecer las fuerzas de ventas en los clientes que pueden dar más
rentabilidad, y el equipo puede tener la información de las ofertas de productos y servicios
de la organización, y a la información de los clientes. Además, ofrece muchas herramientas
10
que ayudan a la administración de las actividades de ventas, como la gestión de contactos,
pedidos, documentos y fuerzas de ventas (Cabello, 2007).
Módulo de servicio
La relación con el cliente es fundamental para la estrategia del CRM porque la satisfacción
del mismo está ligada con los servicios prestados y a la calidad de atención (Mendoza et al.,
2007). En este módulo se gestiona la atención al cliente y permite el manejo de los diferentes
tipos de incidencias con las ofertas de productos o servicios de la organización, también
llamado gestión de casos.
Otras funcionalidades, herramientas y características básicas de un CRM
Los sistemas CRM mejoran la interacción con los clientes a través de muchas herramientas
y funcionalidades para adaptarse a los requerimientos de la organización (Business Software,
2014). En la tabla #2 se muestra un resumen de funcionalidades y herramientas de los
módulos de marketing, ventas y servicios. Se listan también otras funcionalidades y
características comunes de los sistemas CRM.
Módulo de servicio
Módulo de ventas
Módulo de Marketing
Otras funcionalidades,
herramientas y
características básicas
Autoservicio del cliente
Obtener clientes
potenciales
Organizar información
del cliente
Interfaz personalizable
Organizar atención al
cliente
Gestión de contactos
Realizar listas con
diferentes criterios
Seguimiento con redes
sociales
Gestión de casos
Gestión de
oportunidades
Desarrollo de campañas
de marketing
Canales de contacto
Gestión de incidencias
Gestión de cuentas
Relacionar las ventas
con las estrategias de
marketing.
Sincronizar los datos
Gestionar los centros de
llamada
Gestión de propuestas
Gestión de respuestas.
Dar ayuda y soporte al
cliente
Vista de la información
de los clientes
Gestión de comisiones
Supervisión del flujo de
trabajo.
Desarrollo de un
Dashboard
Soporte técnico a los
clientes
Gestionar las cuotas de
ventas
Gestión de eventos.
Informes y reportes
Gestión de devoluciones
Generación de ofertas
Programas para retener
clientes
Indicadores claves de
rendimiento
11
Conocimiento de las
funcionalidades del
producto
Gestión de pedidos
Analizar el mercado y
los clientes
Movilidad
Gestionar la satisfacción
del cliente
Proyección de las ventas
Automatización de
marketing
Agenda, lista de
actividades –
colaboración rápida, los
comentarios en línea y
las funciones de chat.
Sistema de información
competitiva
Identificación de
oportunidades de ventas
cruzadas
Integración con los
sistemas heredados
Gestión de campaña de
tele ventas
Segmentación y análisis
de retención de clientes
Seguridad del software /
Acceso restringido
Gestión de documentos
Segmentación y análisis
de retención de clientes
Entrenamiento y
capacitación
Automatización de
fuerzas de venta
Personalización y
monitoreo de eventos
Soporte técnico
Servicio de soporte /
Help desk
Tabla 1: Cuadro de funcionalidades CRM (Fuente: propia)
Figura 2: Procesos y Arquitectura CRM (Fuente: ON Soluciones 2013)
12
Sistema CRM
El sistema CRM nos permite capturar y registrar toda la información relevante del cliente
cada vez que nos ponemos en contacto con él, sea cual sea la forma en que se logra dicho
contacto. Por ejemplo, cuando me hacen una llamada de un call center para venderme algún
producto y me registran todos los datos posibles en esa llamada, y se puede obtener mucha
información y la organización puede tener estrategias de venta y llegar mejor a sus clientes.
(Fuente: propia)
Cliente
Es lo principal en este sistema, el cliente tiene varios datos y recolectar la mayor cantidad de
datos e intereses que se pueda recopilar.
Lista de Contactos
Cada cliente tiene su lista de contactos.
Figura 3: Contenido de un Sistema CRM Clientes vs contactos (Fuente: propia)
Interacción
Puede ser una llamada de teléfono entrante o saliente.
Motivos y Sub Motivos
Razones por las cuales el cliente puede llamar. Cada “motivo” tiene sus correspondientes
“sub Motivos”. Las tablas están vinculadas con “ID”; no se muestra en la gráfica.
13
Figura 4: Contenido de un Sistema CRM Motivo vs sub motivos (Fuente: propia)
Canal de comunicación
Es la manera como me contacto con el cliente y tiene que quedar registrado.
Referido
Forma en que el cliente nos conoció, sirve para saber cuál medio de publicidad es más
efectivo y hacerlo más fuerte.
Figura 5: Contenido de un Sistema CRM Referidos (Fuente propia)
Usuario del Sistema
Quién ingresó al sistema para cargar todos los datos.
Fecha del Servidor
Es la fecha de la estación de trabajo, siempre donde está la base de datos.
14
Información que brinda un sistema CRM
El sistema muestra los reportes del comportamiento de los clientes con la empresa. Se
registra los comentarios para ver qué pasó con un cliente en particular. Todo esto surge de
las llamadas que entraron al call center.
De los reportes realizados sobre los contactos que tuvo el cliente en la empresa podemos
obtener informes de:
•
Motivo por el cual no se concretaron ventas.
•
Medio de publicidad que fue más efectivo.
•
Motivo por el cual el cliente se va de la empresa.
•
Motivos de quejas del cliente.
•
Tipo de problemas administrativos.
Con estos informes se puede elaborar estrategias y políticas empresariales para:
•
Estrategias de ventas dirigidas a los clientes complejos
•
Publicidad a la medida del medio más efectivo.
•
Políticas de mejor atención al cliente. Capacitación de personal.
•
Fidelización de clientes
Plataforma de programación
La plataforma e interface que se va usar es web, con un lenguaje de programación en php
para más de 100 usuarios conectados a la vez. El Motor de base de datos será MySQL.
El tiempo de programación de este tipo de sistema puede variar desde un año hasta más
dependiendo de la organización, en este caso para la iglesia CCAV sería un año. (Fuente:
propia)
Sistema a medida para la iglesia CCAV
El CRM que se construirá para la organización podrá registrar los datos de los clientes que
vendrán a sus sedes, estos clientes son las personas invitadas que vienen a cada reunión de
fin de semana.
15
Los líderes de la organización quieren tener una visión más cercana a la realidad de lo que
sucede entre los clientes y la organización. A partir de ese contacto en toda la toma de
información que hará el sistema se podrá obtener reportes que permitirán a los directivos y
líderes poder tener una estrategia de mejora para llegar al cliente, una mejora administrativa
de la organización, y un mejor servicio de la iglesia.
Algo importante es que el sistema nos ayudará a cómo recuperar los clientes que se están
alejando de la iglesia por algún motivo.
Con todos los datos tendremos diferentes tipos de estrategias para captar a nuestros clientes
y mantenerlos en la organización.
Figura 6: Contenido de un Sistema CRM Interacción con todas las partes (Fuente: propia)
La organización tendría una tabla de clientes (nuevos creyentes) y sus contactos que pueden
ser sus familiares o amigos, compañeros de trabajo, etc. Que pueden invitar cada fin de
semana a la iglesia.
•
La interacción que puede ser una llamada al nuevo creyente y allí se registra los
motivos por los cuales no puede asistir a un grupo familiar o a la iglesia.
•
La siguiente interacción puede ser la visita al nuevo creyente. Hacer el reporte del
estado del creyente, si asistirá, si ya asistió o no se le encontró para que pueda ir a un
grupo familiar.
•
La organización podrá crecer sostenidamente si logra retener la mayor cantidad de
clientes.
16
1.3
OBJETO DE ESTUDIO
1.3.1
ORGANIZACIÓN OBJETIVO
La Comunidad Cristiana "Agua Viva" CCAV es una iglesia cristiana, con énfasis en el
trabajo por las familias, la formación de líderes y sobre todo de servicio a la comunidad.
La iglesia tiene varias sedes en Lima (En Lince y en el Cercado de Lima) y en los conos
(Los Olivos, San Juan de Lurigancho, San Juan de Miraflores, Villa María del triunfo,
Chosica), también en provincias (Arequipa, Huancayo, Sullana, Tarma, Iquitos, Tacna,
Trujillo, Madre de Dios, Huacho, Ica, Chincha y otros más).
El objeto de estudio de la Organización CCAV y busca crecer en personas y poder ser de
mayor influencia en la sociedad y se ha visto estancada por muchos años y la organización
no ha crecido en cantidad de miembros por no tener una buena gestión de su proceso y
proyección de la información en el desarrollo ministerial en cada una de las sedes.
En el siguiente cuadro se representa la matriz de FODA que fue realizada en la CCAV,
matriz que sirvió para el análisis de la solución de la presente problemática.
FORTALEZAS
DEBILIDADES
Iglesia conocida
No tener una buena retención de las
personas que llegan
Buenas ubicaciones
Falta de un sistema a medida que cubra los
procesos de registro de datos en menos
tiempo, que registre mejor la experiencia
con el cliente, que provea estadísticas para
toma de decisiones.
Buena doctrina
Cierto desorden en gestión de la
información
Cantidad de miembros
No hay líderes bien preparados
OPORTUNIDADES
AMENAZAS
Gente que está buscando ayuda espiritual
Que las personas se vayan a otra iglesia
Personas que se fueron de otras iglesias
No hay interés de pertenecer a la iglesia
por el mal testimonio de sus miembros.
En nuestro país hay libertad de religión
Personas que han querido dividir la iglesia
Tabla 2: Análisis de FODA de la CCAV (Fuente: Propia)
17
1.3.2
DESCRIPCIÓN DE ÁREAS (Fuente: CCAV)
Junta de Administración
Es el área donde están los pastores fundadores y deciden temas administrativos como
contrato de personal, contratos de asesoría legal, creación del algún área funcional o
ministerial en la iglesia, ven la parte legal de la iglesia.
Asesoría Legal
La Junta de administración contrata abogados que asesoran a la organización en temas
legales, contratos de adquisición de nuevos locales, pasar por revisiones de defensa civil,
asesoría legal con los predios actuales que tenemos, etc.
Departamento de Finanzas
Es el área donde se puede hacer la administración de las ofrendas y donativos que llegan a
la organización y de allí se administra para los diferentes gastos que se realizan ya sea para
pagos de servicios, pagos de personal, pagos en seguridad y materiales que se usan en
diferentes sedes de la organización.
Departamento de Contabilidad
La organización tiene su área contable donde los pagos a la SUNAT (Súper Intendencia
Nacional de Administración tributaria) están en regla, el balance general de cada año para
tener todos los registros de costos y gastos actualizados, pago de impuestos y otros que
requieran el área contable.
Departamento de Tecnología e Informática
Es el área donde se realizan labores de soporte técnico en equipos de cómputo en los
diferentes locales que tiene la organización.
Por ejemplo, en el Coliseo Amauta se tienen equipos de pantallas led, cámaras de video,
consolas de sonido y computadoras para la matricula en la escuela de líderes y todos estos
equipos necesitan actualización de software y soporte técnico en antivirus, limpieza física,
etc.
Departamento de Producción
Es el área donde se producen las reuniones de fin de semana y allí hay varias áreas con sus
encargados respectivos que juntamente con sus colaboradores realizan actividades para
18
producir la reunión como por ejemplo las áreas de sonido, comunicaciones, Audio y video,
Librería, redes sociales, servidores, consejeros, ministerio de música, etc.
Departamento de Mantenimiento
Es el área de limpieza y mantenimiento en todas las sedes de la organización, donde el área
de Logística adquiere los insumos y materiales para los trabajadores que realizan esta labor
y siempre tiene en óptimas condiciones de orden y limpieza cada local para recibir a las
personas cada semana.
Departamento de Logística
Es el área donde se realizan las actividades de soporte y medios necesarios para llevar a cabo
el funcionamiento de diferentes procesos de la organización como proveer materiales para
el área de mantenimiento. Proveer materiales de escritorio para las áreas de secretaria y
asistencia pastoral, etc.
Departamento de Comité Creativo
Es el área donde se ve la parte creativa y estratégica para mejorar las actividades que se
producen en las reuniones de fines de semana y eventos en cada local de la organización. Se
ven estrategias, nuevas ideas, mejoras a los procesos en la producción de las reuniones, se
sugieren a qué invitados se podrían traer para el evento internacional que se hace cada año,
etc.
Departamento de Proyectos
Es el área donde se ven los proyectos que tiene la organización, hay varios proyectos por
hacerse como por ejemplo “Proyecto de seguridad”, “Proyectos eléctricos”, “Proyectos de
remodelación e infraestructura”, “Proyectos de estructuras metálicas” para el escenario del
coliseo Amauta, “Inspecciones de Defensa Civil”, etc.
1.3.3
MISIÓN
“La misión es ser una iglesia cristiana que busca la salvación de la persona y la
transformación de la sociedad, formando líderes que viven de acuerdo a los principios y
valores bíblicos, cumpliendo así con el llamado y propósito que Dios nos ha dado.”
(AguaViva, 2015)
19
1.3.4
VISIÓN
“La visión es ser reconocida nacional e internacionalmente como una iglesia de influencia
que trabaja con excelencia, siendo un instrumento para que toda persona pueda
comprometerse con el propósito de Dios para su vida y sea prosperada integralmente.”
(AguaViva, 2015)
1.3.5
OBJETIVOS ESTRATÉGICOS (Fuente: CCAV)
•
Fortalecer la vida espiritual, emocional y mental de las personas para un crecimiento
integral y servicio de calidad a Dios y al prójimo.
•
Realizar eventos evangelísticos para captar mayor cantidad de personas en nuestra
congregación.
•
Formar líderes productivos y desarrollar en cada persona el carácter de Cristo para
realizar con excelencia los grupos familiares.
•
Implementar la estrategia de grupos familiares, para alcanzar con el mensaje de
salvación a las personas de cada distrito de Lima.
•
Establecer estrategias en coordinación con los ministerios de la iglesia para fortalecer
el trabajo en equipo.
INTERNOS
PROCESOS
MIEMBROS
MAPA ESTRATEGICO GENERAL
Fortalecer la
vida espiritual
Seguimiento y
Control
Fortalecer
Trabajo en
Grupos
equipo
CRECIMIENTO
DESARROLLO Y
Familiares
Preparación
Realizar
de los líderes
eventos
Evangelístic
Figura 7: Mapa Estratégico General (Fuente: propia) os
20
1.3.6
ORGANIGRAMA
Figura 8: Organigrama de la Organización CCAV (Fuente: Propia)
21
1.4
CAMPO DE ACCIÓN
1.4.1
BREVE DESCRIPCIÓN
El campo de acción que se quiere cubrir con el desarrollo del proyecto es del proceso
Ministerial [5], el cual se divide y cubre las siguientes áreas:
Comunidad Cristiana
Agua Viva
Llega a la Iglesia
CONSOLIDAR
Afirmar al nuevo
ENCUENTRO
Retiro espiritual
ESCUELA DE LÍDERES
Formación de nuevos líderes
GRUPOS FAMILIARES
Reunión semanal con un líder
Figura 9: Proceso del Desarrollo Ministerial (Fuente: Propia)
Consolidación: Este grupo de actividades tiene como objetivo la inclusión de nuevas
personas a la comunidad cristiana. Los invitados registran sus datos para luego ser
contactados por un líder quien servirá de guía en su desarrollo espiritual.
Encuentro: En esta etapa se invita a las personas que asisten a un grupo familiar a participar
un retiro espiritual que dura tres días donde la persona experimenta un cambio en su forma
de pensar y vivir.
Escuela de Líderes: En este proceso las personas que culminaron su encuentro son invitadas
a iniciar una línea de carrera como líder de la iglesia. Se le brinda una serie de cursos y
seminarios con el fin de desarrollar sus capacidades de líder de nuevos grupos familiares.
22
Grupos Familiares: Estas actividades son una serie de reuniones semanales organizados
por cada líder quien invita a otras personas a que puedan ser parte de la iglesia. El objetivo
es compartir un mensaje de fortaleza y motivación a los invitados por parte del líder y el
grupo mismo, incluir a más personas por medio del proceso de consolidación.
1.4.2
PROCESOS DEL NEGOCIO
En el siguiente gráfico se tiene el mapa de procesos de la empresa en estudio y se resalta el
área que se va a trabajar.
Procesos Estratégicos
Junta de Administración
Área Pastoral
Comité Creativo
Procesos Operativos
Consolidación
Retiros
Escuela de Líderes
Grupos Familiares
ONG
Eventos
Procesos de Soporte
Recursos Humanos
Finanzas
Sistemas TI
Logística
Contabilidad
Figura 10: Mapa de procesos de Comunidad Cristiana Agua Viva (Fuente: Propia)
23
ÁREA DE
CONSOLIDACIÓN
Llega a la Iglesia
Registro de
Datos
Verificación de
datos
Hace una oración
Escucha mensaje
Asiste a Grupo Familiar
Pronto contacto
Figura 11: Área de Consolidación (Fuente: propia)
1.4.3
SISTEMAS AUTOMATIZADOS VINCULADOS CON EL CAMPO DE
ACCIÓN
Actualmente la organización cuenta con un sistema donde se registra el ingreso de
información de las personas, es decir solo ingresan datos y obtienen reportes. El Sistema se
llama Ekklesia y es para la gestión de información, es una aplicación desarrollada en php.
Sin embargo, en los procesos se realizan varias actividades que la trabajan todavía en MS
Excel lo cual es costoso en horas hombre, sin mecanismos de validación para disminuir la
posibilidad de error en el ingreso de información, no permite tener una vista integrada de
todos los procesos. Para optimizar la gestión de la información de las personas, se pretende
desarrollar un sistema web a medida que va ser una mejora del existente.
24
1.5
ANÁLISIS CRÍTICO DE LOS PROBLEMAS DE INFORMACIÓN
1.5.1
SITUACIÓN PROBLEMÁTICA Y PROBLEMAS A RESOLVER
A continuación, se listarán las situaciones problemáticas y los problemas a resolver:
•
En los procesos se realizan varias actividades que la trabajan sin tener una vista
integrada de todos los procesos.
•
Para optimizar la gestión de la información de las personas, se pretende desarrollar
un sistema web a medida.
SITUACIÓN PROBLEMÁTICA
PROBLEMAS A RESOLVER
SP_01: No se tiene a tiempo el listado de las
PR_01: El registro de las personas es a través
personas que llegaron a la iglesia. En algunos de unas hojas que luego son ingresados al
casos la información está disponible en una sistema por un digitador el cual involucra
semana cuando debería estar en dos días.
demoras y el líder al no tener el listado a tiempo
se demora en contactar al nuevo creyente.
SP_02: Los líderes no pueden tomar PR_02: Falta de información y herramientas
decisiones oportunas y eficientes que ayuden que le permita al líder realizar el seguimiento
con el crecimiento de su grupo.
de su grupo y la toma de decisiones oportuna
que permita el crecimiento de su grupo.
SP_03: La organización espera que el líder
PR_03: En muchas ocasiones los líderes
llame y visite a los nuevos creyentes y que olvidan su deber de contactar a las personas o
haga su reporte.
no aprovecha los medios de comunicación
El líder al no reportar no se sabe el estado adecuados para mantener activo el interés.
actual del nuevo creyente.
SP_04: Se presenta demoras en la inscripción PR_04: No se dispone de información
a la escuela de líderes y en ocasiones no se
dispone
oportunamente
del
actualizada y oportuna del historial académico
historial que ayuden con el seguimiento y control del
académico de la persona para determinar sus
desarrollo de la persona.
cursos y talleres hábiles.
SP_05: No se sabe si el nuevo creyente está
PR_05: El creyente no dispone de información
asistiendo a la iglesia, si está yendo a su grupo en tiempo real de las actividades de la iglesia.
familiar, si ya fue a un encuentro o si está Cuando la escucha en las reuniones se olvida,
yendo a sus cursos bíblicos.
25
no las anota y cuando hay un evento no asiste y
se retrasa su crecimiento y desarrollo.
SP_06: No se está llevando un control y PR_06: Falta de herramientas y métricas que
seguimiento
adecuado
del
desarrollo ayuden a las áreas pastorales a tener una mayor
ministerial de cada líder.
visibilidad del desarrollo de cada ministerio y
efectividad de los líderes.
SP_07: No hay campañas de difusión de
PR_07: La información de estos tipos de
grupos familiares, en muchas ocasiones solo
eventos se registra en una base de datos, pero
se registran, pero las personas no saben que
no se difunde apropiadamente para que los
hay uno cerca de su distrito.
nuevos creyentes y nuevas personas puedan
llegar a la iglesia y se queden.
Tabla 3: Situación problemática vs Problemas a resolver (Fuente: propia)
1.6
CONCLUSIONES
En el presente capítulo se procedió a detallar información relevante con respecto a la
organización y al proceso presente en nuestro campo de acción, esto fue necesario para poder
analizar su situación actual y la problemática presente en las actividades de dicho proceso
de gestión ministerial. La organización tiene algunos servicios de ingreso de información a
la base de datos, pero necesita el apoyo de las herramientas y conocimientos que podrían
hacer que el trabajo de los líderes sea más eficiente. Durante las reuniones que se tuvo con
el Jefe de área de Sistemas se observó la predisposición de este para mejorar sus procesos
del área ministerial con el buen uso de la tecnología.
26
2
CAPITULO 2: PROPUESTA DE LA SOLUCIÓN
2.1
INTRODUCCIÓN
En este capítulo se trata la propuesta de solución, con sus respectivos argumentos que
respaldan su desarrollo para resolver el problema del negocio de la organización. Tiene como
finalidad presentar a detalle la solución que se está proponiendo a la problemática detectada
y detallada en el punto anterior, así como también realizar una comparación entre las
tecnologías ya desarrolladas a nivel mundial y la propuesta presentada.
2.2
OBJETIVOS DEL PROYECTO
2.2.1
OBJETIVO GENERAL
El Objetivo general es desarrollar un SISTEMA DE GESTIÓN DE DESARROLLO
MINISTERIAL PARA LOS LÍDERES DE UNA ORGANIZACIÓN CRISTIANA, que es
un sistema informático con la tecnología web y móvil que le permita gestionar de manera
eficiente la información del Proceso Ministerial [5], enfocado en mejorar las áreas de
consolidación, encuentros, escuela de líderes, y grupos familiares. Dicha propuesta permitirá
contar con información en línea vía web y móvil para uso de los líderes y los pastores
principales de la organización.
2.2.2
•
OBJETIVOS ESPECÍFICOS
El sistema deberá permitir registro y actualización de datos de una persona a través
de tecnología web y móvil, manteniéndola centralizada y disponible oportunamente.
•
El sistema deberá permitir el control y seguimiento del desarrollo ministerial de cada
una de las nuevas personas.
•
El sistema deberá permitir la notificación de alertas cuando se produzcan una falta
de comunicación o contacto entre los líderes y su grupo por más de una semana, así
como la falta de actividad de cualquier tipo por más de un mes en la comunidad
cristiana.
•
El sistema deberá permitir la inscripción en línea de los discípulos a los cursos
disponibles en la escuela de líderes.
•
El sistema deberá permitir el control y seguimiento de los cursos que llevan los
estudiantes en la Escuela de Líderes de forma automática.
27
•
El sistema deberá permitir el control y seguimiento del desempeño del líder en base
a métricas que controlen su participación y crecimiento y notifique en caso no se
cumplan.
•
El sistema deberá permitir la gestión de la información y organización de grupos
familiares y de forma automatizada y centralizada.
2.2.3
•
FUNDAMENTACIÓN DE LOS OBJETIVOS
Se automatizará el ingreso de datos de las personas mediante la integración a las
redes sociales, para ello se desarrollará Apps para Facebook y que motiven el registro
de datos de una manera más rápida.
•
Se permitirá la asignación de líderes a los grupos de personas por localización, y se
realizará el control y seguimiento de las personas día a día mediante cuadros de
mando por parte del líder y el envío de notificaciones y resúmenes semanales sobre
el estado de su grupo.
•
Se emitirá mensajes a través de correos y redes sociales la próxima apertura de
nuevos cursos y talleres para su registro en línea, esto agilizará el cálculo de la
demanda y permitirá servir como información relevante acerca del interés de cada
persona.
•
Se facilitará el control y seguimiento del desempeño del líder en su grupo, evaluar
las encuestas de satisfacción y tomar acciones inmediatas por parte de los pastores
en base al crecimiento o decrecimiento.
•
Se permitirá consultar mediante reportes estadísticos los diferentes tipos de la
información de valoración, de líderes, de grupos, de eventos, etc., para que le permita
tomar decisiones a los pastores principales (Gerencia y Junta de Administración) y
proponer mejoras.
28
2.2.4
INDICADORES DE LOGRO DE OBJETIVOS
Indicador
Detalle
Tipo de Documento
Presentación del
Documento detallará a nivel negocio los actores
Documento del
primer modelado de y los casos de uso involucrados, así como el
sistema
negocio.
detalle de cada uno
Este documento incluirá a nivel negocio los
Presentación del
actores, los casos de uso involucrados,
Documento del
modelado de
trabajadores, entidades, diagramas de clases,
sistema
negocio final.
diagramas de actividades y las tareas a
automatizar, así como el detalle de cada uno.
Documentación de Este documento mostrará el análisis y diseño del
Documento del
análisis y diseño del sistema, pruebas, así como las restricciones,
sistema
sistema.
necesidades y funcionalidades del sistema.
Presentación de la
Entrega del sistema con el alcance de los casos
primera versión del
Sistema
de uso del ciclo 0
sistema.
Documento en la cual el autor de la tesis y el
Documento de
interesado de la organización acuerdan y aceptan
conformidad
Carta de
todo lo estipulado en el documento "modelado
modelado de
conformidad
de negocio", cualquier cambio en él se realizará
negocio
previa aceptación de ambas partes.
Documento en la cual el autor de la tesis y el
Documento de
interesado de la organización acuerdan y aceptan
conformidad de
todo lo estipulado en el documento
Carta de
requerimientos del “Requerimientos del sistema", cualquier cambio
conformidad
sistema
en él se realizará previa aceptación de ambas
partes.
Documento en la cual el autor de la tesis y el
Documento de
interesado de la organización acuerdan y aceptan
Carta de
conformidad del
todo lo estipulado en el documento “Proyecto
conformidad
sistema total
Total", cualquier cambio en él se realizará previa
aceptación de ambas partes.
Tabla 4: Indicadores de logro de objetivos (Fuente propia)
29
2.3
BENEFICIOS DEL PROYECTO
2.3.1
BENEFICIOS TANGIBLES
El desarrollo del sistema propuesto traería los siguientes beneficios tangibles para la
empresa:
1. Reducción en un 50% de trabajos operativos, es decir si antes se demoraba ingresar
los datos de las personas en dos días, ahora se ingresará en un día.
2. Los reportes se podrán visualizar en tiempo real, ya que se podrá ver desde un
dispositivo smartphone.
3. Control centralizado de la información en un solo lugar y no de varias fuentes, el
beneficio es que se evitarán errores de información porque en un lugar entra cierta
información y en otro lugar ingresa una diferente entonces así hay ambigüedad de la
información y hay que estar depurando los datos, se evitará duplicidad de
información y también que se ingrese información incompleta.
4. Disponibilidad de la información para los líderes en tiempo real ya que podrán
consultar su información desde el app en su dispositivo smartphone.
2.3.2
BENEFICIOS INTANGIBLES
El desarrollo del sistema propuesto traería los siguientes beneficios intangibles para la
empresa:
5. Mejor orden en los procesos del negocio
6. Ofrecer una mejor imagen a las nuevas personas que llegan a la iglesia
7. Brindar mejor calidad de atención para los creyentes y líderes (los líderes podrán
desempeñar mejor su labor con las personas que tienen a su cargo).
2.4
ANTECEDENTES
En el mercado local, no se encuentran muchas soluciones informáticas para el tipo de
negocio que es la retención de personas en una organización cristiana. Sin embargo, en
Latinoamérica existen algunas soluciones que se asemejan al proyecto propuesto en este
documento. A continuación, se explicarán y compararán las soluciones encontradas en esta
parte de la investigación.
30
2.4.1
SOLUCIONES ENCONTRADAS
Entre las soluciones encontradas más importantes tenemos:
Solución 1
Nombre de la solución
Softlife
País
Colombia
Web
http://misoftlife.com/
Características
✓
✓
✓
✓
✓
✓
✓
✓
Mide el crecimiento espiritual de las personas
Crea múltiples perfiles y usuarios para los líderes
Registro de personas nuevas
Gestión de la membresía
Gestión de las escuelas de líderes
Registro de grupos de casa
Reportes en línea
Seguimiento día a día de las personas(a través de llamadas
y visitas en las casas de los invitados)
Tabla 5: Solución encontrada Softlife (Fuente: http://misoftlife.com)
Solución 2
Nombre de la solución
Administrador Fiel
País
Venezuela
Web
administradorfiel.com
Características
✓
✓
✓
✓
✓
✓
✓
✓
Registro de datos eclesiásticos
Gestión de ministerios
Registro de Observaciones / Incidencias
Registro de datos laborales
Gestión de grupos
Registro y asistencia a eventos
Gestión de finanzas
Registro de inventarios de activos
Tabla 6: Solución encontrada Administrador Fiel (Fuente: administradorfiel.com)
2.4.2
ANÁLISIS COMPARATIVO
Para dar inicio a este proyecto se ha realizado una búsqueda en el mercado de soluciones
que puedan cubrir las necesidades de la organización objetivo. Como resultado se ha
elaborado un cuadro comparativo de las funcionalidades que presentan cada una de ellas,
incluyendo la solución planteada.
Como resultado de la actividad previa se han encontrado 2 soluciones que cumplen
parcialmente lo requerido por la organización, estas se describen a continuación:
31
Cumplimiento del Producto 0-2
moderado: 1
Gran impacto: 2
Cumplimiento del Producto
No cumple: 0
Cumplimiento
Cumplimiento
parcial: 1
total: 2
Productos
Criterios de Evaluación
1 Registro de invitados
2 Integración con redes sociales
3 Admisión de creyentes
4 Asignación de líder
5 Seguimiento y contacto del creyente
6 Invitación selectiva a eventos
7 Campaña de actualización de datos
8 Notificación de eventos e incidencias al líder
9 Encuesta de valoración al líder
10 Registro e invitación a encuentros
11 Publicación de resumen de encuentro
12 Encuesta de valoración del encuentro
13 Cursos/Talleres sugeridos
14 Reserva en línea de curso
15 Publicación de materiales, audios y videos
16 Encuesta de valoración del curso
17 Registro y geolocalización de células(grupos
de familia)
18 Registro de asistencia a la célula
19 Publicación de resumen del tema central de la
célula
20 Encuesta de valoración del evento
21 Reporte de desarrollo del creyente
22 Reporte de crecimiento de grupo del líder
23 Reporte de crecimiento general del líder
24 Reporte de conversiones por evento
25 Reporte de resultados de encuesta al líder
26 Reporte de resultados de encuesta al encuentro
27 Reporte de resultados de encuesta del
curso/talleres
28 Reporte de resultados de encuesta de un grupo
29 Diseño adaptable (responsive) para móviles
Solución Propuesta
Impacto
0
Administrador Fiel
No impacta:
Softlife
Impacto en el Negocio
Impacto en el Negocio
0-2
Leyenda
2
2
2
2
2
2
2
2
2
2
1
2
2
2
2
2
2
0
2
2
1
1
2
0
0
1
0
0
0
0
1
0
2
0
2
2
1
0
2
1
1
1
1
1
0
1
1
1
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
0
2
0
1
2
2
2
2
2
2
2
2
2
2
1
1
1
1
2
2
1
1
0
1
0
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
2
Puntaje:
1
2
1
1
1
1
2
2
2
28
27
58
Tabla 7: Análisis comparativo de otras soluciones (Fuente propia)
32
2.4.3
EVALUACIÓN DE LA MEJOR SOLUCIÓN
Como resultado de la evaluación realizada a los softwares eclesiásticos, ofrecen mayores
funcionalidades básicas de los miembros y orientada a la gestión administrativa, cuando
realmente se busca un sistema que esté orientado a mejorar la experiencia de las personas
que son los invitados y creyentes que llegan por primera vez y un mejor seguimiento al líder,
como por ejemplo:
Integración con redes sociales: Al momento del ingreso de datos de los nuevos creyentes
podremos ingresarlos a través del Facebook y el Gmail.
Seguimiento y contacto del creyente: No solamente se trata de llamar por teléfono sino
visitar al invitado, muchas iglesias no visitan, entonces el sistema ofrecerá programación de
llamadas y visitas, notificaciones al líder para que llame y visite, que le deje mensajes a su
correo electrónico y a su Facebook.
El sistema no dejará de notificar hasta que le líder no haya llevado al invitado a un grupo
familiar y si no lo lleva se le asignará a otro líder.
Invitación selectiva a eventos: De acuerdo a la necesidad de las personas se les invitará a
un evento, por ejemplo si ya asistió al grupo familiar se le invitará a un encuentro. Si ya
terminó un curso bíblico se le invitará a que asista a al siguiente curso. Si ya terminó los
discipulados se le invitará a iniciar su grupo familiar. Si ya está viniendo a la iglesia varias
semanas se le invitará a los eventos internacionales. Hay un caso de uso de sistema de
notificaciones al creyente para que este enterado de los eventos y actividades de la iglesia.
Encuesta de valoración al líder: El creyente podrá valorar al líder a través de encuestas
cuando conteste algunas preguntas y ponga sus sugerencias de si el líder fue bueno, lo visitó
a tiempo, el grupo que dirige lo hace ameno, da buenos mensajes y realmente está siendo
una buena influencia en su vida, o cualquier otro comentario o sugerencia que quiera hacer.
Hay un caso de uso de sistema para que notifique al líder no olvidar hacer su trabajo más
eficientemente.
Registro y geolocalización de Grupos de familiares: El sistema detectará los grupos
familiares que estén cerca a la casa del invitado para que pueda asistir a través de la geo
localización y el sistema le sugerirá a cuál asistir de acuerdo a su necesidad.
33
Encuesta de valoración del evento: Cuando el invitado vaya a un evento de la iglesia podrá
dejar su opinión en un formulario que el sistema le mostrará, podrá sugerir en qué mejorar,
qué le gustó y qué no, y qué influencia tuvo en su vida personal.
Reporte de desarrollo del creyente: El sistema tendrá reportes actualizados del crecimiento
y desarrollo del creyente, cuál es su nivel de curso actual, en que grupo está, si es líder o no,
cuanto tiempo tiene en la iglesia, a cuantos eventos ha ido, etc.
Reporte de crecimiento general del líder: El sistema tendrá reportes actualizados del
crecimiento y desarrollo del líder, cuál es su nivel de liderazgo, en que grupo está, cuantos
ha ganado en el mes, cuantos ha consolidado, cuanto tiempo tiene en la iglesia, a cuantos
eventos ha ido, cuantos discípulos tiene y abrirán sus grupos familiares, cuanta gente se le
quedó y no continuó, ver los radares de cómo van en su crecimiento, etc.
Cursos/Talleres sugeridos: El sistema podrá sugerir a los alumnos los cursos que deberían
llevar de acuerdo a su nivel espiritual y a sus necesidades. Para lograr esto usaremos el
algoritmo a priori que a continuación explicaremos:
La generalidad de un sistema CRM hace complicada la implementación en organizaciones
que tienen particularidades de gestión y son un poco complejas. Por ello, el software que se
propone será más adaptable a una organización cristiana. A continuación, las funciones
previstas:
2.4.4
FUNCIONES PREVISTAS
(WEB)
-
CONSOLIDACIÓN
. Registro de nuevos
. Solicitud de pedido de oración
. Integración con redes sociales
. Asignación de líder
. Seguimiento y contacto del creyente
. Notificación de eventos e incidencias al líder
. Encuesta de valoración al líder
-
ENCUENTROS
. Registro e invitación a encuentros
. Publicación de resumen de encuentro
. Encuesta de valoración
-
ESCUELA DE LÍDERES
. Cursos/Talleres sugeridos en base a perfil
. Reserva en línea de curso
34
. Publicación de materiales, audios y videos
. Encuesta de valoración del curso
-
GRUPOS
. Registro y geo localización de Grupos familiares
. Registro de asistencia a la Grupo familiar
. Publicación de resumen del tema central de la Grupo familiar
. Encuesta de valoración
-
REPORTES ESTADÍSTICOS
. Reporte de desarrollo del miembro
. Reporte de crecimiento de grupo del líder
. Reporte de crecimiento general del líder
. Reporte de conversiones por evento
. Reporte de resultados de encuesta al líder
. Reporte de resultados de encuesta al encuentro
. Reporte de resultados de encuesta del curso/talleres
. Reporte de resultados de encuesta del desarrollo de un grupo
(MÓVIL)
-
CONSOLIDACIÓN
. Registro de nuevos
. Actualización de datos
. Acceso a la comunidad, artículos, cursos y seminarios
. Notificación de próximos eventos de interés
. Rendir encuesta de valoración al líder
-
ENCUENTROS
. Registro a encuentros
. Consulta de programación a encuentros
. Publicación de comentarios y fotos del encuentro
. Rendir encuesta de valoración
-
ESCUELA DE LÍDERES
. Notificación de cursos/talleres sugeridos por apertura
. Consulta e inscripción de cursos y talleres hábiles
. Acceso a los materiales del curso
. Rendir encuesta de valoración
-
GRUPOS
. Actualizar información de grupos del líder
. Compartir información de grupos
. Registrar asistencia a grupo familiar
. Rendir encuesta de valoración
35
2.5
TENDENCIAS Y TECNOLOGÍAS PROPUESTAS
Aplicaciones móviles
Las aplicaciones móviles, en adelante “apps”, son pequeños programas o aplicaciones
informáticas que realizan funciones para las que han sido diseñadas: juegos, calculadoras de
todo tipo, directorios, glosarios, programas formativos, presentaciones o catálogos de
empresas, etc.
Las app’s permite a las empresas ofrecer nuevos servicios a los clientes o personal interno
con multitud de información y contenidos en formatos muy atractivos, todo a través de
teléfonos móviles o tabletas.
Las app’s o aplicaciones se pueden descargar e instalarse en cualquier dispositivo móvil de
última generación (smartphones) y permiten a sus usuarios ejecutarlos con o sin conexión a
internet.
Funcionan para diferentes sistemas operativos: iOS (Apple), Android (Google) Windows
Phone, Symbian (Nokia y otros) BlackBerry, etc. Y ahora también para Windows8 de
Microsoft Surface. (Omnigaea, 2017)
ANDROID
Android es un sistema operativo inicialmente pensado para teléfonos móviles, al igual que
iOS, Symbian y Blackberry OS. Lo que lo hace diferente es que está basado en Linux, un
núcleo de sistema operativo libre, gratuito y multiplataforma.
El sistema permite programar aplicaciones en una variación de Java llamada Dalvik. El
sistema operativo proporciona todas las interfaces necesarias para desarrollar aplicaciones
que accedan a las funciones del teléfono (como el GPS, las llamadas, la agenda, etc.) de una
forma muy sencilla en un lenguaje de programación muy conocido como es Java.
36
Figura 12: Arquitectura del Android (Fuente: Xataka Android)
En este diagrama sencillo, junto a la existencia de herramientas de programación gratuitas,
hace que una de las cosas más importantes de este sistema operativo sea la cantidad de
aplicaciones disponibles, que extienden casi sin límites la experiencia del usuario.
Reglas de Asociación
Estas son usadas para encontrar asociaciones o correlaciones entre los elementos u objetos
(conjunto de ítems) de una base de datos que puede ser de tipo transaccional, relacional o
data-warehouses. (Morales & González, 2012)
En aprendizaje automático y minería de datos, las reglas de asociación pueden descubrir
acciones en común que ocurren dentro de un conjunto de datos. (Menzies & Hu. 2003)
Las reglas de asociación tienen diferentes aplicaciones como soporte para la toma de
decisiones, diagnóstico y predicción de alarmas en telecomunicaciones, web mining,
detección de intrusos, bioinformática, análisis de información de ventas, diseño de catálogos,
37
distribución de mercancías en tiendas, segmentación de clientes en base a patrones de
compra. (Berzal, 2013)
Las reglas de asociación que mostraban las relaciones entre los datos de gran tamaño
obtenidos en los puntos de venta de los mercados, por ejemplo, se pudo encontrar la siguiente
regla:
{zanahorias, vegetales} => {pollo}
Se pudo encontrar en los datos de ventas de un supermercado que un cliente que compra
zanahorias y vegetales a la vez, es probable que también compre pollo. Esta información
puede servir mucho para la toma de decisiones en marketing como para promocionar ciertos
productos y su ubicación dentro del supermercado. (Agrawal & Imielinski, 1993)
Algoritmo A Priori
Este algoritmo permite generar reglas de asociación para detectar los conjuntos de items más
frecuentes en diferentes transacciones, a través de la creación de candidatos y de reglas de
asociación booleanas. (Agrawal & Srikant, 1994)
Está diseñado para trabajar en bases de datos que tienen transacciones como por ejemplo
conjuntos de artículos comprados por clientes o detalles sobre la cantidad de veces que se
visita un sitio web.
Cada transacción se puede ver como un conjunto de ítems. Por ejemplo, tenemos el valor de
un umbral C, el algoritmo puede identificar todos los conjuntos de ítems que son
subconjuntos de C transacciones en una base de datos. A priori usa un ítem a la vez para
ampliar subconjuntos frecuentes y a este proceso se le llama generación de candidatos.
Luego los grupos de candidatos pasan por una validación contra los datos. El algoritmo llega
a su fin cuando ya no se encuentran más ampliaciones de éxito en los conjuntos anteriores.
(Agrawal & Srikant, 1994)
A priori puede generar conjuntos de ítems candidatos de tamaño K basándose en los
conjuntos de ítems de tamaño K – 1, luego saca los candidatos que tienen un patrón que no
es frecuente. Luego el conjunto candidato tiene todos los conjuntos frecuentes de tamaño K.
Luego hace un escaneo de la base de datos para obtener los conjuntos de ítems frecuentes
entre los candidatos.
38
Figura 13: Algoritmo Apriori (Fuente: Agrawal & Srikant, 1994)
Este es el pseudocódigo del algoritmo, en una base de datos transaccional T con un umbral
de confianza € y se puede definir a T como un conjunto multiple. Ck es el conjunto candidato
del nivel k. Luego se obtienen los conjuntos candidatos en base al conjunto de ítems del
nivel precedente. Count [c] ingresa al campo de la estructura de datos del conjunto candidato
c, que inicio con cero. (Agrawal & Srikant, 1994)
Tenemos el siguiente ejemplo:
En una base de datos donde cada transacción es un conjunto de productos que fueron
comprados en grupo. En la siguiente tabla mostramos una base de datos representados por
los siguientes conjuntos:
Conjuntos
{1,2,3,4}
{1,2,4}
{1,2}
{2,3,4}
{2,3}
{3,4}
{2,4}
Tabla 8: Apriori – Ejemplo de conjunto de datos (Fuente: propia)
39
El algoritmo Apriori ayuda a obtener los conjuntos de ítems frecuentes en la base de datos.
Si se decide que un ítem es frecuente si aparece por lo menos en tres transacciones de la base
de datos entonces 3 será denominado como el umbral de confianza. Se cuenta el número de
ocurrencias, que en adelante se le llamará la confianza, de cada ítem por separado, y en un
escaneo primario se muestra los resultados siguientes:
Item
Confianza
{1}
3
{2}
6
{3}
4
{4}
5
Tabla 9: Apriori – Ejemplo de conjunto de resultados (Fuente: propia)
Todos los conjuntos de tamaño 1 su confianza es de al menos 3, porque todos son frecuentes.
Luego se generará la lista de los pares de ítems más frecuentes.
Por ejemplo, si se tiene el par {1,2} de la Tabla 1, se puede ver que los ítems 1 y 2 están
juntos en tres de los conjuntos de ítems de la base de datos, de esta manera se tiene que para
el par {1,2} su confianza es de 3.
Item
Confianza
{1,2}
3
{1,3}
1
{1,4}
2
{2,3}
3
{2,4}
4
{3,4}
3
Tabla 10: Apriori – Ejemplo de conjunto de resultados de pares (Fuente: propia)
Se tiene ahora que los pares {1,2}, {2,3}, {2,4} y {3,4} su confianza es mayor que 3, porque
son frecuentes, en cambio los pares {1,3} y {1,4} no son frecuentes, por lo tanto, los
conjuntos que contengan estos pares son considerados infrecuentes. Y así sucesivamente se
van ajustando los conjuntos.
40
Luego se analiza los conjuntos de ítems frecuentes de tamaño 3 y se muestra lo siguiente:
Item
{2,3,4}
Confianza
2
Tabla 11: Apriori – Ejemplo de conjunto de resultados de triplos (Fuente: propia)
En estos resultados no hay triplos que sean frecuentes, ya que {2, 3,4} al tener 2 está por
debajo del valor de confianza que es 3.
Con estos ejemplos de agrupación de números y sus resultados se logra determinar los
conjuntos frecuentes de ítems presentes en diferentes transacciones de la base de datos.
2.6
CONCLUSIONES
En este capítulo se han determinado los objetivos del proyecto en base a las necesidades que
se encontraron en el análisis del proceso de desarrollo Ministerial que realiza la
organización. Asimismo, se ha detallado los beneficios tangibles e intangibles que el sistema
tendrá y se hace una comparación con soluciones existentes que al evaluarlas no son las
óptimas para satisfacer las necesidades de la organización.
El desarrollo de esta propuesta de solución que se propone, busca generar un valor agregado
en la buena gestión de los datos de las personas y que sea una herramienta útil para los líderes
y miembros de la organización.
Todo esto con el objetivo de llevar una mejor administración y optimización en sus procesos
de negocio y de cumplir con las funcionalidades y necesidades que la organización requiere.
41
3
CAPITULO 3: MODELADO DEL NEGOCIO
3.1
INTRODUCCIÓN
En este capítulo se tratará sobre el modelado del negocio del objeto de estudio a fin de tener
definidas y clasificadas las reglas de negocio que rigen su proceso. Mediante el uso del
lenguaje de modelamiento unificado se procederá a representar los procesos del campo de
acción más relevantes en forma de casos de uso de negocio, así como los actores,
trabajadores y entidades de negocio que participan de ella. También se detallará el flujo de
actividades de cada caso uso de negocio mediante el diagrama de actividades el cual
posteriormente nos servirá para identificar las actividades a automatizar.
3.2
REGLAS DEL NEGOCIO
NOMBRE
RN01_Registro de
datos del nuevo
DESCRIPCIÓN
CLASIFICACIÓN
Si la persona llegó a la iglesia sin que Regla de operación
nadie lo invitara el consejero tiene que – Estímulo y
registrar los datos en un A1.[1]
respuesta
Solo el líder que trajo sus invitados los Regla de operación
RN02_Consolidar a
puede consolidar, ningún otro líder podrá – Estímulo y
los nuevos creyentes
consolidar a esas personas hasta que sea respuesta
reasignado el nuevo creyente.
RN03_Reportar a los
nuevos creyentes
RN04_Asistencia al
pre encuentro
El consolidador debe reportar a las Regla de operación
personas que invitó o que le reasignaron y – Estímulo y
podrá recibir más reasignaciones.
respuesta
Solo se permite la inscripción al encuentro Regla de operación
de quienes asistieron a su clase del pre – De Flujo
encuentro.
RN05_Hacer el pago
Se debe realizar el pago para asistir al Regla de operación
del encuentro
encuentro.
RN06_Dar el código
de Grupo Familiar
– De Flujo
La persona debe tener un código de líder Regla de operación
para inscribirse al encuentro, a la escuela – Estructura
de líderes o a los eventos.
RN07_Incribirse al
Se
debe
asistir
al
encuentro
discipulado 1
inscribirse al Discipulado 1.
(relaciones)
para
Regla de operación
– Estímulo y
respuesta
42
NOMBRE
DESCRIPCIÓN
CLASIFICACIÓN
RN08_Incribirse a
La persona tiene que haber terminado el Regla de operación
los cursos de
curso
discipulado
discipulado 2, 3 o 4.
RN09_Incribirse a
La persona debe terminar el curso de Regla de operación
los cursos de
discipulado 4 para llevar un curso de – Estímulo y
seminario
seminario.
RN010_Aperturar un
Se debe cursar el curso de discipulado 4 Regla de operación
grupo familiar
para iniciar un Grupo Familiar.
RN011_Periodo de
los reportes de radar
anterior
para
inscribirse
al – Estímulo y
respuesta
respuesta
– Inferencia
Los reportes para mostrar los radares Regla de operación
deben contener información de al menos – Estímulo y
un cuatrimestre.
respuesta
Para generar un reporte de radar se Regla de operación
RN012_Data para
necesita información que cumpla con el – Estímulo y
radares
filtro, de lo contrario se mostrará un respuesta
mensaje de alerta.
0 = No definido - En proceso
1 = Asiste a un grupo familiar
2 = Seguimiento
3 = Imposible comunicación
RN013_Resultados
4 = Asiste a otro grupo
5 = No dispone de tiempo
de consolidación
6 = No quiere saber nada
7 = Pendiente
8 = No informó
9 = Asiste a otra iglesia cristiana
10 = Reasignado voluntariamente
RN014_Reporte de la Después de siete días que al consolidador
se le asignó una persona debe hacer un
primera visita
reporte de la primera visita.
RN015_Reporte de la Después de catorce días que al
consolidador se le asignó una persona
segunda visita
debe hacer un reporte de la segunda visita.
El consolidador tiene dos días para hacer
RN016_Realizar la
la llamada telefónica al nuevo creyente
llamada telefónica
para programar la primera visita después
de la fecha de asignación.
Regla de operación
– Estructura
(relaciones)
Regla de operación
– De Flujo
Regla de operación
– De Flujo
Regla de operación
– De Flujo
Tabla 12: Reglas del Negocio (Fuente: propia)
43
3.3
MODELO DE CASOS DE USO DEL NEGOCIO
3.3.1
ACTORES DEL NEGOCIO
ACTOR DEL NEGOCIO
DESCRIPCIÓN
Este actor de negocio es la persona que recibe los
beneficios de ingresar a la iglesia y poder registrar
sus datos.
AV_AN01_Invitado
(from Actores de Negoci o)
Este actor de negocio es la persona que recibe los
beneficios de retener a los creyentes en la iglesia
a través de un grupo familiar.
AV_AN02_Lider
(from Actores de Negoci o)
Este actor de negocio es la persona que recibe los
beneficios de planificar los encuentros.
AV_AN03_Planificador
(from Actores de Negocio)
Este actor de negocio es la persona que recibe las
clases de Escuela de Líderes.
AV_AN04_Alumno
(from Actores de Negocio)
Este actor de negocio es la persona que solicita la
información en cada área que sus discípulos
participan.
AV_AN05_Nuevo Lider
(from Actores de Negoci o)
Tabla 13: Actores del Negocio (Fuente: propia)
44
3.3.2
CASOS DE USO DEL NEGOCIO
ACTOR DEL NEGOCIO
DESCRIPCIÓN
Este caso de uso de negocio inicia cuando el
invitado ingresa a la iglesia. El caso de uso finaliza
cuando el invitado registra sus datos en la iglesia.
AV_CUN01_Gestionar Iniciación
(from Casos de Uso de Negocio)
AV_CUN02_Consolidar Creyentes
Este caso de uso de negocio inicia cuando el
creyente es contactado por un consolidador. El
caso de uso finaliza cuando el creyente asiste a un
grupo familiar y se reporta su asistencia.
(from Casos de Uso de Negocio)
AV_CUN03_Organizar Encuentros
Este caso de uso de negocio inicia cuando el
coordinador de encuentros planifica los
encuentros. El caso de uso termina cuando el
creyente asiste al encuentro y se genera un listado
de asistentes al encuentro.
(from Casos de Uso de Negocio)
AV_CUN04_Realizar Escuela de Lideres
Este caso de uso de negocio inicia cuando el
alumno se inscribe a un curso y asiste a sus clases.
El caso de uso termina cuando el alumno termina
su curso correspondiente.
(from Casos de Uso de Negocio)
AV_CUN05_Organizar Grupos Familiares
Este caso de uso de negocio inicia cuando el
discípulo solicita la apertura de su grupo familiar.
El caso de uso termina cuando el nuevo líder
realiza su grupo familiar.
(from Casos de Uso de Negocio)
Tabla 14: Casos de Uso del Negocio (Fuente: Propia)
45
3.3.3
DIAGRAMA DE CASOS DE USO DEL NEGOCIO
AV_CUN01_Gestionar Iniciación
AV_AN01_Invitado
(from Casos de Uso de Negocio)
(from Actores de Negocio)
AV_CUN02_Consolidar creyentes
AV_AN02_Lider
(from Casos de Uso de Negocio)
(from Actores de Negocio)
AV_CUN03_Organizar Encuentros
AV_AN03_Planificador
(from Casos de Uso de Negocio)
(from Actores de Negocio)
AV_AN04_Alumno
AV_CUN04_Realizar Escuela de Lideres
(from Actores de Negocio)
(from Casos de Uso de Negocio)
AV_AN05_Nuevo Lider
(from Actores de Negocio)
AV_CUN05_Organizar Grupos Familiares
(from Casos de Uso de Negocio)
Figura 14: Diagrama de Casos de Usos del Negocio (Fuente propia)
46
3.4
MODELO DE ANÁLISIS DEL NEGOCIO
3.4.1
TRABAJADORES DEL NEGOCIO
TRABAJADORES
AV_TR01_Guia
AV_TR02_Anfitrión
AV_TR03_Pastor
AV_TR04_Consejero
AV_TR05_Representante
AV_TR06_Consolidador
FUNCIONES
Es el encargado de llevar al invitado al local
de la iglesia. Así mismo lo acompaña a
escuchar el mensaje del pastor en el auditorio
y lo lleva al recinto de consolidación donde le
dan la bienvenida al nuevo creyente.
Es el encargado de dar la bienvenida al
invitado al ingresar al local de la iglesia y
realizar el registro de datos del invitado en la
hoja de datos llamada AMC.
Es el encargado de dar el mensaje bíblico a
todas las personas incluyendo los invitados
que asisten cada fin de semana a los
diferentes locales que tiene la organización
cristiana.
Es el encargado de dar la bienvenida al
invitado en el cuarto de consolidación y de
verificar sus datos que estén correctamente
llenados en la hoja de datos. Así mismo hace
una oración de refuerzo por el invitado y lo
despide con palabras de motivación.
Es el encargado de recoger las hojas de datos
de los invitados. Además, es el encargado de
digitar la información de estas hojas de datos
y de generar el registro de datos. También es
el encargado de registrar el grupo familiar de
un nuevo líder.
Es el encargado de consolidar al nuevo
creyente a través de una llamada y luego una
visita para invitar al nuevo creyente a asistir
al grupo familiar. Además este consolidador
puede ser líder o estar en el proceso.
47
TRABAJADORES
FUNCIONES
AV_TR07_Organizador
Es el encargado de publicitar, de hacer el
cronograma, y de registrar a los que quieren
asistir a un encuentro. Así mismo de generar
el reporte de las personas que asistieron al
encuentro.
AV_TR08_Presentador
Es el encargado de tomar asistencia a las
clases de pre encuentro y de dictar los temas
del mismo. Además genera el listado de pre
encuentro donde están las personas aptas para
asistir a un encuentro.
AV_TR09_Cajero
AV_TR10_Recepcionista
Es el encargado de registrar el pago para
asistir a un encuentro o para matricularse a un
curso bíblico. Así mismo emite un
comprobante que es un recibo por el pago.
Es el encargado de inscribir a una persona al
encuentro o a la escuela de líderes. Antes de
esto verifica si la persona cumple con los
requisitos para poder inscribirse. Además,
hace entrega del recibo de matriculado y
genera un listado de inscritos ya sea para el
encuentro o para un curso bíblico.
AV_TR11_Coordinador Encuentro
Es el encargado de verificar a cada persona
que va al encuentro en el listado de encuentro
y de actualizar ese listado. Además desarrolla
también el contenido del encuentro y genera
un reporte de los asistentes al encuentro.
AV_TR12_Profesor
Es el encargado de dictar los cursos bíblicos
a los alumnos matriculados según un listado
de matrícula y luego genera un reporte de los
alumnos aprobados del curso.
48
TRABAJADORES
FUNCIONES
AV_TR13_Asistente
Es el encargado de coordinar la asignación de
consolidadores, realizar el seguimiento y
control de los mismos y la elaboración de
reportes para el líder.
Tabla 15: Trabajadores del Negocio (Fuente: propia)
3.4.2
ENTIDADES DEL NEGOCIO
ENTIDADES
AV_EN01_Hoja de Datos
AV_EN02_Registro Hoja de Datos
FUNCIONES
Nombre: Hoja de Datos
Descripción: Es la entidad que sirve como
registro de los datos básicos del invitado.
Tipo: Documento
Estados: Invitado, Aceptado.
Atributos:
- Código de Grupo
- Fecha de Conversión
- Nombres y Apellidos del Invitado
- Teléfono
- Dirección
- Referencia de la dirección
- Tipo (AMC/A1)
- Nombres del Guía
Nombre: Registro de Hoja de Datos
Descripción: Es la entidad que sirve como
registro de todas las hojas de datos básicos
del invitado.
Tipo: Documento
Estados: Generado.
Atributos:
- Código de Grupo
- Nombres del Guía
- Tipo
- Nombres y Apellidos del Invitado
- Edad
- Dirección
- Distrito
- Teléfono
- Numero de Reunión
49
ENTIDADES
AV_EN03_Reporte de Visita
AV_EN04_Reporte de Asistencia
AV_EN05_Cronograma
AV_EN06_Hoja de Información
FUNCIONES
Nombre: Reporte de Visita
Descripción: Es la entidad que sirve como
registro de todas las incidencias de las visitas
a los nuevos creyentes.
Tipo: Documento
Estados: Reportado.
Atributos:
- Código de persona
- Código de Grupo
- Nombres y Apellidos del Invitado
- Visita 1
- Visita 2
- Código de Grupo al que asiste
- Observaciones
Nombre: Reporte de Asistencia
Descripción: Es la entidad que sirve como
registro de las asistencias de los nuevos
creyentes a los grupos familiares.
Tipo: Documento
Estados: Asiste.
Atributos:
- Código de persona
- Código de Grupo
- Nombres y Apellidos del Invitado
- Código de Grupo al que asiste
- Observaciones
Nombre: Cronograma
Descripción: Es la entidad que sirve como
registro de la programación mensual de los
encuentros que se realizan.
Tipo: Documento
Estados: Creado.
Atributos:
- Tipo de Encuentro
- Mes
Nombre: Hoja de Información
Descripción: Es la entidad que sirve como
registro de los datos que van en la publicidad
y anuncios para un encuentro.
Tipo: Documento
Estados: Vigente.
Atributos:
- Tipo de Encuentro
- Fecha
- Dirección de Inscripción
50
ENTIDADES
FUNCIONES
-
AV_EN07_Registro Participantes
AV_EN08_Listado Preencuentro
AV_EN09_Recibo de pago
Teléfonos para informes
Links de redes sociales
Nombre: Registro de Participantes
Descripción: Es la entidad que sirve como
registro de los datos de aquellas personas que
quieren asistir a un encuentro.
Tipo: Documento
Estados: Generado.
Atributos:
- Nombres y apellidos
- Sexo
- Dirección
- Distrito
- Teléfonos
- Edad
Nombre: Listado de Pre encuentro
Descripción: Es la entidad que sirve como
registro de los datos de las personas que
asistieron al curso de pree encuentro.
Tipo: Documento
Estados: Registrado.
Atributos:
- Nombres y apellidos
- Sexo
- Dirección
- Distrito
- Teléfonos
- Edad
- Documento de identidad
- Código de grupo
Nombre: Recibo de Pago
Descripción: Es la entidad que sirve como
registro de los datos de las personas que
hacen un pago para inscribirse a un encuentro
o a un curso de escuela de líderes.
Tipo: Documento
Estados: Pagado.
Atributos:
- Nombres y apellidos
- Tipo de pago
51
ENTIDADES
AV_EN10_Listado Encuentro
AV_EN11_Recibo Inscrito
AV_EN12_Listado Asistencia
FUNCIONES
Nombre: Listado Encuentro
Descripción: Es la entidad que sirve como
registro del listado de las personas que se
inscribieron a un encuentro.
Tipo: Documento
Estados: Generado.
Atributos:
- Código de grupo
- Código de Matricula
- Documento de Identidad
- Nombres y Apellidos
- Teléfono
- Edad
Nombre: Recibo Inscrito
Descripción: Es la entidad que sirve como
registro de los datos de las personas que se
inscribieron a un encuentro o aun curso
bíblico.
Tipo: Documento
Estados: Inscrito.
Atributos:
- Código de recibo
- Documento de Identidad
- Fecha de Inicio
- Tipo
- Lugar
- Nombres y Apellidos
- Código de Grupo
- Nombre de su líder
Nombre: Listado de Asistencia
Descripción: Es la entidad que sirve como
registro de los datos de las personas que
asistieron a un encuentro.
Tipo: Documento
Estados: Asistió.
Atributos:
- Código de grupo
- Código de Matricula
- Documento de Identidad
- Nombres y Apellidos
- Teléfono
- Edad
52
ENTIDADES
AV_EN13_Reporte encuentro
AV_EN14_Cursos Aprobados
AV_EN15_Listado Alumnos
AV_EN16_Reporte Aprobados
FUNCIONES
Nombre: Reporte de Encuentro
Descripción: Es la entidad que sirve como
registro de los datos de las personas que
asistieron a un encuentro.
Tipo: Documento
Estados: Asistió.
Atributos:
- Código de grupo
- Documento de Identidad
- Nombres y Apellidos
- Teléfono
- Edad
- Mes
- Tipo
Nombre: Cursos Aprobados
Descripción: Es la entidad que sirve como
registro de los cursos anteriores que
aprobaron los alumnos.
Tipo: Documento
Estados: Matriculado, anulado, reservado.
Atributos:
- Código de matriculo
- Curso
- Periodo
- Resultado
- Nota
Nombre: Listado Alumnos
Descripción: Es la entidad que sirve como
registro de los datos de las personas que se
inscribieron a un curso bíblico.
Tipo: Documento
Estados: Inscrito.
Atributos:
- Código de Horario
- Código de Matricula
- Documento de Identidad
- Nombres y Apellidos
- Código de grupo
Nombre: Reporte Aprobados
Descripción: Es la entidad que sirve como
registro de los datos de las personas que
aprobaron un curso bíblico.
Tipo: Documento
Estados: Aprobado, Desaprobado.
Atributos:
53
ENTIDADES
FUNCIONES
-
AV_EN17_Documento de Identidad
AV_EN18_Grupo Familiar
AV_EN19_Programación Llamada
Código de Horario
Código de Matricula
Documento de Identidad
Nombres y Apellidos
Código de grupo
Nombre: Documento Identidad
Descripción: Es la entidad que sirve como
registro de los datos de las personas que
quieren registrar su grupo familiar.
Tipo: Documento
Estados: Vigente.
Atributos:
- Código de Identidad
- Nombres y Apellidos
- Fecha de Nacimiento
- Código de Barras
- Fecha de caducidad
Nombre: Grupo Familiar
Descripción: Es la entidad que sirve como
registro de los datos de los grupos familiares
de los nuevos líderes.
Tipo: Documento
Estados: Activo, inactivo.
Atributos:
- Código de Grupo
- Fecha de inicio
- Fecha de Termino
- Dirección
- Distrito
- Teléfono
- Código del líder
Nombre: Programación Llamada
Descripción: Es la entidad que contiene la
programación de las llamadas a realizar a los
creyentes.
Tipo: Documento
Estados: Activo, inactivo.
Atributos:
- Código de programación
- Fecha de llamada
- Hora de llamada
- Código de consolidador
- Código de creyente
54
ENTIDADES
AV_EN20_Programación Visita
AV_EN21_Solicitud de exclusión
AV_EN22_Reporte de Consolidación
FUNCIONES
Nombre: Programación Visita
Descripción: Es la entidad que contiene la
programación de las visitas a realizar a los
creyentes.
Tipo: Documento
Estados: Activo, inactivo.
Atributos:
- Código de programación
- Fecha de llamada
- Hora de llamada
- Código de consolidador
- Código de creyente
- Estado de la visita
Nombre: Solicitud de Exclusión
Descripción: Es la entidad que representa a
la solicitud de exclusión a ser contactado.
Tipo: Documento
Estados: Activo, anulado.
Atributos:
- Código de solicitud
- Fecha de solicitud
- Motivo de exclusión
- Código de consolidador
- Código de creyente
Nombre: Reporte de Consolidación
Descripción: Es la entidad que representa al
reporte de consolidación solicitado por el
líder.
Tipo: Documento
Estados: Activo, inactivo.
Atributos:
- Código de reporte
- Fecha de generación
- Código de asistente
- Código de creyente
- Estado de consolidado
Tabla 16: Entidades del Negocio (Fuente: propia)
55
3.4.3
DIAGRAMA DE CLASES DEL NEGOCIO
Figura 15: Diagrama de Clases – Gestionar Iniciación (Fuente propia)
Figura 16: Diagrama de Clases – Consolidar Creyentes (Fuente propia)
56
AV_EN06_Hoja de Información
AV_EN07_Registro Participantes
(from Entidades de Negocio)
(from Entidades de Negocio)
AV_EN10_Listado Encuentro
AV_EN08_Listado Preencuentro
(from Entidades de Negocio)
AV_EN12_Listado Asistencia
(from Entidades de Negocio)
(from Entidades de Negocio)
AV_TR07_Organizador
(from Trabajadores de negocio)
AV_TR08_Presentador
AV_TR10_Recepcionista
AV_TR11_Coordinador Encuentro
(from Trabajadores de negocio)
(from Trabajadores de negocio)
(from Trabajadores de negocio)
AV_EN05_Cronograma
(from Entidades de Negocio)
AV_EN11_Recibo Inscrito
(from Entidades de Negocio)
AV_EN13_Reporte encuentro
(from Entidades de Negocio)
AV_EN09_Recibo de pago
AV_TR09_Cajero
(from Entidades de Negocio)
(from Trabajadores de negocio)
Figura 17: Diagrama de Clases – Organizar Encuentros (Fuente propia)
Figura 18: Diagrama de Clases – Realizar Escuela de Líderes (Fuente propia)
57
Figura 19: Diagrama de Clases – Organizar Grupos Familiares (Fuente propia)
58
3.5
REALIZACIÓN DE LOS CASOS DE USO DEL NEGOCIO
3.5.1
AV_CUN01_GESTIONAR INICIACIÓN
3.5.1.1 ESPECIFICACIÓN DE LOS CASOS DE USO DEL NEGOCIO
Actores del Negocio:
AV_AN01_Invitado.
Propósito:
Proceso que se encarga de gestionar la iniciación de nuevos fieles a la comunidad, obtener
sus datos de contacto para posteriormente realizar las visitas respectivas.
Breve Descripción:
Este caso de uso de negocio inicia cuando el invitado ingresa a la iglesia y contacta al guía.
El caso de uso finaliza cuando el invitado completa su proceso de iniciación y recibe una
constancia de registro.
Flujo básico
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
El Invitado solicita la iniciación en la entrada del local al guía.
El Invitado solicita al Anfitrión la hoja de datos para registrarse. [RN01]
El Anfitrión le ofrece la bienvenida al invitado previamente.
El Anfitrión procede a ayudar al Invitado a llenar sus datos personales en la hoja de
datos.
El Anfitrión entrega una copia de la hoja de datos al Guía y al Invitado
respectivamente.
El Guía lleva al invitado al auditorio para que atienda de la reunión.
El Pastor da inicio a la reunión y comienza brindando el mensaje a todos los
asistentes.
El Pastor invita a ponerse de pie a todos los voluntarios que desean recibir la oración.
El invitado decide ponerse de pie el Pastor brinda una oración por el creyente.
El Guía dirige al Invitado al cuarto de bienvenida.
El Consejero pide al Invitado su hoja de datos.
El Consejero verifica los datos de la hoja que estén completos.
El Consejero actualiza la hoja de datos del Invitado para ser contactado.
EL Consejero entrega la hoja de datos al Representante.
El Representante recibe y consolida la hoja de datos de todos los creyentes del cuarto
de bienvenida.
EL Representante digita cada hoja de datos en un folio.
El Representante genera el registro de hoja de datos para su posterior entrega a sus
respectivos líderes. [RN02]
Consejero brinda al Invitado una última oración de refuerzo y la despedida.
El invitado recibe la constancia de registro y se retira del local.
59
Flujos Alternos
a. Decidir ponerse de pie
1.
En el punto 9, si el Invitado no decide ponerse de pie continúa atendiendo la charla.
2.
El pastor cierra el evento con el rezo de despedida a los invitados.
3.
El invitado finalmente recibe las bendiciones y se retira del local.
b. Datos incompletos
En el punto 12, si los datos están incompletos el Consejero solicita los datos faltantes al
Invitado.
Continúa en el paso 13.
Precondiciones
No Aplica
Post condiciones
Se Generó Reporte de Hojas de Datos
60
3.5.1.2 DIAGRAMAS DE ACTIVIDADES
: AV_AN01_Invitado
: AV_TR01_Guia
Solicitar
iniciación
Solicitar registro en
Hoja de Datos
: AV_TR02_Anfitrión
: AV_TR03_Pastor
: AV_TR04_Conse je ro
: AV_TR05_Re pre se ntante
Tomar datos de
invitado
: AV_EN01_Hoja de Datos
Registrar datos en
la hoja de datos
Recibe una copia de
la hoja de datos
Entrega una copia de hoja de
datos al invitado y al guía
Recibe una copia de
la hoja de datos
Dar el mensaje
Invitar a ponerse de
pie al creyente
¿Decide ponerse de pie?
Recibir las
hojas de datos
Si
Llevar al invitado al
auditorio
Orar por el
creyente
Pedir la hoja de
datos al invitado
No
Invita a pasar al lugar
de Bienvenida
Verificar los datos
del creyente
Continuar con la charla
Digitar las
hojas de datos
¿Datos incompletos?
Recibe las
bendiciones
Brindar rezo de
despedida
Solicitar datos
faltantes
Si
No
Generar registro de
Hojas de datos
Actualizar datos en
la hoja de datos
: AV_EN01_Hoja de Datos
Recibe constancia
de registro
Entregar hoja de datos al
representante
: AV_EN02_Registro Hoja de Datos
Brindar oración de
refuerzo y despedida
Figura 20: Diagrama de Actividades – Gestionar Iniciación (Fuente propia)
Página 61
3.5.1.3 LISTA DE ACTIVIDADES A AUTOMATIZAR
•
Registrar datos en la hoja de datos
•
Verificar los datos del creyente
•
Actualizar los datos en la hoja de datos
•
Generar registro de hoja de datos
Página 62
3.5.2
AV_CUN02_CONSOLIDAR CREYENTES
3.5.2.1 ESPECIFICACIÓN DE LOS CASOS DE USO DEL NEGOCIO
Actores del Negocio:
AV_AN02_Lider.
Propósito:
Proceso que se encarga de gestionar la consolidación (afirmar en su fe al invitado que llegó
a la iglesia invitándolo a asistir a una casa para enseñarle más de la palabra de Dios) de los
creyentes mediante las llamadas y las visitas a su domicilio con el propósito que los creyentes
logren concluir su proceso de consolidación a la comunidad.
Breve Descripción:
Este caso de uso de negocio inicia cuando el líder solicita la consolidación de nuevos
creyentes y asigna un consolidador a los creyentes. El caso de uso finaliza cuando el líder
recibe el reporte de consolidación de los nuevos creyentes.
Flujo básico
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
El Líder da inicio al solicitar la consolidación de nuevos creyentes.
El Asistente recibe el registro de hoja de datos.
El Asistente asigna consolidador a nuevo creyente.
El Asistente notifica la asignación al consolidador.
El Consolidador recibe la hoja de datos del creyente para su seguimiento.
El Consolidador programa fecha de llamada.
El Consolidador hace la llamada al creyente.
El Consolidador coordina fecha y hora de visita al nuevo creyente.
El Consolidador programa fecha y hora de visita al nuevo creyente.
El Consolidador realiza la visita al nuevo creyente.
El Consolidador concreta la visita al nuevo creyente.
El Consolidador coordina con el nuevo creyente para que pueda participar en un
grupo familiar.
El Consolidador registra asistencia del nuevo creyente al grupo familiar.
El Consolidador elabora el reporte de visita del nuevo creyente. [RN03]
El Consolidador emite el reporte de visita del nuevo creyente al Asistente.
El Asistente recibe el reporte de visita del Consolidador.
El Asistente consolida el reporte de visitas de los nuevos creyentes.
El Asistente notifica a los Consolidadores hacer la visita a los grupos familiares.
El Consolidador acompaña al nuevo creyente (invitado) al grupo familiar.
El Consolidador asiste al creyente durante la reunión.
El Consolidador hace el reporte de asistencia del nuevo creyente y se lo envía al
Asistente.
El Asistente genera el reporte de Consolidación y se lo envía al líder.
El líder recibe el reporte de consolidación.
Página 63
Flujos Alternos
c. No responde la llamada
En el punto 8 si el nuevo creyente no responde la llamada el consolidador optará por realizará
una visita directa al nuevo creyente, luego se irá al punto 9.
d. No desea ser contactado
En el punto 9 si el nuevo creyente no desea ser contactado el Consolidador solicitará
exclusión de hoja de datos al Asistente.
El Asistente recibe la solicitud de exclusión de hoja de datos del Consolidador y luego se va
al punto 17.
e. No se encuentra el creyente
En el punto 11 si el nuevo creyente no se encuentra el Consolidador hace un reporte de visita
no concretada y vuelve al punto 6.
f. No acepta asistencia a Grupo Familiar
En el punto 13 si el nuevo creyente no acepta asistir a un grupo familiar el Consolidador
registra el motivo de la no asistencia y regresa al punto 8.
Precondiciones
Existe un Reporte de Hojas de Datos
Post condiciones
Se generó un reporte de visitas, asistencia y consolidación.
Página 64
3.5.2.2 DIAGRAMAS DE ACTIVIDADES
: AV_AN02_Lide r
: AV_TR13_Asis te nte
: AV_TR06_Consolidador
: AV_EN02_Registro Hoja de Datos
Solicitar consolidación de
nuevos creyentes
Recibir Registro
de Hoja de Datos
Asignar consolidador a
nuevo creyente
Recibe hoja de datos de
nuevo creyente
: AV_EN19_Programación Llamada
: AV_EN01_Hoja de Datos
Programa fecha de
llamada
Hace la llamada al
creyente
¿Responde la llamada?
Notificar asignación al
consolidador
Si
No
Coordinar fecha y
hora de visita
Optar visita
directa
¿Desea ser contactado?
Si
: AV_EN21_Solicitud de exclusión
Recibe solicitud de
exclusión
: AV_EN20_Programación Visita
Programar fecha
y hora de visita
No
Solictar exclusión de
hoja de datos
Realiza la visita al
creyente
¿Se encuentra el creyente?
Reportar visita
no concretada
No
Si
Concreta la
visita
Coordinar participación a
un grupo familiar
¿Acepta asistencia a grupo familiar?
Si
Registra participación
a grupo familiar
No
Registrar motivo de no
participación
Elabora el reporte
de visita
: AV_EN03_Reporte de Visita
Consolida reporte
de visitas
Notificar visita a
grupo familiar
Recibe el reporte
de visita
Emite reporte de
visita
Acompaña al creyente al
grupo familiar
Asiste al creyente
durante la reunión
Recibe el reporte
de consolidación
Genera reporte de
consolidación
Hacer el reporte de
asistencia del creyente
: AV_EN22_Reporte de Consolidación
: AV_EN04_Reporte de Asistencia
Figura 21: Diagrama de Actividades – Consolidar Creyentes (Fuente propia)
Página 65
3.5.2.3 LISTA DE ACTIVIDADES A AUTOMATIZAR
•
Consultar hoja de datos
•
Asignar consolidador
•
Consultar Reporte de Contacto
•
Consultar Reporte General de Consolidación
•
Consultar Creyentes Asignados
•
Programar fecha de contacto
•
Generar Reporte de Contacto
Página 66
3.5.3
AV_CUN03_ORGANIZAR ENCUENTROS
3.5.3.1 ESPECIFICACIÓN DE LOS CASOS DE USO DEL NEGOCIO
Actores del Negocio:
AV_AN03_Planificador.
Propósito:
Proceso que se encarga de gestionar las actividades que requiere la organización de cada
encuentro de los creyentes.
Breve Descripción:
Este caso de uso de negocio inicia cuando el coordinador de encuentros planifica los
encuentros. El caso de uso termina cuando el creyente asiste al encuentro y se genera un
listado de asistentes al encuentro.
Flujo básico
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
El Planificador solicita se inicien las actividades de organización de encuentro.
El Organizador recibe la hoja de información y publica el encuentro.
El Organizador elabora el cronograma del encuentro.
El Organizador asigna y genera los participantes en un solo registro.
El Organizador informa del lugar y fecha del pre encuentro.
El Presentador procede a la toma de lista en el día del pre encuentro en base a la
información recibida del formato de pre encuentro que llena el creyente.
El Presentador inicia el desarrollo de los temas del pre encuentro.
Al final, el Presentador actualizad la lista de asistencia de los participantes.
Entrega el carné de asistencia e información de pago pendiente.
El Cajero registra el pago por inscripción.
El Cajero emite el recibo de pago.
El Recepcionista verifica la asistencia al pre encuentro de la lista de pre encuentro.
[RN04], [RN05], [RN06]
El Recepcionista procede a inscribir al nuevo participante a la lista de encuentro.
El Recepcionista entrega el recibo de inscrito y las instrucciones para el encuentro al
participante.
El día del encuentro, el Coordinador del Encuentro verifica si el participante se
encuentra dentro de la lista de participantes al encuentro.
El Coordinador del Encuentro permite el acceso al bus para el viaje al encuentro.
El Coordinador del Encuentro procede con el desarrollo del encuentro.
Al final, el Coordinador del Encuentro actualiza el registro de asistencia del
encuentro.
El Coordinador realiza el cierre del encuentro y retornan a casa.
El Organizador elabora el reporte de encuentro y se la entrega al Planificador.
El Planificador recibe el reporte de encuentro.
Página 67
Flujos Alternos
c.
Validar requisito pre encuentro
En el punto 12, el Recepcionista rechaza la inscripción al encuentro por falta de participación
al pre encuentro que es un requisito., por ello retorna al paso 6.
d.
Validar registro en la lista de participantes al encuentro
En el punto 15, si la persona no se encuentra registrada en la lista, se procede a actualizar la
lista de participantes del encuentro para su regularización.
Precondiciones
Reporte de Hojas de Datos
Post condiciones
Generar Reporte de Encuentro
Página 68
3.5.3.2 DIAGRAMAS DE ACTIVIDADES
: AV_AN03_Planificador
: AV_AN01_Inv itado
: AV_TR07_Organizador
: AV_TR08_Presentador
: AV_TR09_Caj ero
: AV_TR10_Recepcionista
: AV_TR11_Coordinador Encuentro
: AV_EN06_Hoja de Información
Solicita Planificación
del Encuentro
Publicitar el
encuentro
Programar encuentros
Toma de asistencia al
Preencuentro
Verifica si está en la
lista de encuentro
Rechazar
inscripciòn
Registrar
participación
: AV_EN05_Cronograma
Verificar participción a
preencuentro
No
Desarrollo de
los temas
Está en la lista?
Participó de
preencuentro?
: AV_EN08_Listado Preencuentro
No
Si
Informar lugar y
fecha
Actualizar datos del
asistente
Actualiza lista de
asistentes por regularizar
Inscribir al
encuentro
Si
: AV_EN09_Recibo de pago
: AV_EN07_Registro Participantes
Consultar programación
de encuentros
Entrega de carné de asistencia
e información de pago
Permite el
ingreso al bus
Emitir recibo de
pago
Realizar el pago
en caja
: AV_EN10_Listado Encuentro
Registrar pago
por inscripción
Desarrollo del
encuentro
Entrega de recibo de inscrito e
instrucciones para el encuentro
Accede a contenido
del encuentro
Actualiza asistencia
a encuentro
: AV_EN11_Recibo Inscrito
Recibe reporte
de encuentro
: AV_EN13_Reporte encuentro
Publica Contenido
del encuentro
Genera reporte de
encuentro
: AV_EN12_Listado Asistencia
: AV_EN25_Contenido del encuentro
Figura 22: Diagrama de Actividades – Organizar Encuentros (Fuente propia)
Página 69
3.5.3.3 LISTA DE ACTIVIDADES A AUTOMATIZAR
•
Programar encuentros
•
Generar reporte del encuentro
•
Acceder a contenido del encuentro
•
Consultar programación de encuentros
•
Registrar reserva de participación a encuentro
•
Consultar reservas de participación a encuentros
•
Aprobar participación a encuentro
•
Publicar contenido del encuentro
•
Actualizar asistencia a encuentro
Página 70
3.5.4
AV_CUN04_REALIZAR ESCUELA DE LÍDERES
3.5.4.1 ESPECIFICACIÓN DE LOS CASOS DE USO DEL NEGOCIO
Actores del Negocio:
AV_AN04_Alumno.
Propósito:
Proceso que se encarga de gestionar la inscripción de alumnos a los cursos y el resultado
final obtenido por el alumno.
Breve Descripción:
Este caso de uso de negocio inicia cuando el alumno se inscribe a un curso y asiste a sus
clases. El caso de uso termina cuando el alumno termina su curso correspondiente.
Flujo básico
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
El Alumno inicia el proceso de inscripción a un curso. [RN06]
El Cajero registra el pago de la matrícula.
El Cajero genera el recibo de pago y lo emite al alumno.
El Recepcionista con el recibo de pago verifica los cursos aprobados por el alumno.
[RN07], [RN08], [RN09]
El Recepcionista registra la matrícula al curso solicitante.
El Recepcionista genera el recibo de matrícula
El Recepcionista genera el listado de matriculados al curso
En clase, el Profesor recibe la lista de alumnos.
El Profesor desarrolla el dictado del curso bíblico.
Al final del curso el Profesor genera el reporte de aprobados.
El Alumno recibe la calificación final al término del curso.
Flujos Alternos
e.
Validar prerrequisito a los cursos
En el punto 4, si el Alumno ha repetido de algún curso el recepcionista informa al alumno
que debe repetir el curso desaprobado, continúa en el paso 5.
Precondiciones
Reporte de Hojas de Datos
Post condiciones
Generar Reporte de Aprobados
Página 71
3.5.4.2 DIAGRAMAS DE ACTIVIDADES
: AV_AN04_Alumno
Solicita matricula a
un curso
: AV_TR09_Cajero
: AV_TR10_Recepcionista
Reg istrar pag o
de matricula
: AV_EN14_Cursos Aprobados
Generar recibo
de pag o
Verificar cursos aprobados y
recibo de pag o
: AV_TR12_Profeso r
¿Aprobó o no el
curso anterior?
No
: AV_EN09_Recibo de pag o
Repetir Curso
anterior
Si
Reg istrar
matricula
Generar recibo de
matricula
: AV_EN11_Recibo Inscrito
Generar Listado de
matriculados
Recibe Lista de alumnos
Dictar curso
biblico
: AV_EN15_Listado Alumnos
Reg istra ficha de
interes a cursos
Importar información
de cursos
Promocionar
cursos sug eridos
: AV_EN24_Cursos llevados
Figura 23: Diagrama de Actividades – Realizar Escuela de Líderes (Fuente propia)
Página 72
3.5.4.3 LISTA DE ACTIVIDADES A AUTOMATIZAR
•
Importar información de cursos llevados y disponibles
•
Promocionar cursos sugeridos
•
Consultar cursos sugeridos
•
Registrar ficha de interés a cursos
Página 73
3.5.5
AV_CUN05_ORGANIZAR GRUPOS FAMILIARES
3.5.5.1 ESPECIFICACIÓN DE LOS CASOS DE USO DEL NEGOCIO
Actores del Negocio:
AV_AN05_Nuevo Líder.
Propósito:
Proceso que se encarga de gestionar los pasos que conllevan a la creación de nuevos grupos
familiares para aquellos nuevos líderes de la iglesia.
Breve Descripción:
Este caso de uso de negocio inicia cuando el discípulo solicita la apertura de su grupo
familiar. El caso de uso termina cuando el nuevo líder realiza su grupo familiar.
Flujo básico
1.
2.
3.
4.
5.
6.
El Nuevo Líder inicia el proceso con la solicitud de registro de su grupo familiar.
El Representante solicita al Nuevo Líder su documento de identidad.
El Representante verifica si el Nuevo Líder ha culminado el curso que es
prerrequisito. [RN10]
El Representante verifica si ya ha asistido de algún grupo familiar.
El representante procede con el registro del nuevo grupo familiar.
El Representante asigna el código de grupo familiar al Nuevo Líder.
Flujos Alternos
f.
Validar curso prerrequisito
En el punto 3, si el Nuevo Líder aún no ha culminado el/los cursos indicados como
prerrequisitos el Representante informará del problema y cancela el registro.
g.
Validar asistencia a un grupo familiar
En el punto 4, si el Nuevo Líder no ha asistido de ningún grupo familiar aún, el Representante
informará del problema y cancelará el registro.
Precondiciones
Reporte de Hojas de Datos
Post condiciones
Generar nuevo Grupo Familiar
Página 74
3.5.5.2 DIAGRAMAS DE ACTIVIDADES
AN05_Nuevo Lider
Solicitar registro de
grupo familiar
TR05_Representante : AV_EN23_Contenido_Grupo_Familiar
: AV_EN17_Documento de Identidad
Solicitar documento
de identidad
Organizar reunión de
Grupo Familiar
Confirmar asistentes
: AV_EN18_Grupo Familiar
: AV_EN04_Reporte de Asistencia
Publicar contenido
de grupo familiar
Acceder a contenido
de grupo familiar
: AV_EN23_Contenido_Grupo_Familiar
Figura 24: Diagrama de Actividades – Organizar Grupos Familiares (Fuente propia)
Página 75
3.5.5.3 LISTA DE ACTIVIDADES A AUTOMATIZAR
3.6
•
Organizar reunión de grupo familiar
•
Confirmar asistentes
•
Publicar contenido de grupo familiar
•
Aceptar invitación a grupo familiar
•
Consultar reuniones de grupo familiar
•
Acceder a contenido de grupo familiar
CONCLUSIONES
En el presente capítulo se identificó los cinco principales casos de uso de negocio presentes
en el campo de acción y se procedió a identificar en cada uno de ellos a los actores,
trabajadores y entidades, así como la relación que existe entre ellos. También se elaboró el
diagrama de actividades que permite aclarar a detalle el flujo de actividades en cada uno de
los procesos en el cual participan estos artefactos mencionados anteriormente.
La herramienta utilizada, lenguaje de modelado unificado (UML), nos facilita el modelado
y la comprensión de las actividades, interacción de los artefactos y reglas del negocio
presente en los casos de uso para identificar de manera clara las actividades que son
candidatas a ser automatizadas por un sistema de software.
Página 76
4
CAPITULO 4: REQUERIMIENTOS
4.1
INTRODUCCIÓN
En este capítulo se presenta el cronograma de actividades correspondiente a esta primera
etapa del proyecto.
4.2
ESPECIFICACIÓN DE REQUERIMIENTOS DE SOFTWARE
4.2.1
REQUERIMIENTOS FUNCIONALES
• AV_RF0001_Registrar datos en la hoja de datos
El sistema debe permitir al creyente ingresar sus datos o importar sus datos desde las
redes sociales.
• AV_RF0002_Registrar pedidos de oración
El sistema debe permitir al creyente registrar sus pedidos de oración, donde expone
sus necesidades a la comunidad.
• AV_RF0003_Acceder a contenidos y noticias
El sistema debe permitir al creyente acceder a contenidos y noticias que realiza la
iglesia.
• AV_RF0004_Registrar comentarios
El sistema debe permitir al creyente registrar sus comentarios sobre qué opina sobre
los servicios que ofrece la iglesia.
• AV_RF0005_Registrar hoja de datos al Líder
El sistema debe permitir al líder el registro de nuevos creyentes y la asignación
automática como su líder
• AV_RF0006_Consultar Hoja de Datos
El sistema debe permitir al representante consultar la hoja de datos del nuevo
creyente.
• AV_RF0007_Actualizar los datos en la hoja de datos
El sistema debe permitir al representante el registro y actualización de los datos de la
hoja de datos del nuevo creyente.
• AV_RF0008_Publicar contenidos y noticias
El sistema debe permitir al gestor de contenidos publicar contenidos y noticias
referentes a las actividades que hace la iglesia.
Página 77
• AV_RF0009_Asignar líder a nuevo creyente
El sistema debe permitir al representante asignar un líder a un nuevo creyente para
que lo pueda llamar y visitar.
• AV_RF0010_Consultar creyentes asignados
El sistema debe permitir al líder consultar los nuevos creyentes que le asignaron.
• AV_RF0011_Programar fecha de contacto
El sistema debe permitir al líder programar la fecha de la llamada o de la visita al
nuevo creyente.
• AV_RF0012_Elaborar reporte de contacto
El sistema debe permitir al líder registrar su reporte de contacto con los datos y
observaciones de la llamada o visita.
• AV_RF0013_Consultar reporte de contacto
El sistema debe permitir al líder consultar el reporte de contacto hecho por el
consolidador.
• AV_RF0014_Consultar reporte general de consolidación
El sistema debe permitir al líder consultar el reporte general de consolidación de
todos los reportes de contacto con sus observaciones.
• AV_RF0015_Hacer cronograma del encuentro
El sistema debe permitir al organizador hacer el cronograma del encuentro por cada
mes del año.
• AV_RF0016_Generar reporte del encuentro
El sistema debe permitir al organizador generar el reporte del encuentro.
• AV_RF0017_Consultar programación de los encuentros
El sistema debe permitir al creyente consultar la programación de los encuentros del
mes y del año.
• AV_RF0018_Registrar reserva de participación a encuentro
El sistema debe permitir al creyente registrar una reserva de participación al
encuentro.
Página 78
• AV_RF0019_Consultar reserva de participación a encuentro
El sistema debe permitir al recepcionista consultar las reservas de participación al
encuentro.
• AV_RF0020_ Aprobar participación al encuentro
El sistema debe permitir al recepcionista aprobar la participación al encuentro.
• AV_RF0021_Actualizar asistencia a encuentro
El sistema debe permitir al coordinador del encuentro registrar la asistencia de los
creyentes al encuentro.
• AV_RF0022_Publicar contenido del encuentro
El sistema debe permitir al coordinador del encuentro publicar el contenido del
encuentro.
• AV_RF0023_Acceder a contenido del encuentro
El sistema debe permitir al creyente inscrito al encuentro poder acceder a la
información que se publica en el encuentro.
• AV_RF0024_Importar información de cursos llevados y disponibles
El sistema debe permitir al promotor académico importar información de cursos
llevados y disponibles de los sistemas heredados a través de un servicio.
• AV_RF0025_Promocionar cursos sugeridos
El sistema debe permitir al promotor académico promocionar cursos sugeridos, los
cuales se seleccionan en base al perfil de cada creyente y de su historial de
inscripciones obtenidos de los sistemas heredados utilizando reglas de relación.
• AV_RF0026_Consultar cursos sugeridos
El sistema debe permitir al creyente consultar cursos sugeridos en base a su perfil e
historial de inscripciones si los tiene.
• AV_RF0027_Registrar ficha de interés a cursos
El sistema debe permitir al creyente registrar ficha de interés a cursos.
• AV_RF0028_Consultar ficha de interés a cursos
El sistema debe permitir al recepcionista consultar registros de ficha de interés a
cursos y exportarlos en formato de hoja de cálculo.
Página 79
• AV_RF0029_Registrar nuevo grupo familiar
El sistema debe permitir al representante registrar un nuevo grupo familiar y
actualizar los datos utilizando la geolocalización de Google maps.
• AV_RF0030_Actualizar información de grupo familiar
El sistema debe permitir al líder actualizar información de su grupo familiar
utilizando la geolocalización de Google maps y compartir por las redes sociales.
• AV_RF0031_Consultar reuniones de grupo familiar
El sistema debe permitir al creyente consultar reuniones de grupo familiar ubicados
alrededor mediante la geolocalización.
• AV_RF0032_Aceptar invitación a grupo familiar
El sistema debe permitir al creyente aceptar invitación a un grupo familiar.
• AV_RF0033_Confirmar asistentes
El sistema debe permitir al líder confirmar asistentes.
• AV_RF0034_Publicar contenido de grupo familiar
El sistema debe permitir al líder publicar el contenido como fotos y videos de la
última reunión del grupo familiar realizado.
• AV_RF0035_Acceder al contenido de grupo familiar
El sistema debe permitir al creyente acceder a contenidos de fotos y videos de la
última reunión del grupo familiar realizado.
• AV_RF0036_Generar Reporte de crecimiento cuatrimestral
El sistema debe permitir al pastor generar reporte de crecimiento anual que ayude a
comparar el aumento de personas en cada etapa del proceso ministerial anualmente.
• AV_RF0037_Generar Reporte de eficiencia del líder
El sistema debe permitir al pastor generar el reporte de eficiencia del líder que
muestre la cantidad de creyentes consolidados al final de cada cuatrimestre.
• AV_RF0038_Generar Reporte de desarrollo ministerial del creyente
El sistema debe generar al pastor reporte de desarrollo ministerial del creyente que
muestre la evolución de cada creyente al final de cada semestre.
Página 80
• AV_RF0039_Generar Reporte general de satisfacción de servicios
El sistema debe generar el reporte general de satisfacción de servicios en base a las
encuestas tomadas al final de cada evento organizado.
• AV_RF0040_Notificación de seguimiento al líder
El sistema debe hacer notificaciones de seguimiento al líder a través de un Trigger
en caso que algún líder haya descuidado por más de 3 semanas el contacto a algún
creyente asignado.
• AV_RF0041_Notificación de eventos al creyente
El sistema debe hacer notificaciones del próximo encuentro o grupo familiar
organizado al creyente a través de un Trigger.
• AV_RF0042_Actualizar encuesta de satisfacción
El sistema debe permitir al representante actualizar el formato de cada tipo de
encuesta de satisfacción.
• AV_RF0043_Ejecutar encuesta de satisfacción
El sistema debe permitir al representante ejecutar encuesta de satisfacción.
• AV_RF0044_Responder encuesta de satisfacción
El sistema debe permitir al creyente responder encuestas de satisfacción.
• AV_RF0045_Consultar reporte de encuestas de satisfacción
El sistema debe permitir al representante consultar el reporte de encuestas de
satisfacción y comparar los atributos evaluados mediante un gráfico radial.
4.2.2
REQUERIMIENTOS NO FUNCIONALES
Usabilidad
• GEN_RNF001_Mensaje Emergente de Transacción
El sistema debe informar al usuario del éxito o falla de las transacciones que se
realicen en el sistema mediante un mensaje emergente.
• GEN_RNF002_Aspecto Interfaz
El aspecto de las interfaces gráficas del sistema debe facilitar su empleo a usuarios
sin entrenamiento especializado.
Página 81
• GEN_RNF003_Manejo Interfaz
El diseño de la interfaz gráfica se alineará al estándar definido en la organización
para las aplicaciones Web responsiva.
• GEN_RNF004_Acceso al sistema
Se podrá acceder al sistema vía internet desde cualquier dispositivo que cuente con
un navegador compatible.
• GEN_RNF005_Bloqueo Sistema
Si el usuario ingresa una clave secreta que esté incorrecta más de 3 veces
consecutivas, el sistema lo bloqueará impidiendo su acceso desde la IP origen por un
periodo de 30 minutos.
• GEN_RNF006_Inactividade del Sistema
El sistema controlará el tiempo de inactividad al software. Considerando como
máximo 30 minutos de inactividad.
Confiabilidad
• GEN_RNF007_Confidencialidad de contraseña
El sistema debe almacenar las contraseñas de los usuarios de manera encriptada en
la base de datos.
• GEN_RNF008_Mantenimiento del Sistema
El horario de mantenimiento programado regular se realizará los días sábados de
12:00pm a 11:00pm.
• GEN_RNF009_Tiempo promedio de reparación del sistema
El tiempo promedio de reparación de alguna funcionalidad del sistema no debe ser
mayor a 45 min.
• GEN_RNF010_Historial de Operaciones
El sistema deberá mantener almacenado el contenido histórico de todas las
operaciones (Log).
• GEN_RNF011_Protección a fallas
El sistema debe estar protegido contra fallas, por lo cual se debe contar con un
Política de gestión de backup establecida semanalmente (spool) y diarios
(Incremental).
Página 82
Rendimiento
• GEN_RNF012_Tiempo de Generación de Reportes
El tiempo de respuesta promedio del sistema para las operaciones involucradas en el
proceso de generación de Reportes debe ser de 1 minuto.
• GEN_RNF013_Tiempo de Transacciones en el Sistema
El 95% de las transacciones del sistema no deben exceder los 5 segundos en la
actividad diaria (Enfocado al proceso de consolidación).
• GEN_RNF014_Concurrencia de usuarios
El sistema debe permitir 100 usuarios concurrentes sin que se degrade el servicio.
• GEN_RNF015_Tiempo de Carga del Sistema
El tiempo máximo de carga del sistema deberá ser de 10 segundos promedio.
Soporte
• GEN_RNF016_Compatibilidad de navegadores web
La aplicación Web debe ser compatible con los navegadores Internet Explorer 9 o
superior, Chrome 50.0 en adelante, Mozilla Firefox 4.0 en adelante y Safari 9.0 en
adelante.
• GEN_RNF017_Control de Versiones
El controlador de versiones a utilizar será el Git mediante GitLab.com.
• GEN_RNF018_Motor Base datos
El sistema debe usar la base de datos MySQL 5.6 en adelante.
• GEN_RNF019_Servidor Web
El servidor web en el que se aloja la aplicación es Apache 2.4 en adelante sobre el
sistema operativo Linux Debian 8.
• GEN_RNF020_Dispositivo Cliente
El dispositivo cliente debe ser compatible con el sistema operativo Android KitKat
4.4 o superior.
Página 83
Restricciones de diseño.
• GEN_RNF021_Sistema Web
El sistema está realizado en entorno Web mobile HTML5 y una versión nativa en
Android.
• GEN_RNF022 Lenguaje de Programación
El sistema está desarrollado en lenguaje PHP5 y Java/Android.
• GEN_RNF023_Definicion de IDE
El IDE (Entorno de Desarrollo Integrado) que se emplea para el desarrollo del
sistema es Netbeans 8.2 y Android Studio 2.2.
• GEN_RNF024_Arquitectura de Programación
El sistema considerará una arquitectura lógica basada en el patrón de diseño MVC y
bajo el framework PHP Laravel 5.4.
• GEN_RNF025_Software Gestionador de Base de Datos
El software gestor de datos es MySQL será la aplicación PHPMyAdmin 4.7.
• GEN_RNF026_Formato de salida de reportes
Los reportes se exportan en los formatos: EXCEL, PDF, HTML, CSV.
• GEN_RNF027_Arquitectura de Servicios
Se utilizará una arquitectura de servicios RestFul con el estándar de seguridad JWT
(JSON Web Token).
Documentación de usuario y sistema de ayuda
• GEN_RNF028_Manual de Usuario
El sistema contará con manuales de usuario online para su entendimiento y
capacitación en el uso de la herramienta.
Interfaces de usuario
• GEN_RNF029_Formato Interfaz Gráfica Web
El Diseño web del sistema es adaptable o adaptativo (RWD: Responsive Web
Design).
Página 84
• GEN_RNF030_Colores Interfaz Gráfica Web
Las interfaces para el usuario están basadas en un diseño web en el que predominarán
los colores del portal de CCAV, según imagen de muestra.
• GEN _RNF031_Logotipo
El logotipo está siempre presente en la parte superior derecha de todas las interfaces
y reportes. El logotipo a usar es el de la imagen de muestra.
• GEN_RNF032_Organización Diseño Web
El menú de navegación del sistema debe estar siempre disponible en la parte superior
del sistema web y en móviles un menú deslizante al lado izquierdo.
Licenciamiento
• GEN_RNF033_Licencia Servidor Base de Datos
Se cuenta con una licencia gratuita ya que es Open Source para MySQL para ser
usada en el servidor de base de datos del sistema.
• GEN_RNF034_Licencia Servidor Web
Se cuenta con una licencia gratuita para Apache para ser usada en el servidor del
sistema.
Legales y de derecho de autor.
• GEN_RNF035_Patentes
El sistema se encuentra inscrito bajo las patentes legales indicadas por el estado
(INDECOPI).
• GEN _RNF036_Uso de Logotipos
Los logos usados en los formularios son de uso exclusivo y patentado por CCAV.
Página 85
4.3
MODELO DE CASOS DE USO DEL SISTEMA
4.3.1
ESPECIFICACIÓN DE LOS ACTORES DEL SISTEMA
• GEN_AS001_Usuario
Rol que generaliza a todos los actores del sistema con funcionalidades genéricas
como iniciar sesión y cambio de contraseña.
• AV_AS001_Creyente
Rol que se encarga del registro y acceso a sistema, recibe información acerca de
programación de cursos, eventos y otros servicios.
• AV_AS002_Líder
Rol que se encarga de la consolidación del creyente asignado (también llamado
consolidador en esta etapa del proceso), genera la programación de visitas, registra
el reporte correspondiente a la visita, organizar reuniones de grupo familiar y
consulta el reporte general de consolidación.
• AV_AS003_Representante
Rol que se encarga de consultar y actualizar los datos de los nuevos creyentes
registrados en su red, así como la asignación de sus líderes respectivos, además de
actualizar y consultar las encuestas de satisfacción del proceso de consolidación.
• AV_AS004_Pastor
Rol que se encarga de generar reporte de crecimiento anual, generar reporte de
eficiencia del líder, generar reporte de desarrollo ministerial del creyente, y de
generar reporte general de satisfacción de servicios.
• AV_AS005_Promotor Académico
Rol que se encarga de importar la información de los cursos llevados y disponibles,
y promocionar los cursos sugeridos.
• AV_AS006_Gestor de contenidos
Rol que se encarga de actualizar los contenidos del sistema como artículos y noticias
de la comunidad.
• AV_AS007_Organizador
Rol que se encarga de generar el cronograma de encuentros y de generar el reporte
correspondiente al finalizar dicho encuentro.
Página 86
• AV_AS008_Recepcionista
Rol que se encarga de consultar y aprobar las reservas de participación a los
encuentros programados.
• AV_AS009_Coordinador
Rol que se encarga de actualizar la asistencia a los encuentros y publicar el contenido
del encuentro.
• AV_AS010_Trigger
Rol que se encarga de enviar las notificaciones de control y seguimiento al líder,
ejecutar las encuestas de satisfacción al final de las visitas y del envío de
notificaciones de eventos próximos de interés a los creyentes.
• AV_AS011_WebService
Rol que se encarga de proveer de información de los cursos llevados y disponibles
de los sistemas académicos heredados de la organización.
• GEN_AS002_Administrador_sistema
Rol que se encarga de realizar las labores de actualizar la configuración del sistema,
perfiles e información general de los usuarios.
Página 87
4.3.2 DIAGRAMA DE ACTORES DEL SISTEMA
Figura 25: Diagrama de Actores del Sistema (Fuente propia)
Página 88
4.3.3 DIAGRAMA DE PAQUETES
Control de crecimiento y
satisfacción
Organización de
Grupos Familiares
Admisión a la
Comunidad
Promoción de
cursos y talleres
Consolidación
de Creyentes
Organización de
Encuentros
Seguridad
Figura 26: Diagrama de Paquetes del Sistema (Fuente propia)
Página 89
4.3.4 DIAGRAMA DE CASOS DE USO DEL SISTEMA POR PAQUETE
Figura 27: Diagrama de CUS por paquetes – Admisión a la Comunidad (Fuente propia)
Página 90
Figura 28: Diagrama de CUS por paquetes – Consolidar Creyentes (Fuente propia)
Página 91
Figura 29: Diagrama de CUS por paquetes – Organización de Encuentros (Fuente propia)
Página 92
Figura 30: Diagrama de CUS por paquetes – Promoción de Cursos y Talleres (Fuente propia)
Página 93
Figura 31: Diagrama de CUS por paquetes – Organización de Grupos Familiares (Fuente propia)
Página 94
Figura 32: Diagrama de CUS por paquetes – Control de Crecimiento (Fuente propia)
Página 95
Figura 33: Diagrama de CUS por paquetes – Seguridad (Fuente propia)
Página 96
4.3.5 ATRIBUTOS DE LOS CASOS DE USO DEL SISTEMA
NOMENCLATU
RA
AV_CUS001
AV_CUS002
AV_CUS003
AV_CUS004
AV_CUS005
AV_CUS006
AV_CUS007
AV_CUS008
AV_CUS009
AV_CUS010
AV_CUS011
AV_CUS012
AV_CUS013
AV_CUS014
AV_CUS015
AV_CUS016
AV_CUS017
AV_CUS018
AV_CUS019
AV_CUS020
AV_CUS021
AV_CUS022
AV_CUS023
CASO DE USO DE
SISTEMA
COMPLEJI
DAD
ESTADO
Registro de Hoja de
Primario Analizado
Datos
Registrar pedido de
Secundario Analizado
oración
Consultar contenidos
Secundario Analizado
y noticias
Registrar
Secundario Analizado
comentarios
Registrar Ganado
Consultar Hoja de
Datos
Actualizar Hoja de
Datos
Publicar contenidos
y noticias
Asignar Líder
Consultar Creyentes
Asignados
Programar fecha de
contacto
Generar Reporte de
Contacto
Consultar Reporte de
Contacto
Consultar Reporte
General de
Consolidación
Programar
encuentros
Generar reporte del
encuentro
Consultar
programación de
encuentros
Registrar reserva de
participación a
encuentro
Consultar reservas
de participación a
encuentros
Aprobar
participación a
encuentro
Actualizar asistencia
a encuentro
Publicar contenido
del encuentro
Acceder a contenido
del encuentro
DIFIC
ULTAD
PRIORI
DAD
Alta
Ciclo 0
Baja
Ciclo 1
Baja
Ciclo 2
Baja
Ciclo 2
PAQUETE
Primario
Analizado
Media
Ciclo 1
Primario
Analizado
Baja
Ciclo 1
Primario
Analizado
Media
Ciclo 1
Secundario Analizado
Baja
Ciclo 2
Primario
Analizado
Media
Ciclo 0
Admisión a la
comunidad
Admisión a la
comunidad
Admisión a la
comunidad
Admisión a la
comunidad
Admisión a la
comunidad
Admisión a la
comunidad
Admisión a la
comunidad
Admisión a la
comunidad
Consolidar Creyentes
Primario
Analizado
Baja
Ciclo 1
Consolidar Creyentes
Primario
Analizado
Alta
Ciclo 0
Consolidar Creyentes
Primario
Analizado
Media
Ciclo 0
Consolidar Creyentes
Primario
Analizado
Baja
Ciclo 1
Consolidar Creyentes
Secundario Analizado
Media
Ciclo 1
Consolidar Creyentes
Primario
Analizado
Media
Ciclo 1
Secundario Analizado
Media
Ciclo 2
Primario
Analizado
Media
Ciclo 1
Organización de
Encuentros
Primario
Analizado
Media
Ciclo 1
Organización de
Encuentros
Primario
Analizado
Media
Ciclo 1
Organización de
Encuentros
Primario
Analizado
Media
Ciclo 1
Organización de
Encuentros
Primario
Analizado
Media
Ciclo 1
Secundario Analizado
Media
Ciclo 2
Secundario Analizado
Baja
Ciclo 2
Organización de
Encuentros
Organización de
Encuentros
Organización de
Encuentros
Organización de
Encuentros
Organización de
Encuentros
Página 97
AV_CUS024
AV_CUS025
AV_CUS026
AV_CUS027
AV_CUS028
AV_CUS029
AV_CUS030
AV_CUS031
AV_CUS032
AV_CUS033
AV_CUS034
AV_CUS035
Importar
información de
cursos llevados y
disponibles
Promocionar cursos
sugeridos
Consultar cursos
sugeridos
Registrar ficha de
interés a cursos
Consultar fichas de
interés
Registrar nuevo
grupo familiar
Actualizar
información de
grupo familiar
Consultar reuniones
de grupo familiar
Aceptar invitación a
grupo familiar
Confirmar asistentes
Publicar contenido
de grupo familiar
Acceder a contenido
de grupo familiar
Primario
Analizado
Alta
Ciclo 0
Primario
Analizado
Media
Ciclo 1
Primario
Analizado
Media
Ciclo 1
Primario
Analizado
Media
Ciclo 1
Primario
Analizado
Media
Ciclo 1
Primario
Analizado
Alta
Ciclo 0
Primario
Analizado
Alta
Ciclo 0
Primario
Analizado
Media
Ciclo 1
Primario
Analizado
Media
Ciclo 1
Primario
Analizado
Media
Ciclo 1
Secundario Analizado
Baja
Ciclo 2
Secundario Analizado
Baja
Ciclo 2
AV_CUS036
Generar Reporte de
crecimiento anual
Primario
Analizado
Alta
Ciclo 1
AV_CUS037
Generar Reporte de
eficiencia del líder
Primario
Analizado
Alta
Ciclo 0
Alta
Ciclo 1
AV_CUS0038
AV_CUS0039
AV_CUS0040
AV_CUS0041
AV_CUS0042
AV_CUS0043
Generar Reporte de
desarrollo ministerial Primario Analizado
del creyente
Generar Reporte
general de
Secundario Analizado
satisfacción de
servicios
Media
Ciclo 2
Notificación de
seguimiento al líder
Primario
Analizado
Alta
Ciclo 0
Notificación de
eventos al creyente
Primario
Analizado
Alta
Ciclo 1
Actualizar encuesta
de satisfacción
Secundario Analizado
Media
Ciclo 2
Ejecutar encuesta de
satisfacción
Secundario Analizado
Media
Ciclo 2
Promoción de Cursos
y talleres
Promoción de Cursos
y talleres
Promoción de Cursos
y talleres
Promoción de Cursos
y talleres
Promoción de Cursos
y talleres
Organización de
Grupos Familiares
Organización de
Grupos Familiares
Organización de
Grupos Familiares
Organización de
Grupos Familiares
Organización de
Grupos Familiares
Organización de
Grupos Familiares
Organización de
Grupos Familiares
Control de
Crecimiento y
Satisfacción
Control de
Crecimiento y
Satisfacción
Control de
Crecimiento y
Satisfacción
Control de
Crecimiento y
Satisfacción
Control de
Crecimiento y
Satisfacción
Control de
Crecimiento y
Satisfacción
Control de
Crecimiento y
Satisfacción
Control de
Crecimiento y
Satisfacción
Página 98
AV_CUS0044
AV_CUS0045
Responder encuesta
de satisfacción
Secundario Analizado
Media
Ciclo 2
Consultar reporte de
encuestas de
satisfacción
Secundario Analizado
Media
Ciclo 2
Control de
Crecimiento y
Satisfacción
Control de
Crecimiento y
Satisfacción
Tabla 17: Atributos de los Casos del Sistema (Fuente: propia)
4.3.6 ESPECIFICACIONES ALTO NIVEL DE LOS CASOS DE USO DEL SISTEMA
Caso de uso:
Actor(es):
Propósito:
Caso de uso asociado:
Resumen:
Clasificación:
Requerimientos:
AV_CUS001_Registro de Hoja de Datos
AV_AS001_Creyente
Permitir al nuevo creyente el registro al sistema.
Ninguno
El caso de uso inicia cuando el creyente desea registrarse al sistema. El
caso de uso finaliza cuando se completa el registro de datos del creyente.
Primario
AV_RF0001_Registrar datos en la hoja de datos
Tabla 18: AV_CUS001_Registro de Hoja de Datos (Fuente: propia)
Caso de uso:
Actor(es):
Propósito:
Caso de uso asociado:
Resumen:
Clasificación:
Requerimientos:
AV_CUS002_Registrar pedido de oración
AV_AS001_Creyente
Permitir el registro de los datos del pedido de oración del nuevo creyente.
Ninguno
El caso de uso inicia cuando el creyente desea registrar un pedido de
oración. El caso de uso finaliza cuando se ha completado el registro del
pedido de oración del creyente.
Secundario
AV_RF0002_Registrar pedidos de oración
Tabla 19: AV_CUS002_Registrar pedido de oración (Fuente: propia)
Caso de uso:
Actor(es):
Propósito:
Caso de uso asociado:
Resumen
Clasificación:
Requerimientos:
AV_CUS003_Consultar contenidos y noticias
AV_AS001_Creyente
Permitir al creyente el acceso a los contenidos y noticias.
Ninguno
El caso de uso inicia cuando el creyente desea acceder a los contenidos
publicados en el sistema, tales como: videos, audios, predicas de la
semana, comentarios del pastor, noticias, etc. El caso de uso finaliza
cuando se muestra el contenido publicado.
Secundario
AV_RF0003_Acceder a contenidos y noticias
Tabla 20: AV_CUS003_Consultar contenidos y noticias (Fuente: propia)
Página 99
Caso de uso:
Actor(es):
Propósito:
Caso de uso asociado:
Resumen:
Clasificación:
Requerimientos:
AV_CUS004_Registrar comentarios
AV_AS001_Creyente
Permitir el registro de comentarios de los nuevos creyentes.
Ninguno
El caso de uso inicia cuando el creyente desea registrar alguna sugerencia
o comentario dentro del sistema. El caso de uso finaliza cuando se ha
completado el registro de la sugerencia o comentario.
Secundario
AV_RF0004_Registrar comentarios
Tabla 21: AV_CUS004_Registrar comentarios (Fuente: propia)
Caso de uso:
Actor(es):
Propósito:
Caso de uso asociado:
Resumen:
Clasificación:
Requerimientos:
AV_CUS005_Registrar Ganado
AV_AS002_Líder
Permitir al líder el registro y asignación de nuevos creyentes.
Ninguno
El caso de uso inicia cuando el líder desea registrar a un nuevo creyente en
su grupo. El caso de uso finaliza cuando se ha completado el registro de
datos del nuevo creyente.
Primario
AV_RF0005_Registrar hoja de datos al Líder
Tabla 22: AV_CUS005_Registrar Ganado (Fuente: propia)
Caso de uso:
Actor(es):
Propósito:
Caso de uso asociado:
Resumen:
Clasificación:
Requerimientos:
AV_CUS006_Consultar Hoja de Datos
AV_AS003_Representante
Permitir consultar los datos del nuevo creyente.
Ninguno
El caso de uso inicia cuando el representante desea consultar la hoja de
datos del creyente. El sistema le permite ver el contenido de los datos del
creyente para que pueda modificarlos si es necesario. El caso de uso finaliza
cuando el representante visualiza los datos del creyente.
Primario
AV_RF0006_Consultar Hoja de Datos
Tabla 23: AV_CUS006_Consultar Hoja de Datos (Fuente: propia)
Caso de uso:
Actor(es):
Propósito:
Caso de uso asociado:
Resumen:
Clasificación:
Requerimientos:
AV_CUS007_Actualizar Hoja de Datos
AV_AS003_Representante
Permitir actualizar la hoja de datos del nuevo creyente.
AV_CUS006_Consultar Hoja de Datos
El caso de uso inicia cuando el representante desea actualizar la hoja de
datos del nuevo creyente. El sistema muestra el listado de creyentes que
fueron registrados por el representante. El caso de uso finaliza cuando el
representante completa la actualización de datos del creyente.
Primario
AV_RF0007_Actualizar los datos en la hoja de datos
Tabla 24: AV_CUS007_Actualizar Hoja de Datos (Fuente: propia)
Página 100
Caso de uso:
Actor(es):
Propósito:
Caso de uso asociado:
Resumen:
Clasificación:
Requerimientos:
AV_CUS008_Publicar contenidos y noticias
AV_AS006_Gestor de contenidos
Permitir publicar contenidos y noticias de la organización.
Ninguno
El caso de uso inicia cuando el gestor de contenidos desea publicar un
contenido o noticia. El sistema muestra un listado de contenidos con las
opciones para crear, editar y eliminar una publicación. El caso de uso
finaliza cuando el usuario completa el registro de la publicación.
Secundario
AV_RF0008_Publicar contenidos y noticias
Tabla 25: AV_CUS008_Publicar contenidos y noticias (Fuente: propia)
Caso de uso:
Actor(es):
Propósito:
Caso de uso asociado:
Resumen:
Clasificación:
Requerimientos:
AV_CUS009_Asignar Líder
AV_AS003_Representante
Permitir asignar un líder a un nuevo creyente.
AV_CUS006_Consultar Hoja de Datos
El caso de uso inicia cuando el representante desea asignar un líder a cada
creyente anteriormente registrado. El sistema muestra el listado de
creyentes que fueron registrados por el representante y que no cuentan con
un líder asignado. El caso de uso finaliza cuando se completa la asignación
de un líder al nuevo creyente y se envía una notificación al líder.
Primario
AV_RF0009_Asignar líder a nuevo creyente
Tabla 26: AV_CUS009_Asignar Líder (Fuente: propia)
Caso de uso:
Actor(es):
Propósito:
Caso de uso asociado:
Resumen:
Clasificación:
Requerimientos:
AV_CUS010_Consultar Creyentes Asignados
AV_AS002_Líder
Permitir consultar los nuevos creyentes que le asignaron.
Ninguno
El caso de uso inicia cuando el líder desea consultar la lista de creyentes
asignados a su grupo. El caso de uso finaliza cuando el líder visualiza los
datos del creyente asignado.
Primario
AV_RF0010_Consultar creyentes asignados
Tabla 27: AV_CUS010_Consultar Creyentes Asignados (Fuente: propia)
Caso de uso:
Actor(es):
Propósito:
Caso de uso asociado:
Resumen:
Clasificación:
AV_CUS011_Programar fecha de contacto
AV_AS002_Líder
Permitir programar la fecha de la llamada o visita al nuevo creyente.
AV_CUS010_Consultar Creyentes Asignados
El caso de uso inicia cuando el líder desea programar la fecha para la visita
al nuevo creyente asignado. El sistema muestra la lista de creyentes
asignados que aún no han completado su consolidación. El caso de uso
finaliza cuando el líder completa la programación de contacto.
Primario
Página 101
Requerimientos:
AV_RF0011_Programar fecha de contacto
Tabla 28: AV_CUS011_Programar fecha de contacto (Fuente: propia)
Caso de uso:
Actor(es):
Propósito:
Caso de uso asociado:
Resumen:
Clasificación:
Requerimientos:
AV_CUS012_Generar Reporte de Contacto
AV_AS002_Líder
Permitir registrar el reporte de contacto de la llamada o visita.
Ninguno
El caso de uso inicia cuando el líder desea registrar el reporte de contacto
de la vista realizada. El sistema muestra la lista de contactos que presentan
reporte de vistas pendientes de registro. El caso de uso finaliza cuando el
líder completa el registro del reporte de contacto.
Primario
AV_RF0012_Elaborar reporte de contacto
Tabla 29: AV_CUS012_Generar Reporte de Contacto (Fuente: propia)
Caso de uso:
Actor(es):
Propósito:
Caso de uso asociado:
Resumen:
Clasificación:
Requerimientos:
AV_CUS013_Consultar Reporte de Contacto
AV_AS002_Líder
Permitir consultar reportes de contacto registrados por el líder.
Ninguno
El caso de uso inicia cuando el líder desea consultar un reporte de contacto
registrado. El caso de uso finaliza cuando el sistema muestra la información
del reporte de contacto en consulta.
Primario
AV_RF0013_Consultar reporte de contacto
Tabla 30: AV_CUS013_Consultar Reporte de Contacto (Fuente: propia)
Caso de uso:
Actor(es):
Propósito:
Caso de uso asociado:
Resumen:
Clasificación:
Requerimientos:
AV_CUS014_Consultar Reporte General de Consolidación
AV_AS002_Líder
Permitir consultar el reporte general de la consolidación.
Ninguno
El caso de uso inicia cuando el líder desea consultar reporte general de
consolidación para el cuatrimestre actual. El caso de uso finaliza cuando el
sistema muestra el reporte general de consolidación del cuatrimestre actual
incluyendo el porcentaje de eficiencia en la consolidación de su ganado.
Secundario
AV_RF0014_Consultar reporte general de consolidación
Tabla 31: AV_CUS014_Consultar Reporte General de Consolidación (Fuente: propia)
Caso de uso:
Actor(es):
Propósito:
Caso de uso asociado:
Resumen:
AV_CUS015_Programar encuentros
AV_AS007_Organizador
Permitir elaborar el cronograma del encuentro por cada mes del año.
Ninguno
El caso de uso inicia cuando el organizador desea actualizar la
programación de encuentros. El sistema muestra un cuadro con campos
como mes del encuentro, fecha del encuentro, tipo de encuentro y columnas
Página 102
para poder actualizar el cronograma de encuentro del año en curso. El caso
de uso finaliza cuando el organizador completa el registro del nuevo
cronograma de los encuentros.
Primario
AV_RF0015_Hacer cronograma del encuentro
Clasificación:
Requerimientos:
Tabla 32: AV_CUS015_Programar encuentros (Fuente: propia)
Caso de uso:
Actor(es):
Propósito:
Caso de uso asociado:
Resumen:
Clasificación:
Requerimientos:
AV_CUS016_Generar reporte del encuentro
AV_AS007_Organizador
Permitir generar el reporte del encuentro.
Ninguno
El caso de uso inicia cuando el organizador desea generar el reporte del
encuentro realizado. El sistema muestra un cuadro con los creyentes que
asistieron al encuentro y registrará las observaciones que se presentaron en
el encuentro. El caso de uso finaliza cuando el sistema genera el reporte del
encuentro en el sistema.
Secundario
AV_RF0016_Generar reporte del encuentro
Tabla 33: AV_CUS016_Generar reporte del encuentro (Fuente: propia)
Caso de uso:
Actor(es):
Propósito:
Caso de uso asociado:
Resumen:
Clasificación:
Requerimientos:
AV_CUS017_Consultar programación de encuentros
AV_AS001_Creyente
Permitir consultar la programación de los encuentros en el año.
Ninguno
El caso de uso inicia cuando el creyente desea consultar la programación
de los próximos encuentros. El caso de uso finaliza cuando sistema muestra
un cuadro con campos como mes, fecha y tipo de encuentro en el año en
curso.
Primario
AV_RF0017_Consultar programación de los encuentros
Tabla 34: AV_CUS017_Consultar programación de encuentros (Fuente: propia)
Caso de uso:
Actor(es):
Propósito:
Caso de uso asociado:
Resumen:
Clasificación:
Requerimientos:
AV_CUS018_Registrar reserva de participación a encuentro
AV_AS001_Creyente
Permitir registrar una reserva de participación al encuentro.
Ninguno
El caso de uso inicia cuando el creyente desea reservar la participación a
uno de los encuentros programados. El sistema muestra un cuadro con los
datos de los próximos encuentros disponibles. El caso de uso finaliza
cuando se completa el registro de la reserva de participación al encuentro.
Primario
AV_RF0018_Registrar reserva de participación a encuentro
Tabla 35: AV_CUS018_Registrar reserva de participación a encuentro (Fuente: propia)
Página 103
Caso de uso:
Actor(es):
Propósito:
Caso de uso asociado:
Resumen:
Clasificación:
Requerimientos:
AV_CUS019_Consultar reservas de participación a encuentros
AV_AS008_Recepcionista
Permitir consultar las reservas de participación al encuentro.
Ninguno
El caso de uso inicia cuando el recepcionista desea consultar las reservas
de participación a un encuentro a realizarse. El caso de uso finaliza cuando
el sistema muestra la lista de creyentes que reservaron su participación.
Primario
AV_RF0019_Consultar reserva de participación a encuentro
Tabla 36: AV_CUS019_Consultar reservas de participación a encuentros (Fuente: propia)
Caso de uso:
Actor(es):
Propósito:
Caso de uso asociado:
Resumen:
Clasificación:
Requerimientos:
AV_CUS020_Aprobar participación a encuentro
AV_AS008_Recepcionista
Permitir aprobar la participación del creyente al encuentro.
Ninguno
El caso de uso inicia cuando el recepcionista desea aprobar las reservas de
participación a encuentros. El caso de uso finaliza cuando el recepcionista
valida el pago de la inscripción y confirma su participación al encuentro.
Primario
AV_RF0020_Aprobar participación al encuentro
Tabla 37: AV_CUS020_Aprobar participación a encuentro (Fuente: propia)
Caso de uso:
Actor(es):
Propósito:
Caso de uso asociado:
Resumen:
Clasificación:
Requerimientos:
AV_CUS021_Actualizar asistencia a encuentro
AV_AS009_Coordinador
Permitir registrar la asistencia de los creyentes al encuentro.
Ninguno
El caso de uso inicia cuando el coordinador desea registrar la asistencia al
encuentro en el sistema. El sistema muestra una lista con los datos de los
creyentes inscritos al encuentro, si algún creyente no figura en la lista, pero
tiene su recibo de inscrito el usuario podrá actualizar su información en el
sistema. El caso de uso finaliza cuando se completa el registro de asistencia
de cada participante al encuentro.
Primario
AV_RF0021_Actualizar asistencia a encuentro
Tabla 38: AV_CUS021_Actualizar asistencia a encuentro (Fuente: propia)
Caso de uso:
Actor(es):
Propósito:
Caso de uso asociado:
Resumen:
AV_CUS022_Publicar contenido del encuentro
AV_AS009_Coordinador
Permitir publicar el contenido del encuentro.
Ninguno
El caso de uso inicia cuando el coordinador desea publicar un contenido
del encuentro. El sistema permite publicar los temas del encuentro, algunos
videos y audios referentes al encuentro, como testimonios, etc. El caso de
uso finaliza cuando se completa el registro y publicación del contenido del
encuentro.
Página 104
Clasificación:
Requerimientos:
Secundario
AV_RF0022_Publicar contenido del encuentro
Tabla 39: AV_CUS022_Publicar contenido del encuentro (Fuente: propia)
Caso de uso:
Actor(es):
Propósito:
Caso de uso asociado:
Resumen:
Clasificación:
Requerimientos:
AV_CUS023_Acceder a contenido del encuentro
AV_AS001_Creyente
Permitir acceder a la publicación del encuentro.
Ninguno
El caso de uso inicia cuando el creyente desea acceder al contenido del
encuentro. El caso de uso finaliza cuando el sistema muestra el contenido
del encuentro.
Secundario
AV_RF0023_Acceder a contenido del encuentro
Tabla 40: AV_CUS023_Acceder a contenido del encuentro (Fuente: propia)
Caso de uso:
Actor(es):
Propósito:
Caso de uso asociado:
Resumen:
Clasificación:
Requerimientos:
AV_CUS024_Importar información de cursos llevados y disponibles
AV_AS005_Promotor Académico, AV_AS011_WebService
Permitir importar información de inscripciones de los sistemas heredados
a través del WebServie para el recalculo de las reglas de asociación.
Ninguno
El caso de uso inicia cuando el promotor académico desea actualizar la
información de la regla de asociación de cursos. El sistema extrae de la
WebService la información de las nuevas inscripciones de los sistemas
heredados. El caso de uso finaliza cuando el sistema muestra la lista de
cursos importados y se actualiza la regla de asociación de cursos sugeridos.
Primario
AV_RF0024_Importar información de cursos llevados y disponibles
Tabla 41: AV_CUS024_Importar información de cursos llevados y disponibles (Fuente: propia)
Caso de uso:
Actor(es):
Propósito:
Caso de uso asociado:
Resumen:
Clasificación:
Requerimientos:
AV_CUS025_Promocionar cursos sugeridos
AV_AS005_Promotor Académico
Permitir promocionar cursos sugeridos a iniciar mediante el envío de
correos masivos en base a las preferencias de los creyentes.
Ninguno
El caso de uso inicia cuando el promotor académico requiere enviar la
información de nuevos cursos y talleres a abrirse. El sistema envía la
información selectivamente a los creyentes en base a la regla de asociación
de cursos. El caso de uso finaliza cuando el sistema genera los cursos
sugeridos y se lo envía la información al correo del creyente.
Primario
AV_RF0025_Promocionar cursos sugeridos
Tabla 42: AV_CUS025_Promocionar cursos sugeridos (Fuente: propia)
Página 105
Caso de uso:
Actor(es):
Propósito:
Caso de uso asociado:
Resumen:
Clasificación:
Requerimientos:
AV_CUS026_Consultar cursos sugeridos
AV_AS001_Creyente
Permitir consultar información de cursos sugeridos por el sistema.
Ninguno
El caso de uso inicia cuando el creyente desea consultar los cursos
sugeridos disponibles. El caso de uso finaliza cuando el sistema muestra la
información de los cursos sugeridos.
Primario
AV_RF0026_Consultar cursos sugeridos
Tabla 43: AV_CUS026_Consultar cursos sugeridos (Fuente: propia)
Caso de uso:
Actor(es):
Propósito:
Caso de uso asociado:
Resumen:
Clasificación:
Requerimientos:
AV_CUS027_Registrar ficha de interés a cursos
AV_AS001_Creyente
Permitir registrar ficha de interés a cursos.
Ninguno
El caso de uso inicia cuando el creyente desea registrar una ficha de interés
a un curso o taller. El sistema muestra un formulario con sus nombres y
DNI pres establecidos y los campos de sede y horario a seleccionar. El caso
de uso finaliza cuando se completa el registro de la ficha de interés.
Primario
AV_RF0027_Registrar ficha de interés a cursos
Tabla 44: AV_CUS027_Registrar ficha de interés a cursos (Fuente: propia)
Caso de uso:
Actor(es):
Propósito:
Caso de uso asociado:
Resumen:
Clasificación:
Requerimientos:
AV_CUS028_Consultar fichas de interés
AV_AS008_Recepcionista
Permitir consultar registros de ficha de interés a cursos.
Ninguno
El caso de uso inicia cuando el recepcionista desea consultar las fichas de
interés registradas en sistema. El caso de uso finaliza cuando el sistema
devuelve las fichas de interés a cursos en formato excel.
Primario
AV_RF0028_Consultar ficha de interés a cursos
Tabla 45: AV_CUS028_Consultar fichas de interés (Fuente: propia)
Caso de uso:
Actor(es):
Propósito:
Caso de uso asociado:
Resumen:
Clasificación:
Requerimientos:
AV_CUS029_Registrar nuevo grupo familiar
AV_AS003_Representante
Permitir registrar un nuevo grupo familiar y actualizar los datos utilizando
la geolocalización.
Ninguno
El caso de uso inicia cuando el representante requiere registrar un nuevo
grupo familiar para un líder nuevo. El caso de uso finaliza cuando se
completa el registro de un nuevo grupo familiar.
Primario
AV_RF0029_Registrar nuevo grupo familiar
Tabla 46: AV_CUS029_Registrar nuevo grupo familiar (Fuente: propia)
Página 106
Caso de uso:
Actor(es):
Propósito:
Caso de uso asociado:
Resumen:
Clasificación:
Requerimientos:
AV_CUS030_Actualizar información de grupo familiar
AV_AS002_Líder
Permitir actualizar información de su grupo familiar utilizando la
geolocalización y compartir por las redes sociales.
Ninguno
El caso de uso inicia cuando el líder desea actualizar la información de su
grupo familiar. El sistema muestra la lista de grupos familiares que le
corresponde con la opción para editar, mostrar en el mapa y compartir la
información de sus grupos familiares. El caso de uso finaliza se completa
la actualización de los datos del grupo familiar.
Primario
AV_RF0030_Actualizar información de grupo familiar
Tabla 47: AV_CUS030_Actualizar información de grupo familiar (Fuente: propia)
Caso de uso:
Actor(es):
Propósito:
Caso de uso asociado:
Resumen:
Clasificación:
Requerimientos:
AV_CUS031_Consultar reuniones de grupo familiar
AV_AS001_Creyente
Permitir consultar reuniones de grupo familiar ubicados alrededor
mediante la geolocalización.
Ninguno
El caso de uso inicia cuando el creyente desea consultar las reuniones de
grupo familiar. El caso de uso finaliza cuando el sistema muestra mediante
geolocalización los grupos familiares cercanos a su zona.
Primario
AV_RF0031_Consultar reuniones de grupo familiar
Tabla 48: AV_CUS031_Consultar reuniones de grupo familiar (Fuente: propia)
Caso de uso:
Actor(es):
Propósito:
Caso de uso asociado:
Resumen:
Clasificación:
Requerimientos:
AV_CUS032_Aceptar invitación a grupo familiar
AV_AS001_Creyente
Permitir aceptar invitación a un grupo familiar.
Ninguno
El caso de uso inicia cuando el creyente desea confirmar su participación
de algún grupo familiar en su zona. El sistema muestra por cada grupo
familiar la información de su dirección y horarios. El caso de uso finaliza
cuando el creyente confirma la participación al grupo en un horario.
Primario
AV_RF0032_Aceptar invitación a grupo familiar
Tabla 49: AV_CUS032_Aceptar invitación a grupo familiar (Fuente: propia)
Caso de uso:
Actor(es):
Propósito:
Caso de uso asociado:
Resumen:
AV_CUS033_Confirmar asistentes
AV_AS002_Líder
Permitir confirmar a los asistentes al grupo familiar.
Ninguno
El caso de uso inicia cuando el líder desea confirmar la asistencia de un
creyente a su grupo familiar. El caso de uso finaliza cuando se confirma la
asistencia de los creyentes al grupo familiar dentro del sistema.
Página 107
Clasificación:
Requerimientos:
Primario
AV_RF0033_Confirmar asistentes
Tabla 50: AV_CUS033_Confirmar asistentes (Fuente: propia)
Caso de uso:
Actor(es):
Propósito:
Caso de uso asociado:
Resumen:
Clasificación:
Requerimientos:
AV_CUS034_Publicar contenido de grupo familiar
AV_AS002_Líder
Permitir publicar el contenido de la última reunión del grupo familiar.
Ninguno
El caso de uso inicia cuando el líder desea publicar contenido del grupo
familiar realizado. El sistema permite publicar los temas del grupo familiar,
algunos videos y audios referentes al grupo, como testimonios, etc. El caso
de uso finaliza cuando se completa el registro y publicación del contenido.
Secundario
AV_RF0034_Publicar contenido de grupo familiar
Tabla 51: AV_CUS034_Publicar contenido de grupo familiar (Fuente: propia)
Caso de uso:
Actor(es):
Propósito:
Caso de uso asociado:
Resumen:
Clasificación:
Requerimientos:
AV_CUS035_Acceder a contenido de grupo familiar
AV_AS001_Creyente
Permitir acceder a contenidos de la última reunión del grupo familiar.
Ninguno
El caso de uso inicia cuando el creyente desea acceder a los contenidos en
del grupo familiar que ha asistido. El caso de uso finaliza el sistema muestra
el contenido de las publicaciones del grupo familiar.
Secundario
AV_RF0035_Acceder al contenido de grupo familiar
Tabla 52: AV_CUS035_Acceder a contenido de grupo familiar (Fuente: propia)
Caso de uso:
Actor(es):
Propósito:
Caso de uso asociado:
Resumen:
Clasificación:
Requerimientos:
AV_CUS036_Generar_Reporte de crecimiento anual
AV_AS004_Pastor
Permitir generar reporte de crecimiento anual.
Ninguno
El caso de uso inicia cuando el pastor desea generar el reporte de
crecimiento anual. El caso de uso finaliza cuando el sistema genera el
reporte de crecimiento anual agrupados por cuatrimestres.
Primario
AV_RF0036_Generar Reporte de crecimiento cuatrimestral
Tabla 53: AV_CUS036_Generar_Reporte de crecimiento anual (Fuente: propia)
Caso de uso:
Actor(es):
Propósito:
Caso de uso asociado:
Resumen:
AV_CUS037_Generar Reporte de eficiencia del líder
AV_AS004_Pastor
Permitir generar el reporte de eficiencia del líder.
Ninguno
El caso de uso inicia cuando el pastor desea generar el reporte de eficiencia
del líder en cada área. El caso de uso finaliza cuando el sistema genera el
reporte de eficiencia mensual del líder agrupado por cuatrimestre.
Página 108
Clasificación:
Requerimientos:
Primario
AV_RF0037_Generar Reporte de eficiencia del líder
Tabla 54: AV_CUS037_Generar Reporte de eficiencia del líder (Fuente: propia)
Caso de uso:
Actor(es):
Propósito:
Caso de uso asociado:
Resumen:
Clasificación:
Requerimientos:
AV_CUS038_Generar Reporte de desarrollo ministerial del creyente
AV_AS004_Pastor
Permitir generar el reporte de desarrollo ministerial del creyente.
Ninguno
El caso de uso inicia cuando el pastor desea generar el reporte de desarrollo
ministerial del creyente en cada área. El caso de uso finaliza cuando el
sistema genera el reporte de desarrollo ministerial del creyente.
Primario
AV_RF0038_Generar Reporte de desarrollo ministerial del creyente
Tabla 55: AV_CUS038_Generar Reporte de desarrollo ministerial del creyente (Fuente: propia)
Caso de uso:
Actor(es):
Propósito:
Caso de uso asociado:
Resumen:
Clasificación:
Requerimientos:
AV_CUS039_Generar Reporte general de satisfacción de servicios
AV_AS004_Pastor
Permitir generar el reporte general de satisfacción de servicios.
Ninguno
El caso de uso inicia cuando el pastor desea generar el reporte general de
satisfacción de servicios. El pastor podrá hacer un análisis escrito de los
gráficos que el sistema le muestra para la toma de decisiones y ver un nivel
de satisfacción del crecimiento o del decrecimiento, si se encontró un punto
débil en algún área. El caso de uso finaliza cuando el sistema genera el
reporte general de satisfacción de servicios.
Secundario
AV_RF0039_Generar Reporte general de satisfacción de servicios
Tabla 56: AV_CUS039_Generar Reporte general de satisfacción de servicios (Fuente: propia)
Caso de uso:
Actor(es):
Propósito:
Caso de uso asociado:
Resumen:
Clasificación:
Requerimientos:
AV_CUS040_Notificación de seguimiento al líder
AV_AS010_Trigger
Permitir enviar notificaciones de seguimiento al líder.
Ninguno
El caso de uso inicia cuando el usuario Trigger inicia el proceso de
Notificación al Líder al comienzo del día. Obtiene la lista de líderes que
presentan algún creyente que no ha recibido un contacto últimamente y
procede una notificación. El caso de uso finaliza cuando Trigger envía la
notificación por correo y al celular al líder.
.
Primario
AV_RF0040_Notificación de seguimiento al líder
Tabla 57: AV_CUS040_Notificación de seguimiento al líder (Fuente: propia)
Página 109
Caso de uso:
Actor(es):
Propósito:
Caso de uso asociado:
Resumen:
Clasificación:
Requerimientos:
AV_CUS041_Notificación de eventos al creyente
AV_AS010_Trigger
Permitir enviar notificaciones de próximos eventos organizados.
Ninguno
El caso de uso inicia cuando el usuario Trigger de acuerdo a su
programación del día empieza a enviar notificaciones de próximos eventos
al creyente. El caso de uso finaliza cuando el sistema envía una notificación
por correo y al celular al creyente.
Primario
AV_RF0041_Notificación de eventos al creyente
Tabla 58: AV_CUS041_Notificación de eventos al creyente (Fuente: propia)
Caso de uso:
Actor(es):
Propósito:
Caso de uso asociado:
Resumen:
Clasificación:
Requerimientos:
AV_CUS042_Actualizar encuesta de satisfacción
AV_AS003_Representante
Permitir actualizar el formato de encuesta de satisfacción.
Ninguno
El caso de uso inicia cuando el representante desea actualizar la encuesta
de satisfacción. El sistema permite crear la encuesta de Consolidación,
Encuesta de Cursos y Talleres, Encuesta de Encuentros, Encuesta de
Grupos Familiares donde el creyente podrá poner su nivel de satisfacción
de los eventos en los que haya participado. El caso de uso finaliza cuando
se completa la actualización del formato la encuesta de satisfacción.
Secundario
AV_RF0042_Actualizar encuesta de satisfacción
Tabla 59: AV_CUS042_Actualizar encuesta de satisfacción (Fuente: propia)
Caso de uso:
Actor(es):
Propósito:
Caso de uso asociado:
Resumen:
Clasificación:
Requerimientos:
AV_CUS043_Ejecutar encuesta de satisfacción
AV_AS010_Trigger
Permitir ejecutar las encuestas de satisfacción.
Ninguno
El caso de uso inicia cuando el usuario Trigger inicia la ejecución de
encuesta de satisfacción en el momento en el que se concluya la
participación a uno de los eventos registrados por el creyente. El caso de
uso finaliza cuando se completa la ejecución de las encuestas de
satisfacción mediante el envío de un correo y notificación al app.
Secundario
AV_RF0043_Ejecutar encuesta de satisfacción
Tabla 60: AV_CUS043_Ejecutar encuesta de satisfacción (Fuente: propia)
Caso de uso:
Actor(es):
Propósito:
Caso de uso asociado:
AV_CUS044_Responder encuesta de satisfacción
AV_AS001_Creyente
Permitir responder las encuestas de satisfacción.
Ninguno
Página 110
Resumen:
Clasificación:
Requerimientos:
El caso de uso inicia cuando el creyente desea responder la encuesta de
satisfacción. El caso de uso finaliza cuando se completa la respuesta a la
encuesta de satisfacción.
Secundario
AV_RF0044_Responder encuesta de satisfacción
Tabla 61: AV_CUS044_Responder encuesta de satisfacción (Fuente: propia)
Caso de uso:
Actor(es):
Propósito:
Caso de uso asociado:
Resumen:
Clasificación:
Requerimientos:
AV_CUS045_Consultar reporte de encuestas de satisfacción
AV_AS003_Representante
Permitir consultar el reporte de encuestas de satisfacción.
Ninguno
El caso de uso inicia cuando el representante desea consultar el reporte de
encuestas de satisfacción. El sistema permite consultar las encuestas de
satisfacción como la encuesta de Consolidación, Encuesta de Cursos y
Talleres, Encuesta de Encuentros y Encuesta de Grupos Familiares donde
el creyente califica el nivel de satisfacción de los eventos en las que ha
participado. El caso de uso finaliza cuando el sistema genera el reporte de
encuestas de satisfacción.
Secundario
AV_RF0045_Consultar reporte de encuestas de satisfacción
Tabla 62: AV_CUS045_Consultar reporte de encuestas de satisfacción (Fuente: propia)
Página 111
4.3.7 ESPECIFICACIONES DETALLADAS DE LOS CASOS DE USO DEL NÚCLEO
CENTRAL
4.3.7.1 AV_CUS024_Importar información de cursos llevados y disponibles
1. Actores del sistema
AV_AS005_Promotor Académico
2. Propósito
Importar la lista de nuevos cursos programados e inscripciones de los sistemas heredados de la
organización para actualizar la tabla de asociación de cursos.
3. Breve descripción
El caso de uso inicia cuando el usuario selecciona la opción “Escuela de Líderes/Importar
información de cursos”. El sistema permite al actor importar la información de programaciones
de cursos e inscripciones confirmadas de los sistemas heredados. El caso de uso finaliza cuando
el sistema muestra la regla de asociación de cursos actualizada en base a la información.
4. Flujo de eventos
a. Flujo básico
1.
El promotor académico selecciona la opción Escuela de “Líderes/Importar información de
cursos” del menú principal.
2.
El sistema muestra la interfaz “Importar Información de Cursos” con el botón “Importar
cursos e inscripciones”, seguido de una tabla con últimas importaciones ejecutadas
ordenadas por fecha de inicio de la más reciente a la más antigua y con los campos
relacionados a la importación como el código, fecha de inicio, fecha de término, total de
cursos importados, total de inscripciones importadas y estado, cuyos valores pueden ser:
•
Completo: Si el proceso de importación culminó correctamente y se actualizaron las
reglas de asociación.
•
Sin Cambios: Si el proceso de importación no recibió información de registros nuevos
desde el servicio Rest de los sistemas heredados. No se afectan las reglas de asociación.
•
Fallido: Si el proceso de importación finaliza a causa de un error inesperado en los
servicios web. No se afectan las reglas de asociación a causa del error.
3.
El promotor académico selecciona el botón “Importar cursos e inscripciones”.
Página 112
4.
El sistema realiza el proceso de importación, obtiene a través del servicio la lista de cursos
programados que aún no han sido importados al sistema y la lista de inscripciones por cada
curso programado.
5.
El sistema graba los datos de cursos programados e inscripciones.
6.
El sistema actualiza la tabla de cursos asociados para cada curso mediante el algoritmo
Apriori en base a la información de cursos e inscripciones importados.
7.
El sistema muestra al usuario el resumen de cursos e inscripciones importados indicando
para cada uno la cantidad y los botones “Ver cursos importados” y “Ver inscripciones
importadas” respectivamente. Seguido del resumen donde se visualiza la nueva regla de
asociación generada, indicando el código y nombre del curso adquirido seguido del código
y nombre de curso(s) sugeridos seguido del nivel de confianza para esta relación.
8.
El caso de uso finaliza.
b. Subflujos
Ver cursos importados
1.
En el paso 7, si el promotor académico desea visualizar la lista de cursos importados
selecciona la opción “Ver cursos importados”.
2.
El sistema muestra la tabla de cursos importados con los campos: código de programación,
código de curso, nombre de curso, término, fecha de inicio y fecha fin.
3.
El caso de uso continúa en el paso 8 del flujo básico.
Ver inscripciones importadas
1.
En el paso 7, si el promotor académico desea visualizar la lista de inscripciones importadas
selecciona la opción “Ver inscripciones importadas”.
2.
El sistema muestra la tabla de inscripciones importadas con los campos: código de
inscripción, código de creyente, código de programación, fecha de registro y estado.
3.
El caso de uso continúa en el paso 8 del flujo básico.
c. Flujos alternos
En el punto 4, si no existe registro nuevo a importar el sistema mostrará un mensaje indicando
que se encontraron registros nuevos en los servicios, y no se aplicará cambios en las reglas de
asociación actual. El caso de uso continúa en el paso 8 del flujo básico.
Página 113
5. Precondiciones
Ninguno
6. Postcondiciones
Se actualizó la lista de cursos programados.
Se actualizó la lista de inscripciones.
Se actualizó la tabla de reglas de asociación de cursos.
7. Información adicional
Ninguna
8. Prototipo
Figura 34: AV_CUS024 - Pantalla 01 de Mockup (Elaborado con Justinmind)
Página 114
Figura 35: AV_CUS024 - Pantalla 02 de Mockup (Elaborado con Justinmind)
Página 115
4.3.7.2 AV_CUS040_Notificación de seguimiento al líder
1. Actores del sistema
AV_AS010_Trigger
2. Propósito
Notificar al líder cuando algún miembro de su ganado no ha recibido ningún tipo de atención
como una llamada o visita en un periodo determinado.
3. Breve descripción
El caso de uso inicia cuando el usuario Trigger inicia el proceso de Notificación al Líder al
comienzo del día. Obtiene la lista de líderes a ser notificados que presentan algún creyente que
no ha recibido un contacto en la última semana si son nuevos, 2 meses desde su última visita o
aún no se ha registrado el informe de visita. El caso de uso finaliza cuando Trigger envía la
notificación por correo y al celular de cada líder en lista.
4. Flujo de eventos
a. Flujo básico
1.
El usuario Trigger inicia al comienzo del día (6:00am) su proceso de Notificación al Líder.
2.
El usuario Trigger comienza la consulta y selecciona a los líderes que presentan creyentes
que fueron asignados hace una semana y aún no han sido contactados por primera vez.
3.
El usuario Trigger hace una siguiente consulta y selecciona a los líderes que presentan una
programación de visita o llamada vencida y que aún no han registrado un informe.
4.
El usuario Trigger hace una siguiente consulta y selecciona a los líderes que presentan
creyentes que no se les ha programado una visita desde hace más de 2 meses.
5.
El caso de uso termina cuando el usuario Trigger no tiene más líderes pendientes de
notificación.
b. Subflujos
Ninguno
c. Flujos alternos
Creyentes recientemente asignados
1.
En el paso 2, si se encuentra resultados de creyentes recientemente asignados y que aún no
han sido contactados el usuario Trigger envía una notificación vía Firebase Cloud Message
Página 116
y otro mediante correo electrónico a sus respectivos líderes asignados. El caso de uso
continúa en el paso 3 del flujo básico.
Reporte de visita pendiente
1.
En el paso 3, si se encuentra resultados de líderes que tienen pendiente el ingreso de sus
reportes de visita el usuario Trigger envía una notificación vía Firebase Cloud Message y
otro mediante correo electrónico a cada líder. El caso de uso continúa en el paso 4 del flujo
básico.
Creyentes abandonados
1.
En el paso 4, si se encuentra resultados de creyentes que no han sido atendidos desde hace
más de dos meses el usuario Trigger envía una notificación vía Firebase Cloud Message y
otro mediante correo electrónico a sus respectivos líderes asignados. El caso de uso continúa
en el paso 5 del flujo básico.
5. Precondiciones
Líderes registrados en sistema
Cada líder debe tener asignado un creyente
6. Postcondiciones
Se registró la notificación a cada líder
7. Información adicional
Ninguna
8. Prototipo
Página 117
Figura 36: AV_CUS040 - Pantalla 01 de Mockup (Elaborado con Justinmind)
Página 118
4.3.7.3 AV_CUS001_Registro de Hoja de Datos
1. Actores del sistema
AV_AS001_Creyente
2. Propósito
Registrar un creyente a través de las redes sociales para que pueda acceder a los beneficios
ofrecidos por el proceso ministerial. (Se tiene las opciones de Ingresar con DNI, Ingresar con
Google, Ingresar con Facebook y Regístrate)
3. Breve Descripción
El Caso de uso comienza cuando el Creyente ingresa a la interfaz de Ingreso al Sistema. El
Creyente hace el ingreso de datos personales, el sistema genera la cuenta del Creyente. El caso
de uso finaliza cuando el sistema registra a un creyente.
4. Flujo de Eventos
a. Flujo Básico
1.
El Creyente ingresa a la interfaz de Ingreso al sistema.
2.
El sistema muestra en la interfaz de INGRESO AL SISTEMA los siguientes campos:
a. Campo Ingrese dni: Permite ingresar el número de documento de identidad del Creyente.
b. Campo Ingrese clave: Permite ingresar la clave que el Creyente ingresó en el sistema.
c. Botón Ingresar con DNI: Permite ingresar con las credenciales anteriores.
d. Botón Ingresar con Google: Permite ingresar con las credenciales de Google.
e. Botón Iniciar sesión con Facebook: Permite ingresar con las credenciales de Facebook.
f. Enlace Regístrate: Permite abrir un formulario de registro de datos.
3.
El Creyente ingresa el número de documento de identidad.
4.
El Creyente ingresa la clave que registró en el sistema.
5.
El Creyente selecciona la opción de “Ingresar con DNI”
6.
Si el Creyente selecciona la opción de “Ingresar con Google” - ver subflujo “Ingresar con
Google”
7.
Si el Creyente selecciona la opción de “Ingresar con Facebook” – ver subflujo “Ingresar con
Facebook”
Página 119
8.
Si el Creyente selecciona la opción “Regístrate” – ver subflujo “Regístrate”
9.
El sistema muestra la interfaz inicial de ingreso al sistema con todos los menús que contiene.
10. El caso de uso finaliza.
b. Subflujos
Ingresar con Google
1.
En el paso 6 del flujo básico, el Creyente selecciona la opción “Ingresar con Google”.
2.
El sistema muestra la interfaz ELEGIR UNA CUENTA de Google con las cuentas actuales
que están registradas.
3.
Google pedirá que se ingrese la contraseña de la cuenta y se elige la opción “Siguiente”
4.
El sistema da acceso satisfactoriamente.
5.
El caso de uso finaliza.
Ingresar con Facebook
1.
En el paso 7 del flujo básico, el Creyente selecciona la opción “Ingresar con Facebook”.
2.
El sistema muestra la interfaz con el título “Inicia sesión para utilizar tu cuenta de Facebook
con Agua Viva”.
3.
Facebook pedirá que se ingrese el correo electrónico o el teléfono, luego la contraseña y se
elige la opción “iniciar sesión”.
4.
El sistema da acceso satisfactoriamente.
5.
El caso de uso finaliza.
Regístrate
1.
En el paso 8 del flujo básico, el Creyente selecciona la opción “Regístrate”.
2.
El sistema muestra la interfaz FORMULARIO DE REGISTRO con los siguientes campos:
•
Nombres: Se debe ingresar los nombres completos del creyente.
•
Apellidos: Se debe ingresar los apellidos completos del creyente.
•
DNI: Se debe ingresar el número de documento de identidad del creyente.
•
Correo: Se debe ingresar el correo electrónico del creyente.
Página 120
•
Contraseña: Se debe ingresar la contraseña o clave para que el creyente pueda acceder al
sistema.
•
Fecha de nacimiento: Se debe ingresar la fecha de nacimiento del creyente.
•
Sexo: Se debe ingresar el sexo del creyente.
•
Teléfono Fijo: Se debe ingresar el número del teléfono fijo del creyente.
•
Teléfono Celular: Se debe ingresar el número del teléfono celular del creyente.
•
Departamento: Se debe seleccionar el departamento del creyente.
•
Provincia: Se debe seleccionar la provincia del creyente.
•
Distrito: Se debe seleccionar el distrito del creyente.
•
Dirección: Se debe ingresar la dirección del creyente.
•
Ley de protección de datos: Se debe seleccionar la opción “Acepto”.
•
Tipo de petición: Se puede ingresar el tipo de pedido de oración.
•
Detalle de petición: Se puede ingresar el detalle del pedido de oración.
•
No soy un robot: Se debe seleccionar con un check esta opción.
3.
El sistema muestra la opción de “Obtener datos de Google”
4.
El sistema muestra la opción de “Obtener datos de Facebook”
5.
El creyente ingresa los datos personales en los siguientes campos:
•
Nombres: Se debe ingresar los nombres completos del creyente.
•
Apellidos: Se debe ingresar los apellidos completos del creyente.
•
DNI: Se debe ingresar el número de documento de identidad del creyente.
•
Correo: Se debe ingresar el correo electrónico del creyente.
•
Contraseña: Se debe ingresar la contraseña o clave para que el creyente pueda acceder al
sistema.
•
Fecha de nacimiento: Se debe ingresar la fecha de nacimiento del creyente.
•
Sexo: Se debe ingresar el sexo del creyente.
•
Teléfono Fijo: Se debe ingresar el número del teléfono fijo del creyente.
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•
Teléfono Celular: Se debe ingresar el número del teléfono celular del creyente.
•
Departamento: Se debe seleccionar el departamento del creyente.
•
Provincia: Se debe seleccionar la provincia del creyente.
•
Distrito: Se debe seleccionar el distrito del creyente.
•
Dirección: Se debe ingresar la dirección del creyente.
•
Ley de protección de datos: Se debe seleccionar la opción “Acepto”.
•
Tipo de petición: Se puede ingresar el tipo de pedido de oración.
•
Detalle de petición: Se puede ingresar el detalle del pedido de oración.
•
No soy un robot: Se debe seleccionar con un check esta opción.
6.
El sistema valida los datos personales ingresados.
7.
El creyente selecciona la opción “Guardar”
8.
El sistema guarda los datos personales del creyente y genera el usuario y contraseña para el
ingreso al sistema.
9.
El caso de uso finaliza.
c. Flujo Alternativo
Validar DNI del creyente
1.
En el paso 5 del flujo básico, si el DNI ingresado es incorrecto el sistema muestra el siguiente
mensaje: “Los datos ingresados son incorrectos”
2.
Si solo se ingresa la clave, pero no se ingresa el DNI el sistema muestra el mensaje “Ingrese
usuario”
3.
El caso de uso finaliza.
Validar contraseña del creyente
1.
En el paso 5 del flujo básico, si la contraseña ingresada es incorrecta el sistema muestra el
siguiente mensaje: “Los datos ingresados son incorrectos”
2.
Si solo se ingresa el DNI, pero no se ingresa la clave el sistema muestra el mensaje “Ingrese
clave”
3.
El caso de uso finaliza.
Página 122
Validar datos de Google
1.
En el paso 6 del flujo básico, si el creyente ya registró sus datos en el sistema a través del
Google se mostrará un mensaje de bienvenida y el sistema le dará acceso.
2.
El caso de uso finaliza.
Validar datos de Facebook
1.
En el paso 7 del flujo básico, si el creyente ya registró sus datos en el sistema a través del
Facebook se mostrará un mensaje de bienvenida y el sistema le dará acceso.
2.
El caso de uso finaliza.
Validar datos obligatorios del creyente
1.
En el paso 5 del subflujo “Regístrate”, el sistema verificará que todos los campos
obligatorios se encuentren con datos, de no encontrarse llenados el sistema mostrará un
mensaje de notificación y no permitirá grabar.
2.
Si no completa los campos Nombres, Apellidos, DNI, correo, Contraseña, fecha de
nacimiento, Sexo, teléfono fijo, teléfono celular, departamento, provincia, distrito y
dirección se mostrará el mensaje “Completa este campo”. Si se ingresa el DNI con menos
dígitos saldrá el mensaje “Haz coincidir el formato”. Si al ingresar el correo electrónico no
se digita “@” se muestra el mensaje “Incluye un signo @ en la dirección de correo
electrónico”. Si ingresa una contraseña menor a 8 caracteres y que no incluya una
combinación entre minúsculas, mayúsculas y números se muestra el mensaje “Debe elegir
una contraseña fuerte”. Si se selecciona el campo “Tipo de petición” y se deja en blanco el
campo “Detalle de petición” se muestra el mensaje “Debe completar el detalle de su pedido
de oración”, y en caso viceversa se muestra el mensaje “Debe indicar el tipo de pedido de
oración”.
3.
El caso de uso retorna al paso 2 del subflujo “Regístrate”.
Validar DNI único del creyente
1.
En el paso 7 del subflujo “Regístrate”, el sistema verificará que el DNI ingresado no se
encuentre registrado a otro creyente en base de datos, caso contrario mostrará el mensaje “El
DNI indicado ya existe” y no permitirá grabar.
2.
El caso de uso retorna al paso 2 del subflujo “Regístrate”.
Página 123
Validar correo único del creyente
1.
En el paso 7 del subflujo “Regístrate”, el sistema verificará que el correo ingresado no se
encuentre registrado a otro creyente en base de datos, caso contrario mostrará el mensaje “El
correo indicado ya existe” y no permitirá grabar.
2.
El caso de uso retorna al paso 2 del subflujo “Regístrate”.
Ingreso con Google
1.
En el paso 3 del subflujo “Regístrate”, si el creyente opta por registrarse con Google el
sistema lo llevará al subflujo “Ingresar con Google”
2.
El caso de uso finaliza.
Ingreso con Facebook
1.
En el paso 4 del subflujo “Regístrate”, si el creyente opta por registrarse con Facebook el
sistema lo llevará al subflujo “Ingresar con Facebook”
2.
El caso de uso finaliza.
5. Precondiciones
No aplica
6. Postcondiciones
Se realiza el registro de los datos del creyente satisfactoriamente.
Se crea un usuario y contraseña para el acceso del creyente al sistema.
7. Información Adicional
Ninguno
8. Prototipo
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Figura 37: AV_CUS001 - Pantalla 01 de Mockup (Elaborado con Justinmind)
Figura 38: AV_CUS001 - Pantalla 02 de Mockup (Elaborado con Justinmind)
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Figura 39: AV_CUS001 - Pantalla 03 de Mockup (Elaborado con Justinmind)
Página 126
4.3.7.4 AV_CUS009_Asignar Líder
1. Actores del sistema
AV_AS003_Representante
2. Propósito
Asignar un líder responsable del proceso consolidación del nuevo creyente.
3. Breve Descripción
El caso de uso inicia cuando el representante requiere asignar un líder a cada nuevo creyente
registrado. El sistema muestra la lista de creyentes registrados pendientes de asignación de líder
y las opciones para buscar, ver hoja de datos y asignar un líder. El caso de uso finalizar cuando
se completa la asignación de un líder a un nuevo creyente y se le notifica al líder vía correo.
4. Flujo de Eventos
a.
Flujo Básico
1.
El representante ingresa a la opción “Conexión/Asignar Líder” del menú principal.
2.
El sistema presenta la interfaz Asignar Líder con un formulario para buscar creyentes con
los filtros Nombres, Departamento, Provincia y Distrito y el botón Buscar y Limpiar; además
por defecto se muestran una lista de creyentes registrados pendiente de asignación de líder
con las columnas de ID, DNI, Nombres, Distrito y Dirección y las opciones para ver la Hoja
de Datos, Ingresar nombre de líder y el botón de asignar para asignar el líder.
3.
Si el representante selecciona la opción “Ver Hoja de Datos” del creyente irá al subflujo Ver
Hoja de Datos.
4.
El representante ingresa parte del nombre del líder y el sistema mostrará una lista de
autocompletado con los nombres de líderes que coincidan con lo digitado.
5.
El representante selecciona el nombre completo del líder de la lista y se completa los
nombres del líder seleccionado en la caja de texto.
6.
El representante selecciona la opción de “Asignar”.
7.
El sistema solicita la confirmación para efectuar la asignación del líder al nuevo creyente.
8.
El representante selecciona la opción “Ok” para proceder con la confirmación.
9.
El sistema graba la asignación del líder y muestra un mensaje de confirmación “Conector
asignado y notificado”. Además, el sistema envía una notificación de asignación al líder.
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10. El caso de uso finaliza.
b. Sub Flujo
Ver Hoja de Datos
1.
En el paso 3 del flujo básico, el representante selecciona la opción Ver Hoja de Datos.
2.
El sistema muestra una ventana modal con siguientes los datos del creyente: foto de perfil,
nombres, dirección, correo electrónico, teléfono fijo, celular, fecha de nacimiento, siempre
y cuando existan cada uno de estos datos.
3.
El líder selecciona la opción “Cerrar”.
4.
El sistema cierra la ventana modal y retorna a la interfaz Asignar Líder.
c.
Flujo Alternativo
No aplica
5. Precondiciones
Deben existir registros de nuevos creyentes con datos completos y pendientes de asignación de
líderes.
6. Postcondiciones
Creyente con un Líder asignado y notificado.
7. Información Adicional
Ninguno
8. Prototipos
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Figura 40: AV_CUS009 - Pantalla 01 de Mockup (Elaborado con Justinmind)
Figura 41: AV_CUS009 - Pantalla 02 de Mockup (Elaborado con Justinmind)
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Figura 42: AV_CUS009 - Pantalla 03 de Mockup (Elaborado con Justinmind)
Figura 43: AV_CUS009 - Pantalla 04 de Mockup (Elaborado con Justinmind)
Página 130
4.3.7.5 AV_CUS011_Programar fecha de contacto
1. Actores del sistema
AV_AS002_Líder
2. Propósito
Programar las fechas de visitas de los creyentes y mantener informado al líder sobre visitas aún
no programadas, el día de visita y reportes pendientes por subir.
3. Breve Descripción
El caso de uso inicia cuando el líder selecciona la opción de menú “Mis Visitas”. El líder
selecciona las visitas pendientes de programación. El caso de uso termina cuando el líder graba
la programación de una visita.
4. Flujo de Eventos
a.
Flujo Básico
1.
El líder ingresa a la opción “Mis Visitas” del Menú lateral.
2.
El sistema presenta la interfaz MIS VISITAS. Por defecto se muestra un listado de todos los
nuevos creyentes que el líder tiene asignados para poder contactarlos.
3.
El líder puede visualizar la lista de tarjetas de información de los nuevos creyentes con los
siguientes datos:
•
Nombres y apellidos, permite visualizar los nombres y apellidos del nuevo creyente.
•
Dirección, permite visualizar la dirección del nuevo creyente.
•
Número de celular permite visualizar el número celular del nuevo creyente.
•
El estado de la vista se representa a través de tres íconos, ícono bronce de primera visita,
ícono de plata por la segunda visita y el ícono dorado si el creyente ya asistió a una célula.
4.
El líder selecciona el creyente que desea programar una visita, dándole un tap sobre una de
las tarjetas de información del creyente.
5.
El líder visualiza la información del creyente, como la foto, nombres y apellidos, dirección,
número telefónico, email y las opciones de llamar, enviar un correo y la de programar visita.
6.
Si el líder selecciona la opción “Llamar” - Ver subflujo Llamar creyente.
7.
Si el líder selecciona la opción “Escribir” - Ver subflujo Escribir a creyente.
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8.
Si el líder selecciona la opción “Programar 1era visita”, “Programar 2da visita” o
“Programar vista a célula” - Ver subflujo Programar visita.
9.
El caso de uso finaliza.
b. Sub Flujo
Llamar creyente:
1.
En el paso 5 del flujo básico, el líder selecciona la opción Llamar.
2.
El sistema inicia la llamada al número del creyente mediante la aplicación de llamadas del
dispositivo móvil.
3.
El usuario puede cancelar o terminar la llamada.
4.
El sistema retorna al punto 5 del flujo básico.
Escribir creyente:
1.
En el paso 5 del flujo básico, el líder selecciona la opción Escribir.
2.
El sistema inicia la aplicación de envío de correo.
3.
El usuario puede cancelar o enviar el mensaje al correo del creyente.
4.
El sistema retorna al punto 5 del flujo básico.
Programar visita:
1.
En el paso 5 del flujo básico, el líder selecciona la opción “Programar 1era visita”,
“Programar 2da visita” o “Programar vista a célula”.
2.
El sistema muestra el calendario semanal ubicando en la fecha actual.
3.
El líder selecciona el día y la hora en el que desea programar la visita.
4.
El sistema muestra un cuadro de confirmación.
c.
Flujo Alternativo
Programar segunda visita [RN014_Reporte de la primera visita]
1.
En el paso 5 del flujo básico, si la primera visita fue concluida se mostrará en lugar de la
opción de programar primera visita la opción de programar segunda visita.
Programar visita a célula [RN015_Reporte de la segunda visita]
Página 132
1.
En el paso 5 del flujo básico, si la segunda visita fue concluida se mostrará en lugar de la
opción de programar primera y segunda visita el botón de programar visita a célula.
Alertar programación pendiente [RN016_Realizar la llamada telefónica]
1.
En el paso 5 si el usuario no programa la vista el sistema lanzará una notificación como
recordatorio cada seis horas hasta que se programe la visita, para la primera visita debe de
programarse en una semana luego de la asignación del creyente, para la segunda visita deberá
ser programa dentro de la segunda semana luego de la asignación y la consolidación dentro
de las dos siguientes semanas.
5. Precondiciones
Tener nuevos creyentes asignados.
Tener un grupo familiar al que asiste el consolidador
Ser un líder de grupo
6. Postcondiciones
Primera visita programada.
Segunda visita programa.
Visita a célula programada.
7. Información Adicional
Ninguno
8. Prototipos
Página 133
Figura 44: AV_CUS011 - Pantalla 01 de Mockup (Elaborado con Justinmind)
Figura 45: AV_CUS011 - Pantalla 02 de Mockup (Elaborado con Justinmind)
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Figura 46: AV_CUS011 - Pantalla 03 de Mockup (Elaborado con Justinmind)
Figura 47: AV_CUS011 - Pantalla 04 de Mockup (Elaborado con Justinmind)
Página 135
4.3.7.6 AV_CUS012_Generar Reporte de Contacto
1. Actores del sistema
AV_AS002_Líder
2. Propósito
Registrar el reporte de las visitas a los nuevos creyentes para tener la evidencia y el control del
seguimiento en su proceso de consolidación.
3. Breve Descripción
El caso de uso inicia cuando el líder requiere registrar el reporte de la visita realizada. El líder
visualiza la lista de creyentes que tienen pendiente el registro del reporte de vista, con las
opciones de Mostrar, Primera visita, Segunda visita. El caso de uso termina cuando el líder
completa el registro del reporte de vista.
4. Flujo de Eventos
a.
Flujo Básico
2.
El líder ingresa a la opción “Mis Reportes” del Menú lateral.
3.
El sistema presenta la interfaz MIS REPORTES (ver imagen 02- Prototipos). El sistema
muestra un listado de todos los nuevos creyentes que el líder tiene asignados y que tienen
pendiente el registro del reporte de contacto.
4.
5.
El líder puede visualizar la información de los nuevos creyentes con los siguientes datos:
•
Nombres y apellidos, permite visualizar los nombres y apellidos del nuevo creyente.
•
Dirección, permite visualizar la dirección del nuevo creyente.
•
Número de celular permite visualizar el número celular del nuevo creyente.
El líder selecciona en el menú de tres puntos y se muestra un menú contextual con tres
opciones.
6.
Si el líder selecciona la opción “Mostrar” - ver subflujo Mostrar.
7.
Si el líder selecciona la opción “Primera visita” - Ver subflujo Primera visita.
8.
Si el líder selecciona la opción “Segunda visita” - Ver subflujo Segunda Visita.
9.
El caso de uso finaliza.
b. Sub Flujo
Página 136
Mostrar
1.
En el paso 5 del flujo básico, el líder selecciona la opción Mostrar.
2.
El sistema muestra la interfaz DATOS DEL CREYENTE, mostrando los datos registrados
del nuevo creyente.
3.
El líder podrá visualizar los siguientes datos del nuevo creyente:
•
La foto del creyente si es que lo ha subido.
•
Nombres y apellidos.
•
Correo electrónico, es el correo que se registró
•
DNI: Número de documento de identidad
•
Fecha de nacimiento
•
Teléfono fijo.
•
Teléfono celular
•
Departamento
•
Provincia
•
Distrito
•
Dirección, donde vive actualmente el nuevo creyente
4.
El caso de uso finaliza.
5.
El líder selecciona la opción “Regresar”.
6.
El sistema retorna al punto 2 del flujo básico y muestra la interfaz MIS REPORTES.
Primera visita
1.
En el paso 6 del flujo básico, el líder selecciona la opción Primera visita.
2.
El sistema muestra la interfaz REGISTRO DE REPORTES, auto cargando los datos
registrados del nuevo creyente.
3.
El sistema muestra los siguientes campos:
•
Código de grupo, es el código de líder al que se le asignó el nuevo creyente.
•
Nombres y apellidos, del nuevo creyente.
Página 137
•
Visita 1, el líder puede seleccionar alguna de las diez opciones de resultado de
consolidación.
•
Grupo al que asiste, el líder puede ingresar el código de grupo al que asiste el nuevo
creyente.
•
Observaciones, el líder puede ingresar los detalles de la visita.
4.
El líder ingresa datos en los registros y selecciona “Guardar”.
5.
El sistema valida los campos requeridos:
•
“Primera visita” se debe seleccionar un código de resultado de consolidación.
•
“Código del grupo” se debe ingresar el código de grupo al que asiste el nuevo creyente.
6.
El sistema muestra mensaje de “Reporte actualizado”.
7.
El caso de uso finaliza.
8.
El sistema retorna al punto 2 del flujo básico y muestra la interfaz MIS REPORTES.
Segunda visita
1.
En el paso 6 del flujo básico, el líder selecciona la opción Primera visita.
2.
El sistema muestra la interfaz REGISTRO DE REPORTES, auto cargando los datos
registrados del nuevo creyente.
3.
El sistema muestra los siguientes campos:
•
Código de grupo, es el código de líder al que se le asignó el nuevo creyente.
•
Nombres y apellidos, del nuevo creyente.
•
Visita 1, el líder puede seleccionar alguna de las diez opciones de resultado de
consolidación.
•
Grupo al que asiste, el líder puede ingresar el código de grupo al que asiste el nuevo
creyente.
•
Observaciones, el líder puede ingresar los detalles de la visita.
4.
El líder ingresa datos en los registros y selecciona “Guardar”.
5.
El sistema valida los campos requeridos:
•
“Primera visita” se debe seleccionar un código de resultado de consolidación.
Página 138
•
“Código del grupo” se debe ingresar el código de grupo al que asiste el nuevo creyente.
6.
El sistema muestra mensaje de “Reporte actualizado”.
7.
El caso de uso finaliza.
8.
El sistema retorna al punto 2 del flujo básico y muestra la interfaz MIS REPORTES.
c.
Flujo Alternativo
No tiene personas por visitar
1.
En el paso 2 del flujo básico, si el líder no tiene personas asignadas para hacer visitas el
sistema mostrará un mensaje “Usted no tiene personas asignadas, debe invitar personas para
el fin de semana”.
5. Precondiciones
Tener nuevos creyentes asignados.
Tener un grupo familiar al que asiste el consolidador
Ser un líder de grupo
6. Postcondiciones
Se listó los nuevos creyentes asignados.
Se reportó la primera visita.
Se reportó la segunda visita.
7. Información Adicional
Ninguno
8. Prototipos
Página 139
Figura 48: AV_CUS012 - Pantalla 01 de Mockup (Elaborado con Justinmind)
Figura 49: AV_CUS012 - Pantalla 02 de Mockup (Elaborado con Justinmind)
Página 140
4.3.7.7 AV_CUS029_Registrar nuevo grupo familiar
9. Actores del sistema
AV_AS003_Representante
10. Propósito
Realizar la actualización de los grupos familiares. (Se tiene las opciones de Buscar, Mostrar,
Editar, Eliminar y Nuevo)
11. Breve Descripción
El caso de uso permite realizar la actualización de los grupos familiares. El líder del grupo podrá
buscar, registrar, modificar y eliminar sus grupos familiares a fin de tenerlos actualizados y los
creyentes puedan acceder algún grupo.
12. Flujo de Eventos
d. Flujo Básico
2.
El líder ingresa a la opción “Grupo Familiar/Actualizar Grupo Familiar” del menú principal.
3.
El sistema presenta la interfaz ACTUALIZAR GRUPOS FAMILIARES (ver imagen001Prototipos). Por defecto se muestran todos los grupos existentes en páginas de 10 registros.
4.
El Líder selecciona y tiene la posibilidad de elegir una se las siguientes opciones:
•
Código Célula, permite hacer la búsqueda de un grupo por el código del grupo.
•
Tipo Célula, permite hacer la búsqueda de un grupo por el tipo de grupo.
•
Tipo Reunión, permite hacer la búsqueda de un grupo por el tipo de reunión.
•
Departamento, permite hacer la búsqueda de un grupo por el departamento.
•
Provincias, permite hacer la búsqueda de un grupo por la provincia.
•
Distritos, permite hacer la búsqueda de un grupo por el distrito
5.
El líder hace clic en el botón Buscar.
6.
El sistema presenta la lista de registros filtrados según la configuración realizada.
7.
Si el líder selecciona la opción “Mostrar” ver subflujo Mostrar.
8.
Si el líder selecciona la opción “Editar” – Ver subflujo Editar.
9.
Si el líder elige la opción “Eliminar” – Ver subflujo Eliminar.
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10. Si el líder elige la opción “Nuevo” – Ver subflujo Nuevo.
11. El caso de uso finaliza.
e.
Sub Flujo
Mostrar
12. En el paso 6 del flujo básico, el líder selecciona el botón Editar.
13. El sistema muestra la interfaz DETALLE DE GRUPO FAMILIAR, mostrando los datos
registrados del grupo familiar.
14. El líder podrá visualizar los siguientes datos del grupo familiar:
•
La foto del mapa con el punto de geolocalización de Google maps del grupo familiar
•
La dirección del grupo familiar donde se muestra el departamento, la provincia, el distrito
del grupo y la calle/jirón con el número que ingresó el líder.
•
Teléfono fijo de la casa donde se realiza el grupo familiar.
•
Tipo Célula que puede ser de líderes o de nuevas personas.
•
Tipo Reunión que puede ser de jóvenes o de adultos.
•
Código de Célula, se debe ingresar el código de grupo que lo identifica de los demás.
•
El horario que se realiza el grupo familiar.
•
El líder que dirige el grupo familiar.
15. El líder selecciona la opción “Regresar”.
16. El sistema retorna a la interfaz ACTUALIZAR GRUPOS FAMILIARES.
Editar
1.
En el paso 7 del flujo básico, el líder selecciona el botón Editar.
2.
El sistema muestra la interfaz CREAR GRUPO, auto cargando los datos registrados del
grupo familiar a editar.
3.
El líder completa los datos del grupo familiar:
•
Localización, se debe seleccionar en el mapa de Google Maps el punto de geolocalización
donde se encuentra el grupo familiar y mostrará las coordenadas de la ubicación.
Página 142
•
Departamento, se debe seleccionar el nombre del departamento donde se encuentra el
grupo familiar.
•
Provincia, se debe seleccionar el nombre de la provincia donde se encuentra el grupo
familiar.
•
Distrito, se debe seleccionar el nombre del distrito donde se encuentra el grupo familiar.
•
Dirección, se debe ingresar la dirección donde se encuentra el grupo familiar.
•
Teléfono Fijo, se debe ingresar el número de teléfono fijo de la casa donde se realiza el
grupo familiar.
•
Líder, se selecciona el nombre del líder de una lista que aparecerá cuando empecemos a
ingresar los nombres del líder.
•
Tipo Célula, se debe seleccionar si es de líderes o es de nuevas personas.
•
Tipo Reunión, se debe seleccionar si es de jóvenes o adultos.
•
Código de Célula, se debe ingresar el código de grupo que lo identifica de los demás.
•
Día de Reunión, se debe selecciona el día que se realiza el grupo familiar.
•
Hora de Reunión, se debe seleccionar la hora de que se realiza el grupo familiar.
4.
El líder modifica los registros y selecciona “Guardar”.
5.
El sistema valida los campos requeridos:
•
Localización, se debe seleccionar en el mapa de Google Maps el punto de geolocalización
donde se encuentra el grupo familiar y mostrará las coordenadas de la ubicación.
•
Departamento, se debe seleccionar el nombre del departamento donde se encuentra el
grupo familiar.
•
Provincia, se debe seleccionar el nombre de la provincia donde se encuentra el grupo
familiar.
•
Distrito, se debe seleccionar el nombre del distrito donde se encuentra el grupo familiar.
•
Dirección, se debe ingresar la dirección donde se encuentra el grupo familiar.
•
Teléfono Fijo, se debe ingresar el número de teléfono fijo de la casa donde se realiza el
grupo familiar.
Página 143
•
Líder, se selecciona el nombre del líder de una lista que aparecerá cuando empecemos a
ingresar los nombres del líder.
•
Tipo Célula, se debe seleccionar si es de líderes o es de nuevas personas.
•
Tipo Reunión, se debe seleccionar si es de jóvenes o adultos.
•
Código de Célula, se debe ingresar el código de grupo que lo identifica de los demás.
6.
El sistema muestra mensaje de Registro guardado correctamente.
7.
El caso de uso finaliza.
Eliminar
1.
En el paso 8 del flujo básico, el líder selecciona el botón Eliminar.
2.
El sistema muestra una ventana de confirmación con dos botones: OK y Cancel.
3.
El líder selecciona “OK”.
4.
El sistema elimina el grupo familiar satisfactoriamente.
5.
El caso de uso finaliza.
Nuevo
1.
En el paso 9 del flujo básico, el sistema muestra la interfaz CREAR GRUPO (ver imagen002
- Prototipos) que permite registrar información del grupo familiar, las coordenadas de
geolocalización y al líder que dirige el grupo.
2.
El líder ingresa los datos solicitados en la interfaz.
3.
Información del grupo familiar:
•
Localización, se debe seleccionar en el mapa de Google Maps el punto de geolocalización
donde se encuentra el grupo familiar y mostrará las coordenadas de la ubicación.
•
Departamento, se debe seleccionar el nombre del departamento donde se encuentra el
grupo familiar.
•
Provincia, se debe seleccionar el nombre de la provincia donde se encuentra el grupo
familiar.
•
Distrito, se debe seleccionar el nombre del distrito donde se encuentra el grupo familiar.
•
Dirección, se debe ingresar la dirección donde se encuentra el grupo familiar.
Página 144
•
Teléfono Fijo, se debe ingresar el número de teléfono fijo de la casa donde se realiza el
grupo familiar.
•
Líder, se selecciona el nombre del líder de una lista que aparecerá cuando empecemos a
ingresar los nombres del líder.
•
Tipo Célula, se debe seleccionar si es de líderes o es de nuevas personas.
•
Tipo Reunión, se debe seleccionar si es de jóvenes o adultos.
•
Código de Célula, se debe ingresar el código de grupo que lo identifica de los demás.
•
Día de Reunión, se debe selecciona el día que se realiza el grupo familiar.
•
Hora de Reunión, se debe seleccionar la hora de que se realiza el grupo familiar.
4.
El líder selecciona la opción “Guardar”.
5.
El sistema valida que los datos sean correctos, muestra mensaje de Registro guardado
correctamente y retorna a la interfaz ACTUALIZAR GRUPOS FAMILIARES.
6.
El sistema muestra el último registro ingresado en la lista de grupos.
f.
Flujo Alternativo
Registro de Grupo Familiar No satisfactorio
1.
En el paso 9 del flujo básico, si no se cumple con las validaciones para registrar el Grupo
Familiar. El sistema mostrará un mensaje con el título “Completa este campo” en los campos
Dirección, Teléfono Fijo, Líder, y Código de Célula. Asimismo, el sistema mostrará otro
mensaje con el título “Selecciona un elemento de la lista” en los campos Departamento,
Provincia, Distrito, Tipo Reunión. Finalmente, si no se valida el campo de “Localización”
se selecciona para ser llenado y su contorno se resalta en color rojo.
Registros no encontrados
1.
En el paso 3 del subflujo – Buscar, Si la búsqueda del filtro no tiene coincidencias. El sistema
no mostrará ningún resultado en la interfaz de “Lista de Grupos Familiares”.
13. Precondiciones
Tener líderes, departamentos, provincias, distritos, tipos de grupos y tipos de reunión registrados,
y por lo menos un grupo familiar registrado.
Página 145
14. Postcondiciones
Se listó los grupos familiares disponibles.
Se registró un nuevo grupo familiar.
Se actualizó un grupo familiar.
Se eliminó un grupo familiar.
Se visualizó el mapa de la ubicación de un grupo familiar.
15. Información Adicional
Ninguno
16. Prototipos
Figura 50: AV_CUS029 - Pantalla 01 de Mockup (Elaborado con Justinmind)
Página 146
Figura 51: AV_CUS029 - Pantalla 02 de Mockup (Elaborado con Justinmind)
Figura 52: AV_CUS029 - Pantalla 03 de Mockup (Elaborado con Justinmind)
Página 147
Figura 53: AV_CUS029 - Pantalla 04 de Mockup (Elaborado con Justinmind)
Figura 54: AV_CUS029 - Pantalla 05 de Mockup (Elaborado con Justinmind)
Página 148
Figura 55: AV_CUS029 - Pantalla 06 de Mockup (Elaborado con Justinmind)
Página 149
4.3.7.8 AV_CUS030_Actualizar información de grupo familiar
1. Actores del sistema
AV_AS002_Líder
2. Propósito
Realizar la actualización de los grupos familiares para que el creyente pueda asistir a una de ellas.
(Se tiene las opciones de Mostrar, Ver Mapa, y Editar)
3. Breve Descripción
El caso de uso permite realizar la consulta de los grupos familiares. El líder podrá buscar,
modificar y ver el mapa de sus grupos familiares a fin de tenerlos actualizados y para que los
nuevos creyentes puedan acceder algún grupo.
4. Flujo de Eventos
a.
Flujo Básico
1.
El líder ingresa a la opción “Mis Grupos” del Menú lateral.
2.
El sistema presenta la interfaz MIS GRUPOS (ver imagen 02- Prototipos). Por defecto se
muestran todos los grupos existentes que tiene el líder a su cargo.
3.
4.
El Líder puede visualizar la información de los grupos con los siguientes datos:
•
Tipo Grupo, permite visualizar el tipo de grupo (evangelística / líderes) [16]
•
Código Grupo, permite visualizar el código del grupo. [14]
•
Dirección, permite visualizar la dirección del grupo.
•
Fecha permite visualizar el día y la hora que se realiza el grupo.
El líder hace un TAB [18] en el menú de tres puntos y se muestra un menú contextual con
tres opciones.
5.
Si el líder selecciona la opción “Mostrar” ver subflujo Mostrar.
6.
Si el líder selecciona la opción “Editar” – Ver subflujo Editar.
7.
Si el líder elige la opción “Ver en Mapa” – Ver subflujo Ver en mapa.
8.
El caso de uso finaliza.
Página 150
b. Sub Flujo
Mostrar
1.
En el paso 5 del flujo básico, el líder selecciona la opción Mostrar.
2.
El sistema muestra la interfaz DETALLE DE GRUPO, mostrando los datos registrados del
grupo familiar.
3.
El líder podrá visualizar los siguientes datos del grupo familiar:
•
La foto del mapa con el punto de geolocalización de Google Maps del grupo familiar
•
Código del grupo, se debe ingresar el código de grupo que lo identifica de los demás.
•
La dirección del grupo familiar donde se muestra el departamento, la provincia, el distrito
del grupo y la calle/jirón con el número que ingresó el líder.
•
El horario que se realiza el grupo familiar.
•
Tipo de grupo que puede ser de líderes o de nuevas personas.
•
Teléfono, de la casa donde se realiza el grupo familiar o el número celular del líder.
4.
El líder selecciona la opción “Regresar”.
5.
El sistema retorna al punto 2 del flujo básico y muestra la interfaz MIS GRUPOS.
6.
El caso de uso finaliza.
Editar
1.
En el paso 6 del flujo básico, el líder selecciona LA opción Editar.
2.
El sistema muestra la interfaz EDITAR GRUPO, auto cargando los datos registrados del
grupo familiar a editar.
3.
El líder completa los datos del grupo familiar:
•
Departamento, se debe seleccionar el nombre del departamento donde se encuentra el
grupo familiar.
•
Provincia, se debe seleccionar el nombre de la provincia donde se encuentra el grupo
familiar.
•
Distrito, se debe seleccionar el nombre del distrito donde se encuentra el grupo familiar.
•
Dirección, se debe ingresar la dirección donde se encuentra el grupo familiar.
Página 151
•
Geolocalización, se debe seleccionar en el mapa de Google Maps el punto de
geolocalización donde se encuentra el grupo familiar y mostrará las coordenadas de la
ubicación para actualizar la nueva dirección.
•
Teléfono, se debe ingresar el número de teléfono fijo de la casa donde se realiza el grupo
familiar o el teléfono del líder que dirige el grupo.
•
Tipo de Grupo, se debe seleccionar si es de líderes o es evangelística [16].
•
Tipo Reunión, se debe seleccionar si es de jóvenes o adultos.
•
Día de Reunión, se debe seleccionar el día que se realiza el grupo familiar.
•
Hora de Reunión, se debe seleccionar la hora de que se realiza el grupo familiar.
•
Descripción, se debe ingresar algún detalle del grupo familiar.
4.
El líder modifica los registros y selecciona “Guardar”.
5.
El sistema valida los campos requeridos:
•
Departamento, se debe seleccionar el nombre del departamento donde se encuentra el
grupo familiar.
•
Provincia, se debe seleccionar el nombre de la provincia donde se encuentra el grupo
familiar.
•
Distrito, se debe seleccionar el nombre del distrito donde se encuentra el grupo familiar.
•
Dirección, se debe ingresar la dirección donde se encuentra el grupo familiar.
•
Geolocalización, se debe seleccionar en el mapa de Google Maps el punto de
geolocalización donde se encuentra el grupo familiar y mostrará las coordenadas de la
ubicación para actualizar la nueva dirección.
•
Teléfono, se debe ingresar el número de teléfono fijo de la casa donde se realiza el grupo
familiar o el teléfono del líder que dirige el grupo.
•
Tipo de Grupo, se debe seleccionar si es de líderes o es evangelística [16].
•
Tipo Reunión, se debe seleccionar si es de jóvenes o adultos.
•
Día de Reunión, se debe seleccionar el día que se realiza el grupo familiar.
•
Hora de Reunión, se debe seleccionar la hora de que se realiza el grupo familiar.
Página 152
•
Descripción, se debe ingresar algún detalle del grupo familiar.
6.
El sistema muestra mensaje de “Registro actualizado”.
7.
El sistema retorna al punto 2 del flujo básico y muestra la interfaz MIS GRUPOS.
8.
El caso de uso finaliza.
Ver en Mapa:
1.
En el paso 7 del flujo básico, el líder selecciona la opción “Ver en Mapa”.
2.
El sistema muestra el mapa de Geolocalización donde está ubicado el grupo familiar.
3.
El sistema muestra el logo de la organización con los siguientes datos del grupo familiar:
4.
•
Tipo Grupo, permite visualizar el tipo de grupo (evangelística / líderes) [16]
•
Código Grupo, permite visualizar el código del grupo. [14]
•
Dirección, permite visualizar la dirección del grupo.
•
Fecha permite visualizar el día y la hora que se realiza el grupo.
Si se agranda el mapa el sistema muestra todos los grupos geolocalizados que tiene bajo su
cobertura el líder.
5.
El caso de uso finaliza.
c.
Flujo Alternativo
Edición de Grupo Familiar No satisfactorio
1.
En el paso 6 del flujo básico, si no se cumple con las validaciones para registrar el Grupo
Familiar. El sistema mostrará un mensaje con los títulos “Selecciona un Departamento”,
“Selecciona una provincia”, “Selecciona un distrito”, “Ingrese una dirección” en los campos
Departamento, Provincia, Distrito, Dirección. Finalmente, al ingresar a la opción
“Geolocalización” si no se selecciona la ubicación del grupo se muestra un mensaje “Debe
seleccionar una ubicación”.
5. Precondiciones
Tener líderes, departamentos, provincias, distritos, tipos de grupos y tipos de reunión registrados,
y por lo menos un grupo familiar registrado.
6. Postcondiciones
Se listó los grupos familiares disponibles.
Página 153
Se actualizó un grupo familiar.
Se visualizó el mapa de la ubicación de un grupo familiar.
7. Información Adicional
Ninguno
8. Prototipos
Ver el Menú de la App
Figura 56: AV_CUS030 - Pantalla 01 de Mockup (Elaborado con Justinmind)
Página 154
Figura 57: AV_CUS030 - Pantalla 02 de Mockup (Elaborado con Justinmind)
Figura 58: AV_CUS030 - Pantalla 03 de Mockup (Elaborado con Justinmind)
Página 155
Figura 59: AV_CUS030 - Pantalla 04 de Mockup (Elaborado con Justinmind)
Figura 60: AV_CUS030 - Pantalla 05 de Mockup (Elaborado con Justinmind)
Página 156
4.3.7.9 AV_CUS037_Generar Reporte de eficiencia del líder
1. Actores del sistema
AV_AS004_Pastor
2. Propósito
Generar el reporte de los radares que brinda información importante de los logros y avances en
porcentajes y números de cada área ministerial y esto sirve para la toma de decisiones de los
pastores en sus escuelas y ver en qué área mejorar.
3. Breve Descripción
El Caso de uso comienza cuando el actor Pastor ingresa al menú “Control y Seguimiento” y luego
a la opción “Reporte de Eficiencia del Líder”, luego selecciona los filtros necesarios para
visualizar los radares. El caso de uso termina cuando el reporte visual ha sido generado.
4. Flujo de Eventos
a.
Flujo Básico
1.
El Pastor ingresa a la opción Control y Seguimiento / Reporte de Eficiencia del Líder del
menú principal.
2.
El sistema muestra la interfaz BUSCAR LÍDER con el filtro “Ingrese código de líder”
3.
El Pastor escribe el “código del líder” que desea visualizar su radar y presiona el botón
“Buscar”.
4.
El sistema muestra la interfaz “Resultado de búsqueda de líder” con el año más actual y los
siguientes campos:
•
Código: es el código jerárquico del líder [14].
•
Nombres: aparece los nombres y apellidos del líder
•
Año: es el año que se van a visualizar los radares
•
Diagrama cartesiano: son diagramas donde se muestra un polígono con puntos en sus
vértices donde se representan las áreas ministeriales con su logro porcentual de cada
cuatrimestre [RN011_Periodo de los reportes de radar].
5.
El sistema también muestra debajo del diagrama cartesiano una tabla de efectividad
numérica y porcentual con los siguientes campos:
Página 157
•
Fórmula: es la métrica para sacar el resultado numérico y porcentual de cada área
ministerial.
•
Área: es el área ministerial
•
Logro: es el logro numérico que obtuvo el líder en esa área ministerial en el respectivo
cuatrimestre.
•
Meta: es la meta numérica que debería lograr el líder en esa área ministerial en el
respectivo cuatrimestre
•
6.
Efectividad: es el número de porcentaje de efectividad lograda con respecto a la meta.
Si el Pastor selecciona un año en la opción “Seleccione un año” ver subflujo “Seleccione un
año”.
7.
Si el Pastor selecciona el menú contextual del diagrama cartesiano, ver subflujo “Imprimir
diagrama”.
8.
Si el Pastor selecciona el menú contextual del diagrama cartesiano, ver subflujo “Descargar
imagen PNG”.
9.
Si el Pastor selecciona el menú contextual del diagrama cartesiano, ver subflujo “Descargar
imagen JPEG”.
10. Si el Pastor selecciona el menú contextual del diagrama cartesiano, ver subflujo “Descargar
documento PDF”.
11. Si el Pastor selecciona el menú contextual del diagrama cartesiano, ver subflujo “Descargar
imagen SVG vector”.
12. El caso de uso finaliza.
b. Sub Flujos
Seleccione un año
1.
El sistema muestra la interfaz “Resultado de búsqueda de líder” con el año que seleccionó
el actor y los siguientes campos:
•
Código: es el código jerárquico del líder.
•
Nombres: aparece los nombres y apellidos del líder
•
Año: es el año que se van a visualizar los radares
Página 158
•
Diagrama cartesiano: son diagramas donde se muestra un polígono con puntos en sus
vértices donde se representan las áreas ministeriales con su logro porcentual de cada
cuatrimestre.
2.
El sistema también muestra debajo del diagrama cartesiano una tabla de efectividad
numérica y porcentual con los siguientes campos:
•
Fórmula: es la métrica para sacar el resultado numérico y porcentual de cada área
ministerial.
•
Área: es el área ministerial [9]
•
Logro: es el logro numérico que obtuvo el líder en esa área ministerial en el respectivo
cuatrimestre.
•
Meta: es la meta numérica que debería lograr el líder en esa área ministerial en el
respectivo cuatrimestre
•
3.
Efectividad: es el número de porcentaje de efectividad lograda con respecto a la meta.
El caso de uso retorna al punto 2 del flujo básico.
Imprimir diagrama
1.
En el paso 7 del flujo básico, el Pastor selecciona la opción “Imprimir diagrama”
2.
El sistema carga la interfaz predeterminada de impresión del navegador en uso.
3.
El caso de uso retorna al punto 2 del flujo básico.
Descargar imagen PNG
1.
En el paso 8 del flujo básico, el Pastor selecciona la opción “Descargar imagen PNG”
2.
El sistema descarga el archivo de imagen a la computadora a través del navegador en uso.
3.
El caso de uso retorna al punto 2 del flujo básico.
Descargar imagen JPEG
1.
En el paso 9 del flujo básico, el Pastor selecciona la opción “Descargar imagen JPEG”
2.
El sistema descarga el archivo de imagen a la computadora a través del navegador en uso.
3.
El caso de uso retorna al punto 2 del flujo básico.
Página 159
Descargar documento PDF
1.
En el paso 10 del flujo básico, el Pastor selecciona la opción “Descargar documento PDF”
2.
El sistema descarga el archivo de PDF a la computadora a través del navegador en uso.
3.
El caso de uso retorna al punto 2 del flujo básico.
Descargar imagen SVG vector
1.
En el paso 11 del flujo básico, el Pastor selecciona la opción “Descargar imagen SVG
vector”
2.
El sistema descarga el archivo de imagen a la computadora a través del navegador en uso.
3.
El caso de uso retorna al punto 2 del flujo básico.
c.
Flujo Alternativo
Completa este campo [RN012_Data para radares]
1.
En el paso 3 del flujo básico, si el sistema no contiene información para el filtro de “Código
de líder” se mostrará el mensaje resaltado con un signo de admiración “Completa este
campo”.
5. Precondiciones
Tener información de los logros de las diferentes áreas ministeriales [9] por cada cuatrimestre.
6. Postcondiciones
Reporte de Eficiencia del líder generado.
7. Información Adicional
No aplica.
8. Prototipo
Página 160
Figura 61: AV_CUS037 - Pantalla 01 de Mockup (Elaborado con Justinmind)
Figura 62: AV_CUS037 - Pantalla 02 de Mockup (Elaborado con Justinmind)
Página 161
4.4
MODELO CONCEPTUAL
4.4.1 DIAGRAMA DEL MODELO CONCEPTUAL
Figura 63: Diagrama del Modelo Conceptual
Página 162
4.4.2 DICCIONARIO DEL MODELO CONCEPTUAL
Creyente
Representa a los creyentes que forman parte de la comunidad cristiana.
Nombre del atributo
Descripción
Tipo de dato
código
Código del creyente asignado
Integer
nombres
Nombres del creyente
String
apellidos
Apellidos del creyente
String
sexo
Género del creyente
String
fnacimiento
Fecha de nacimiento del creyente
Date
dni
Documento de identidad del creyente
String
correo
Correo electrónico del creyente
String
teléfono
Teléfono del creyente
String
celular
Número de celular del creyente
String
departamento
Nombre del departamento
String
provincia
Nombre de la provincia
String
distrito
Nombre del distrito
String
referencia
Datos de la referencia de domicilio
String
direccion
Dirección del creyente
String
Tabla 63: Diccionario del Modelo Conceptual – Creyente (Fuente: propia)
Líder
Representa a los líderes que forman parte de la comunidad cristiana.
Nombre del atributo
Descripción
Tipo de dato
carnet
Código de carné del líder
Integer
fecregistro
Fecha de registro
Date
tipo
Tipo de rol del líder (Líder o Consolidador)
String
cédula
Número de cédula del líder
String
Tabla 64: Diccionario del Modelo Conceptual – Líder (Fuente: propia)
Página 163
Consolidación
Representa al registro de consolidación que se ejecuta para para creyente con un consolidador.
Nombre del atributo
Descripción
Tipo de dato
código
Código de consolidación
Integer
fecasignación
Fecha de asignación de consolidador
Date
Feccierre
Fecha de cierre de la consolidación
Date
estado
Estado actual del proceso de consolidación
String
Tabla 65: Diccionario del Modelo Conceptual – Consolidación (Fuente: propia)
ProgramaciónContacto
Representa a la programación de la llamada, visita o acompañamiento que hace los consolidadores.
Nombre del atributo
Descripción
Tipo de dato
código
Código de la programación
Integer
fecha
Fecha del evento
Date
hora
Hora del evento
Time
tipo
Tipo de evento (llamada, visita o acompañamiento)
String
estado
Estado del evento (Programada, Cancelada, Cumplida, Sin
éxito)
String
Tabla 66: Diccionario del Modelo Conceptual – Programación Contacto (Fuente: propia)
ReporteContacto
Representa al reporte que registran los consolidadores al final del evento.
Nombre del atributo
Descripción
Tipo de dato
código
Código del reporte
Integer
fecha
Fecha de registro
Date
observaciones
Observaciones encontradas durante la ejecución del evento
String
sugerencias
Sugerencias que el líder propone como mejora
String
Tabla 67: Diccionario del Modelo Conceptual – Reporte Contacto (Fuente: propia)
Página 164
ReporteConsolidacion
Representa al reporte general de consolidación que resume semestralmente varios reportes de contacto.
Nombre del atributo
Descripción
Tipo de dato
número
Código del reporte
Integer
fecha
Fecha de registro
Date
observaciones
Observaciones encontradas durante la ejecución del evento
String
Tabla 68: Diccionario del Modelo Conceptual – Reporte Consolidación (Fuente: propia)
CursoBíblico
Representa al catálogo de cursos que son ejecutadas cada cierto periodo.
Nombre del atributo
Descripción
Tipo de dato
código
Código del curso bíblico
Integer
nombre
Nombre del curso bíblico
String
área
Área al cual pertenece el curso bíblico
String
estado
Estado del curso bíblico
String
frecuencia
Número de veces que ha sido adquirido el curso
Integer
Tabla 69: Diccionario del Modelo Conceptual – Curso Bíblico (Fuente: propia)
CursoProgramado
Representa a los cursos que se han programado en una fecha y tienen inscripciones abiertas.
Nombre del atributo
Descripción
Tipo de dato
código
Código del curso programado
Integer
descripción
Descripción del curso programado
String
periodo
Nombre del periodo al cual pertenece el curso programado
String
fecinicio
Fecha de inicio del curso programado
Date
fecfin
Fecha de fin del curso programado
Date
estado
Estado actual del curso programado
String
Tabla 70: Diccionario del Modelo Conceptual – Curso programado (Fuente: propia)
Página 165
Inscripción
Representa a las inscripciones que realizan los creyentes a un curso programado.
Nombre del atributo
Descripción
Tipo de dato
fecregistro
Feccha de registro al curso programado
Date
estado
Estado actual de la inscripción
String
Tabla 71: Diccionario del Modelo Conceptual – Inscripción (Fuente: propia)
CursoAsociación
Representa a la relación entre cursos en base al algoritmo a priori.
Nombre del atributo
confianza
Descripción
Grado de confianza de la relación
Tipo de dato
Integer
Tabla 72: Diccionario del Modelo Conceptual – Curso Asociación (Fuente: propia)
GrupoFamiliar
Representa a un evento de grupo familiar organizado por el líder.
Nombre del atributo
Descripción
Tipo de dato
código
Código del evento de grupo familiar
Integer
fecinicio
Fecha de inicio del grupo familiar
Date
hora
Hora de inicio del grupo familiar
Time
direccion
Dirección del evento de grupo familiar
String
latitud
Valor de la latitud en el mapa
String
longitud
Valor de la longitud en el mapa
String
estado
Estado del evento de grupo familiar
String
Tabla 73: Diccionario del Modelo Conceptual – Grupo Familiar (Fuente: propia)
Encuentro
Representa a un evento de grupo familiar organizado por el líder.
Nombre del atributo
Descripción
Tipo de dato
código
Código del encuentro
Integer
asunto
Motivo relacionado al encuentro
String
fecinicio
Fecha de inicio del encuentro
Date
fecfin
Fecha de término del encuentro
Date
Página 166
lugar
Lugar del encuentro
String
capacidad
Capacidad del encuentro
Integer
estado
Estado del encuentro
String
Tabla 74: Diccionario del Modelo Conceptual – Encuentro (Fuente: propia)
Asistente
Representa a los asistentes al grupo familiar.
Nombre del atributo
Descripción
Tipo de dato
fecregistro
Fecha de registro al grupo familiar
Integer
estado
Estado actual del registro al grupo familiar
String
calificación
Calificación que el asistente valora al grupo familiar
Integer
Tabla 75: Diccionario del Modelo Conceptual – Asistente (Fuente: propia)
Encuentrista
Representa a los creyentes que participan del encuentro.
Nombre del atributo
Descripción
Tipo de dato
fecregistro
Fecha de registro al encuentro
Integer
prerrequisito
Indica si cumple los prerequisitos
String
estado
Estado actual del registro al encuentro
String
observaciones
Observaciones que el creyente anota del encuentro
String
Tabla 76: Diccionario del Modelo Conceptual – Encuentrista (Fuente: propia)
Página 167
4.5
CONCLUSIONES
En el presente capítulo se detalló la lista de requerimientos funcionales del proceso de negocio y los no
requerimientos funcionales para la arquitectura del software, los cuales permitieron establecer casos de
uso de sistema que satisfagan cada uno de los requerimientos funcionales y mecanismos que permitan
alcanzar el logro de los requerimientos no funcionales.
En general se definieron seis paquetes de sistema que representa a los procesos más importantes dentro
del campo de acción. También se describió la especificación de dos casos de uso de ciclo cero que están
considerados dentro de la prueba de concepto. Estas especificaciones nos ayudan a tener claro el
comportamiento de que debería tener cada uno de los casos de uso dentro de nuestro sistema de software,
sus precondiciones y pos condiciones, quiénes son los actores que intervienen cada una de ella, las reglas
de negocio presente en el flujo de actividades y las entidades que se leen o graban dentro de ellas.
Finalmente se elabora un modelo conceptual que describe las distintas entidades y relación entre ellas
que servirán de soporte para la persistencia de la información del sistema.
Con lo todo lo descrito anteriormente se tiene claro lo que se espera del software y se ha desarrollado
dos casos de uso como prueba de concepto.
Página 168
5
CAPITULO 5: ARQUITECTURA DE SOFTWARE
5.1
INTRODUCCIÓN
En el presente capítulo se presenta la arquitectura de software del sistema. Se ha identificado los casos
de uso más significativos por cada paquete. Luego, se han definido las metas de la arquitectura en base
a los requerimientos no funcionales. Después, se han analizado las restricciones que tiene la arquitectura
del software tales como confidencialidad de contraseña, compatibilidad con navegadores web, formato
de interfaz gráfica web, límite mínimo de conexiones y mensajes emergentes de transacciones.
Acto seguido, se definieron los mecanismos arquitecturales tales como el algoritmo md5, el CSS3, el
diseño web adaptable o adaptativo (RWD: Responsive Web Design), se utilizará Bootstrap, y
formularios en JavaScript para abrir ventanas emergentes. Seguido de lo anterior, se muestra la vista
lógica la cual contiene las capas de la aplicación y la vista de componentes de los casos de uso más
significativos.
Finalmente se muestra la vista de implementación y la vista de despliegue.
Página 169
5.2
DIAGRAMA DE CASOS DE USO MAS SIGNIFICATIVOS PARA LA ARQUITECTURA
Figura 64: Diagrama de CUS más significativos para la Arquitectura
Página 170
5.3
METAS DE LA ARQUITECTURA DE SOFTWARE
RNF
META
GEN_RNF001_Mensaje Emergente de Transacción
Mostrar mensajes de error detallados al usuario.
GEN_RNF005_Confidencialidad de contraseña
Garantizar la confidencialidad de las claves del
sistema.
GEN_RNF006_Inactividade del Sistema
Controlar la inactividad del sistema.
GEN_RNF010_Historial de Operaciones
Permite que las actividades en el sistema sean
detectadas y guardadas.
GEN_RNF012_Tiempo de Generación de Reportes
Reducir el tiempo de respuesta de reportes.
GEN_RNF013_Tiempo de Transacciones en el Sistema
Reducir el tiempo de respuesta en las transacciones del
sistema.
GEN_RNF014_Concurrencia de usuarios
Permitir el soporte de concurrencia de usuarios.
GEN_RNF027_Arquitectura de Servicios
Garantizar el intercambio de información en tiempo
real con los sistemas de la organización de forma
rápida y segura.
GEN_RNF029_Formato Interfaz Gráfica Web
Las interfaces del sistema deberán adaptarse al tamaño
y resolución del dispositivo.
GEN_RNF030_Colores Interfaz Gráfica Web
Predominan los colores corporativos.
Tabla 77: Metas de la Arquitectura de Software (Fuente: propia)
Página 171
5.4
RESTRICCIONES DE LA ARQUITECTURA DE SOFTWARE
RNF
RESTRICCIÓN
GEN_RNF001_Mensaje Emergente de Transacción
Presentar los mensajes de error relacionados a la
validación de las reglas de negocio.
GEN_RNF005_Confidencialidad de contraseña
Cifrado irreversible de las contraseñas almacenadas.
GEN_RNF006_Inactividade del Sistema
Inactividad del Sistema.
GEN_RNF010_Historial de Operaciones
Sistema en ejecución de las principales operaciones de
Distribución.
GEN_RNF012_Tiempo de Generación de Reportes
Tiempo de generación del reporte menor a 1 minuto.
GEN_RNF013_Tiempo de Transacciones en el Sistema
En el 95% de las transacciones el sistema no deberá
sobrepasar los 5 segundos entre el Request y Response.
GEN_RNF014_Concurrencia de usuarios
Máximo 100 usuarios concurrentes.
GEN_RNF027_Arquitectura de Servicios
Plataformas tecnológicas variadas e incompatibles.
GEN_RNF029_Formato Interfaz Gráfica Web
Resolución variante por dispositivo.
GEN_RNF030_Colores Interfaz Gráfica Web
Utilizar los colores corporativos.
Tabla 78: Restricciones de la Arquitectura de Software (Fuente: propia)
Página 172
5.5
MECANISMOS ARQUITECTURALES
META
RESTRICCIÓN
SOLUCIÓN
Mostrar mensajes de error
detallados al usuario.
[GEN_RNF001_Mensaje Emergente
de Transacción]
Presentar los mensajes de error
relacionados a la validación de las
reglas de negocio.
Utilizar estructuras de control y clases
validadoras que validen las reglas de
negocio del Software.
Controlar la inactividad del
sistema.
[GEN_RNF006_Inactividade del
Sistema]
Inactividad del Sistema.
Se utilizará la configuración del time
out de la sesión mediante el archivo de
configuración de la aplicación.
Permite que las actividades en
el sistema sean detectadas y
guardadas.
[GEN_RNF010_Historial de
Operaciones]
Sistema en ejecución de las
principales operaciones de
Distribución.
Utilizar un LOG que maneje las
actividades de la sesión.
Garantizar el intercambio de
información en tiempo real
con los sistemas de la
organización de forma rápida
y segura.
[GEN_RNF027_Arquitectura de
Servicios]
Plataformas tecnológicas variadas e
incompatibles.
Desarrollar servicios que permita el
intercambio de información entre
sistemas de plataformas distintas,
utilizando RESTFul por su rapidez y
simplicidad y JWT como capa de
seguridad.
Garantizar la
confidencialidad de las claves
del sistema.
[GEN_RNF005_Confidencialidad de
contraseña]
Cifrado irreversible de las
contraseñas almacenadas.
Desarrollar un componente que se
encargue de cifrar las contraseñas con el
algoritmo sha1 en el momento del
registro.
Reducir el tiempo de
respuesta de reportes.
[GEN_RNF012_Tiempo de
Generación de Reportes]
Tiempo de generación del reporte
menor a 1 minuto.
Desarrollar Stored Procedures que
realice las consultas a las tablas
transaccionales y su información las
migre a tablas plana.
Reducir el tiempo de
respuesta en las transacciones
del sistema.
[GEN_RNF013_Tiempo de
Transacciones en el Sistema]
En el 95% de las transacciones el
sistema no deberá sobrepasar los 5
segundos entre el Request y
Response.
Implementar en los formularios web
una llamada ajax para la ejecución
asíncrona de las transacciones del
sistema. Se utilizara las librerías
JavaScript de Jquery.
Permitir el soporte de
concurrencia de usuarios.
[GEN_RNF014_Concurrencia de
usuarios]
Máximo 100 usuarios concurrentes.
Implementación de algoritmos de
control de concurrencia en el Gestor de
Base de datos que haga uso de los
bloqueos a los recursos compartidos por
el sistema.
Las interfaces del sistema
deberán adaptarse al tamaño
y resolución del dispositivo.
[GEN_RNF029_Formato Interfaz
Gráfica Web]
Resolución variante por dispositivo.
Diseñar las interfaces Web aplicando la
técnica Responsive Web Design. Esto
será posible utilizando el framework
CSS3 bootstrap 3.3.7.
Tabla 79: Mecanismos de la Arquitectura de Software (Fuente: propia)
Página 173
5.6
VISTA LÓGICA DE LA ARQUITECTURA DE SOFTWARE
Figura 65: Vista Lógica (Fuente: propia)
Capa
Descripción
Vista
Contiene a los componentes responsables de mostrar la información.
Controlador
Contiene aquellos componentes que se encargar de recibir las solicitudes
del cliente, intermediar entre los componentes modelo y vista y
responder un a la solicitud inicial.
Modelo
Contiene componentes responsables de la persistencia de la información
y la lógica de negocio.
Tabla 80: Capas de la Vista Lógica (Fuente: propia)
Página 174
5.7
VISTA DE IMPLEMENTACIÓN DE LA ARQUITECTURA DE SOFTWARE
Figura 66: Vista de Implementación (Fuente: propia)
Página 175
5.8
VISTA DE DESPLIEGUE DE LA ARQUITECTURA DE SOFTWARE
Figura 67: Vista de Despliegue (Fuente: propia)
Página 176
5.9
CONCLUSIONES
En el presente capítulo se identificó las principales metas que demanda la arquitectura de
software de la solución, así como de las restricciones técnicas presentes en cada una de ellas,
esto nos permite identificarlas y proponer mecanismos claros que nos permita superar estas
metas que son partes de los requerimientos no funcionales. Además, hasta este capítulo, se
tiene detallado el escenario en el cual se desplegará los componentes de software como el
diagrama de despliegue y el diagrama de implementación respectivamente. También se tiene
el diagrama de vista lógica la cual nos permitirá conocer la distribución de capas dentro del
software propuesto.
Página 177
6
CAPÍTULO 6: CALIDAD Y PRUEBAS DE SOFTWARE
6.1
INTRODUCCIÓN
En este capítulo se va a desarrollar el plan de calidad del software, en donde se encuentran la
política y los objetivos de calidad de la organización Comunidad Cristiana Agua Viva.
Asimismo, se identificará cada una de las normas de calidad establecidas que se aplican al
producto de software que se está desarrollando y se detallará las principales métricas de calidad
usadas en el software construido.
Finalmente, se mostrará el plan de pruebas del software desarrollado a dos casos de uso de los
nueve del núcleo central del sistema, así como también el análisis de los resultados obtenidos.
6.2
PLAN DE CALIDAD DE SOFTWARE
6.2.1 Políticas De Calidad
La organización Comunidad Cristiana Agua Viva no tiene una política de calidad definida que
se pueda aplicar al desarrollo de software; por lo tanto, se propone tener en cuenta las siguientes
consideraciones y políticas que ayudarán con el aseguramiento y control de la calidad de los
diferentes productos de software que se puedan desarrollar para la organización.
Comunidad Cristiana agua Viva tiene el compromiso de brindar servicios de calidad a las
personas que asisten a sus sedes. Una de las formas de brindar calidad es mediante el uso de
herramientas tecnológicas como los sistemas web y apps que permitan ayudar con éxito al
desarrollo y crecimiento numérico de los líderes. Comunidad Cristiana Agua Viva considera a
los creyentes como centro de todas las actividades que realiza, es por eso que en el proceso de
desarrollo ministerial se gestiona el uso y desarrollo de software con valor y que se presente
como la mejor solución tecnológica existente con una mejora continua.
6.2.2 Objetivos De Calidad
Los objetivos de calidad aplicados al desarrollo de software que se propone al presente proyecto
son los siguientes:
•
Desarrollar productos de software usando procedimientos y estándares reconocidos
para el análisis, diseño, construcción y pruebas del producto; garantizando la
confiabilidad y mantenibilidad del mismo.
•
Todo producto de software desarrollado debe suplir las necesidades del usuario para
cumplir con cada uno de los requerimientos previamente establecidos.
Página 178
•
El 90% de todas las pruebas ejecutadas para los casos de uso tengan un resultado
aceptable.
•
La organización brindará una capacitación constante a los usuarios en cuanto al uso de
herramientas tecnológicas, por lo menos una vez al año en las áreas de ministeriales,
como son Consolidación, Encuentros, Escuela de Líderes, y Grupos Familiares.
6.2.3 Normatividad Aplicable
La organización CCAV no tiene normas de calidad que pueda aplicar a sus procesos de negocio
ni para el software que se está proponiendo o los sistemas que se puedan adquirir, entonces se
propone la siguiente normativa para la evaluación de la calidad del software:
6.2.3.1 Norma ISO/IEC 25000
Esta norma provee una guía para el uso de normas internacionales llamadas Requisitos y
Evaluación de Calidad de Productos de Software (SQuaRE). El propósito de la norma ISO /
IEC 25000 es dar una visión general de los contenidos de SQuaRE, los modelos y definiciones,
así como la relación entre los documentos, lo que permite a los usuarios la comprensión de esas
series de estándares, de acuerdo con su propósito de uso. Asimismo da una explicación sobre
la transición entre la antigua ISO / IEC 9126 y la serie ISO / IEC 14598 y SQuaRE. (ISO, 2017)
La serie de normas SQuaRE está compuesta de las siguientes divisiones:
1. ISO / IEC 2500n, División de Gestión de Calidad.
2. ISO / IEC 2501n, División del modelo de calidad.
3. ISO / IEC 2502n, División de medición de calidad.
4. ISO / IEC 2503n, División de requisitos de calidad.
5. ISO / IEC 2504n, División de Evaluación de Calidad.
Una de las normas contenidas en la división del modelo de calidad es la ISO / IEC 25010 que
a continuación describiremos:
6.2.3.2 Norma ISO/IEC25010
Esta norma provee de un modelo de calidad como base en la que se establece el sistema para
la evaluación de la calidad del producto. En este modelo se detallan las características de
calidad que se verán a la hora de evaluar las cualidades de un producto software.
La calidad del software se puede entender como la satisfacción de los requerimientos de los
usuarios dando de esta manera un valor. Estos requisitos (funcionalidad, seguridad,
Página 179
mantenibilidad, etc.) son los que se encuentran detallados en el modelo de calidad, el cual
clasifica la calidad del producto en características y sub características. (iso25000.com, 2017)
Modelo de Calidad del Producto Software (Calidad Interna y Externa)
Este modelo tiene ocho características para la calidad interna y externa de un software:
Adecuación Funcional, Fiabilidad, Eficiencia de desempeño, Compatibilidad, Usabilidad,
Fiabilidad, Seguridad, mantenibilidad y Portabilidad, y estos tienen sub características que se
pueden apreciar en la ilustración y que a su vez pueden ser medidas con métricas internas o
externas. (ISO, ISO/IEC 25010, 2008)
Página 180
Figura 68: Modelo de calidad del Producto de Software (Fuente http://iso25000.com)
Página 181
Modelo de Calidad en Uso
Este modelo de define cinco características que son: Efectividad, Eficiencia, Satisfacción,
Libertad de Riesgo y Cobertura de Contexto, y estas tienen sub características que pueden ser
medidas con métricas de calidad en uso.
El resultado de la calidad en uso depende de los resultados de la calidad externa y este a su vez
depende de la calidad interna. (ISO, ISO/IEC 25010, 2008)
Figura 69: Modelo de calidad de Uso (Fuente ISO/IEC 25010)
6.2.3.3 Normas ISO/IEC 25022 y 25023
Estas normas proveen un conjunto de métricas para la calidad interna, externa y de uso, que
están en base al modelo de calidad ISO/IEC 25010. Los usuarios pueden seleccionar las
características y sub características de calidad para ser evaluadas identificando las métricas más
apropiadas para luego poder interpretar los resultados de una manera objetiva. (ISO, ISO/IEC
25023, 2011)
En la ilustración se puede apreciar la relación que existe entre las etapas de calidad, sus métricas
y la dependencia que hay entre ellos.
Página 182
Figura 70: Relación de los tipos de métricas de calidad (Fuente ISO/IEC 25023)
Las métricas de calidad interna ayudan al usuario a identificar los problemas y errores de
calidad en la etapa de desarrollo del sistema donde se encuentran los requerimientos, la
especificación del diseño o código fuente y poder corregir a tiempo el software.
Las métricas de calidad externa miden el comportamiento del software y se puede usar en la
etapa de pruebas y el sistema tiene que estar en ejecución.
Las métricas de calidad en uso miden si el software suple las necesidades de los usuarios y para
esto se debe ejecutar el sistema en un ambiente real de trabajo donde se va usar el software de
una manera continua.
Página 183
6.2.4 MÉTRICAS DE CALIDAD DEL SOFTWARE
6.2.4.1 MÉTRICA DE USO
ISO 25010
ISO 25022
Característica
Uso
Descripción de la métrica
Sub Característica
Satisfacción
Responsable
Categoría Indicador
Métrica de Uso
Unidad de medida
Fuente de Información
Grado de satisfacción del usuario
Pablo Guevara
%
Encuestas a Usuarios
Código
MU01
Objetivo
Meta
Medir la satisfacción del uso de las funcionalidades del módulo de Consolidación de Creyentes.
Comprueba si las funcionalidades satisfacen con los requerimientos de los usuarios.
>90%
Forma de Cálculo
Tipo Medida
X = ((A*0 +B*0.25 +C*0.50 +D*0.75 +E*1.00) / F)*100
A = Cantidad de respuestas marcadas con A
B= Cantidad de respuestas marcadas con B
C= Cantidad de respuestas marcadas con C
D= Cantidad de respuestas marcadas con D
E= Cantidad de respuestas marcadas con E
F = Suma total de las anteriores (A+B+C+D+E)
X = cantidad / cantidad
A = cantidad
B = cantidad
C= cantidad
D= cantidad
E= cantidad
F= cantidad
Interpretación
Umbrales
Mientras el resultado esté más cerca al 100% se puede asegurar que el
cliente está satisfecho.
SLA
<= 70%
No aceptable
> 70% y <= 90% Parcialmente aceptable
> 90%
Aceptable
Tabla 81: Métrica de Uso (Fuente: propia)
Página 184
6.2.4.2 MÉTRICAS INTERNAS
ISO 25010
Tabla 28 (ISO 25023) Pag 28
Característica
Mantenibilidad
Descripción de la métrica
Sub Característica
Testeabilidad
Responsable
Cobertura de pruebas según funcionalidad.
Pablo Guevara
Categoría Indicador
Métrica Interna
Unidad de medida
Ratio
Código
MI01
Fuente de Información
Especificación de Caso de Uso
Escenarios de Caso de Uso
Buenas Prácticas Calidad de SW
Objetivo
Meta
Comprueba si los casos de prueba planteados cubren las funcionalidades presentadas en la especificación de casos de Uso.
1.00
Forma de Cálculo
Tipo Medida
X = A/B
A = Número de escenarios especificados
B = Número de flujo de eventos en la ECU (Flujo básico+Flujo alternativo+subflujos)
X = cantidad / cantidad
A = cantidad
B = cantidad
Interpretación
SLA
Umbrales
0<= X <= 1
Entre más cercano a 1, más completa
Significa que tanto se ha cubierto con escenarios los diferentes flujos de
eventos que puede seguir un caso de uso.
<= 0.7 No aceptable
> 0.7
Parcialmente aceptable
=1
Aceptable
Tabla 82: Métrica Interna 01 (Fuente: propia)
Página 185
ISO 25010
Tabla 1 (ISO 25023) Pag 8
Característica
Adecuación Funcional
Descripción de la métrica
Sub Característica
Completitud de Implementación Funcional
Responsable
¿Qué tan completa está la implementación funcional?
Pablo Guevara
Categoría Indicador
Métrica Interna
Unidad de medida
Ratio
Código
MI02
Fuente de Información
Requerimientos funcionales
Casos de Prueba
Casos de uso
Objetivo
Meta
Contar las funciones faltantes detectadas en la evaluación y comparar con el número de funciones consideradas en el caso de uso.
>0.85
Forma de Cálculo
X = 1 – A/B
A = número de funciones faltantes o problemáticas en la evaluación
B = número de funciones consideradas
Interpretación
Umbrales
Tipo Medida
X = cantidad / cantidad
A = cantidad
B = cantidad
SLA
<= 0.6 No aceptable
>0.6 y <=0.85 Parcialmente aceptable
>0.85 Aceptable
0 <= X <= 1
Mientras X esté más cercano a 1, más completa.
Tabla 83: Métrica Interna 02 (Fuente: propia)
Página 186
6.2.4.3 MÉTRICAS EXTERNAS
ISO 25010
Tabla 15 (ISO 25023) Pag 20
Característica
Sub Característica
Confiabilidad
Madurez
Descripción de la métrica
Responsable
Categoría Indicador
Métrica Externa
Unidad de medida
Corrección de fallos
Pablo Guevara
%
Objetivo
Código
ME01
Fuente de Información
Aplicativo SW-Gestión de desarrollo
ministerial
Casos de Prueba Ejecutados
Meta
Verificar cuántos fallos han sido corregidos
>95%
Forma de Cálculo
Tipo Medida
X = A/B *100
A = número de fallas corregidos en la fase de pruebas
B = número de fallas detectada en la fase de pruebas
X = cantidad / cantidad
A = cantidad
B = cantidad
Interpretación
Umbrales
Mientras el resultado esté más cercano al 100% se asegura que se tiene un
producto confiable, ya que logró levantar sus errores satisfactoriamente.
SLA
<= 70%
No aceptable
> 70% y <= 95% Parcialmente aceptable
> 95%
Aceptable
Tabla 84: Métrica Externa 01 (Fuente: propia)
Página 187
ISO 25010
Tabla 04 (ISO 25023) Pag10
Característica
Sub Característica
Eficiencia
Comportamiento de tiempo
Descripción de la métrica
Responsable
Categoría Indicador
Métrica Externa
Unidad de medida
Tiempo de respuesta
Segundos (s)
Pablo Guevara
Objetivo
Código
ME02
Fuente de Información
Aplicativo SW-Gestión de desarrollo
ministerial
Meta
Evaluar la eficiencia de las llamadas al software.
Estimar el tiempo de respuesta basado en ello.
<= 5s
Forma de Cálculo
X = (A+B+C+D+E) / N
A = Tiempo de respuesta 1
B= Tiempo de respuesta 2
C= Tiempo de respuesta 3
D= Tiempo de respuesta 4
E= Tiempo de respuesta 5
N = Cantidad de respuestas medidas
Interpretación
Umbrales
0s <= X
Mientras el resultado sea más cercano a 0 segundos se asegura que el
software tiene un tiempo de respuesta favorable.
Tipo Medida
X = segundos
A = segundos
B = segundos
C= segundos
D= segundos
E= segundos
N= cantidad
SLA
>= 8s
No aceptable
>= 5s y < 8s Parcialmente aceptable
<= 5s
Aceptable
Tabla 85: Métrica Externa 02 (Fuente: propia)
Página 188
6.2.5 ANÁLISIS DE RESULTADOS DE LA MEDICIÓN
6.2.5.1 Métrica de Uso (Satisfacción)
CUS Grupo 01(Módulo Propio)
AV_CUS040_Notificación
de seguimiento al líder
AV_CUS024_Importar
información de cursos
llevados y disponibles
A
B
C
D
E
Sum/F
*0
*0.25
*0.5
*0.75
*1.0
Sum
0
0
0
9
28
37
#A
#B
#C
#D
#E
F
0
0
0
12
28
40
*0
*0.25
*0.5
*0.75
*1.0
Sum
0
0
0.5
9.75
26
36.25
#A
#B
#C
#D
#E
F
0
0
1
13
26
40
Puntuación
Total
0.925
0.906
RESULTADO
(X)
Umbral
Análisis
92.5%
Se tiene un resultado del
92.5%, lo cual es un valor
Aceptable aceptable para el nivel de
satisfacción del usuario en el
uso del sistema.
90.6%
Se tiene un resultado del
90.6%, lo cual es un valor
Aceptable aceptable para el nivel de
satisfacción del usuario en el
uso del sistema.
Tabla 86: Resultados de Métrica de Uso (Fuente: propia)
Total de encuestados: 10 personas
Total de preguntas por encuesta: 4 preguntas (Modelo de encuesta en Anexo 5)
Encuesta de
satisfacción de uso del SGDM (respuestas).xlsx
Página 189
6.2.5.2 Métrica Interna - Mantenibilidad (Testeabilidad)
CUS
AV_CUS040_Notificación de
seguimiento al líder
AV_CUS024_Importar
información de cursos llevados
y disponibles
Flujos(A)
3
4
Escenarios(B)
3
4
RESULTADO (X)
1.00
1.00
Umbral
Análisis
Aceptable
Se obtiene un valor aceptable de 1.00 en la
cobertura de todos los flujos presentes en el
caso de uso con respecto a los escenarios para
sus respectivos casos de prueba.
Aceptable
Se obtiene un valor aceptable de 1.00 en la
cobertura de todos los flujos presentes en el
caso de uso con respecto a los escenarios para
sus respectivos casos de prueba.
Tabla 87: Resultados de Métrica Interna 01 (Fuente: propia)
Matriz Flujos vs
Escenarios.xlsx
Página 190
6.2.5.3 Métrica Interna – Adecuación Funcional (Completitud funcional)
CUS
Funciones
Faltantes(A)
Funciones
consideradas(B)
RESULTADO (X)
Umbral
AV_CUS040_Notificación de
seguimiento al líder
0.13
3
0.96
Aceptable
Esto significa que si está cerca de 1
entonces, está más completa la
implementación funcional.
Aceptable
Esto significa que si está cerca de 1
entonces, está más completa la
implementación funcional.
AV_CUS024_Importar
información de cursos llevados
y disponibles
0.10
4
0.98
Análisis
Tabla 88: Resultados de Métrica Interna 02 (Fuente: propia)
Informe - Adecuacion
funcional.xlsx
Página 191
6.2.5.4 Métrica Externa - Confiabilidad (Madurez)
CUS
NÚMERO DE FALLAS
CORREGIDAS(A)
NÚMERO DE
FALLAS (B)
RESULTADO
(X)
Umbral
AV_CUS040_Notificación
de seguimiento al líder
10
10
100%
Aceptable
83.3%
Se ha corregido el 80% de los fallos
Parcialmente detectados en la primera ejecución del
Aceptable SW. Es parcialmente aceptable, ya que se
espera solucionar los defectos en un 95%.
AV_CUS024_Importar
información de cursos
llevados y disponibles
5
6
Análisis
Se ha logrado levantar los defectos
reportados en la primera ejecución.
Tabla 89: Resultados de Métrica Externa 01 (Fuente: propia)
Informe de Errores
en Pruebas.xlsx
Página 192
6.2.5.5 Métrica Externa - Eficiencia (Comportamiento de tiempo)
CUS
A
B
C
D
AV_CUS040_Notificac
ión de seguimiento al
4.13 3.53 3.67 3.8
líder
E
3.8
AV_CUS024_Importar
información de cursos 1.38 1.34 1.31 1.28 1.36
llevados y disponibles
Cantidad
Tiempo Resultado (X)
Ejecuciones
Promedio en segundos
(N)
5
5
3.79
1.33
Umbral
Análisis
3.79s
Se concluye que el resultado obtenido de
3.79 segundos como tiempo promedio de
Aceptable
respuesta es aceptable por encontrarse
por debajo de los 5 segundos.
1.33s
Se concluye que el resultado obtenido de
1.33 segundos como tiempo promedio de
Aceptable
respuesta es aceptable por encontrarse
por debajo de los 5 segundos.
Tabla 90: Resultados de Métrica Externa 02 (Fuente: propia)
Informe de Tiempo
de respuesta.xlsx
Página 193
6.3
PRUEBAS DEL SOFTWARE
6.3.1 PLAN DE PRUEBAS
El propósito de este plan es planificar, estructurar y documentar la planificación de las pruebas de
aceptación del sistema a realizar, así como la estrategia a utilizar para su ejecución.
6.3.2 OBJETIVO
El objetivo del presente documento es la certificación las funcionalidades del sistema de gestión de
desarrollo ministerial de la organización cristiana.
Se probará que la aplicación cumpla con los requerimientos de alto nivel que fueron especificados
previamente, verificando que se cumple satisfactoriamente con las funcionalidades y características
necesarias.
6.3.3 ROLES Y RESPONSABILIDADES DEL EQUIPO DE PRUEBAS
Recursos humanos
Cargo
Recursos
necesarios
Administrador
Erick Benites
de pruebas
mínimos Responsabilidades específicas / comentarios
Proporcionar atención especial al funcionamiento
correcto de las tareas principales del sistema.
Responsabilidades:
Diseñador
pruebas
de Erick Benites
Proporcionar dirección técnica.
Adquirir los recursos apropiados.
Administración de reportes.
Identificar, asignar la prioridad, e implementar los
casos de la prueba a ejecutar.
Responsabilidades:
Ejecutores
prueba
de Pablo Guevara
Administrador
Erick Benites
del sistema de
pruebas
Generar el plan de prueba.
Generar la especificación de los distintos tipos de
prueba.
Generar el modelo de prueba.
Evaluar la eficacia del esfuerzo en la prueba.
Realizar las pruebas
Responsabilidades:
Ejecutar pruebas.
Registrar resultados.
Recuperación después de errores.
Documentación de errores.
Asegurar el ambiente de prueba, así como mantener y
manejar sus ventajas.
Responsabilidades:
Administrar el manejo de pruebas del sistema.
Página 194
Administrador
Erick Benites
de la Definición
de Datos
Controlar el acceso de los integrantes del equipo a los
sistemas de prueba.
Asegurar el ambiente de los datos de prueba, así como
manejar y mantener sus ventajas.
Responsabilidades:
Administrar los datos de prueba.
Tabla 91: Roles y responsabilidades del equipos de Pruebas (Fuente: propia)
6.3.4 PRERREQUISITOS
Especificaciones de Casos de Uso
6.3.5 ALCANCE
DENTRO DEL ALCANCE
Para las pruebas de certificación se validará el funcionamiento de los casos de uso desarrollados en la
prueba de concepto.
FUERA DEL ALCANCE
Toda aquella funcionalidad no definida en las especificaciones de casos de Uso.
6.3.6 ESTRATEGIA DE PRUEBAS
La funcionalidad del aplicativo se validará siguiendo los flujos identificados, teniendo en cuenta los
requerimientos especificados en las especificaciones de caso de uso.
Las pruebas a ejecutar serán de tipo manual y se usará como herramienta un archivo Excel para guardar
las evidencias de las observaciones encontradas.
6.3.7 DOCUMENTACIÓN DE RESPALDO
Las evidencias se guardarán en el archivo Excel de Pruebas adjunto.
6.3.8 FUNCIONALIDAD DEL SISTEMA
Se verificará la funcionalidad del sistema según los siguientes Casos de Pruebas:
•
AV_CUS040_Notificación de seguimiento al líder
•
AV_CUS024_Importar información de cursos llevados y disponibles
Página 195
6.3.9 CASOS DE PRUEBA
Caso de Uso
Escenario
Resultado Esperado
Persona
probó
que
Escenario 040-01
Recibe una notificación y/o
Erick Benites /
Notificar miembros correo de miembro nuevo a
Equipo Calidad
asignados
contactar.
Escenario 040-02
Recibe una notificación y/o
AV_CUS040_Notificación Notificar reporte de correo de reporte de visita Erick Benites /
Equipo Calidad
de seguimiento al líder
visita pendiente
pendiente.
Escenario 040-03
Recibe una notificación y/o
Erick Benites /
Notificar miembro correo de miembro no
Equipo Calidad
abandonado
contactado por mucho tiempo.
Escenario 024-01
Informar al usuario que no
Erick Benites /
Validar
datos existe datos nuevos a
Equipo Calidad
existentes a importar importar.
Escenario 024-02
Reglas
de
asociación Erick Benites /
Importar datos y
recalculadas.
Equipo Calidad
generar reglas
AV_CUS024_Importar
información de cursos Escenario 024-03
Mostrar lista de cursos Erick Benites /
llevados y disponibles
Consultar
cursos
recientemente importados.
Equipo Calidad
importados
Escenario 024-04
Consultar
Mostrar lista de inscripciones Erick Benites /
inscripciones
recientemente importadas.
Equipo Calidad
importadas
Fecha de Prueba
04/11/2017
04/11/2017
04/11/2017
04/11/2017
04/11/2017
04/11/2017
04/11/2017
Tabla 92: Casos de Prueba (Fuente: propia)
Matriz de Flujos vs Escenarios
AV_CUS040_Notificación de seguimiento al líder
Escenario
040-01
Flujo1
Paso 1, Paso 2, Flujo Alterno (creyentes
recientemente asignados),Paso 3, Paso 4, Paso 5
Escenario
040-02
Escenario
040-03
X
Flujo2
Paso 1, Paso 2, Paso 3, Flujo Alterno (reportes de
visita pendiente), Paso 4, Paso 5
Flujo3
Paso 1, Paso 2, Paso 3, Paso 4, Flujo Alterno
(creyentes abandonados), Paso 5
X
X
Tabla 93: Matriz caso de uso AV_CUS040 (Fuente: propia)
AV_CUS024_Importar información de cursos llevados y disponibles
Escenario
024-01
Flujo1
Paso 1, Paso 2, Paso 3, Paso 4, Paso
Escenario
024-02
Escenario
024-03
Escenario
024-04
X
Página 196
5, Paso 6, Paso 7, Paso 8, Paso 9,
Paso 10
Flujo2
Paso 1, Paso 2, Paso 3, Paso 4, Paso
5, Paso 6, Paso 7, Subflujo 1 (Ver
cursos...), Paso 4, Paso 5
Flujo3
Paso 1, Paso 2, Paso 3, Paso 4, Paso
5, Paso 6, Paso 7, Subflujo 2 (Ver
inscripciones…), Paso 4, Paso 5
Flujo4
Paso 1, Paso 2, Paso 3, Paso 4,
Flujo Alterno, Paso 8, Paso 9, Paso
10
X
X
X
Tabla 94: Matriz caso de uso AV_CUS024 (Fuente: propia)
Estos casos de prueba se han generado cumpliendo dos criterios:
•
Análisis: Determinando el número de casos de prueba y variedad a considerar contando para ello
con la información obtenida de los Casos de Uso
•
Desarrollo de los casos de prueba: se elaboraron los casos de prueba teniendo en cuenta la Matriz
y descripción de los distintos Caso de Pruebas
6.3.10 AMBIENTE DE PRUEBAS
REQUISITOS DE HARDWARE Y SOFTWARE
Para la ejecución de los casos de prueba, se utilizará una PC como estación de trabajo, configurada con
las especificaciones mínimas, las cuales son las siguientes:
•
Procesador: Intel Core I3 CPU o superior.
•
Sistema Operativo: Microsoft Windows 7 o superior.
•
Ram: 4 GB
•
Video: 800 x 600, High Color (16 bit) y 1024 x 768, 256 colores
•
Navegador Google Chrome o Microsoft Internet Explorer 11
•
Último Service Pack y actualizaciones críticas de Windows
•
Acceso a internet
Página 197
REQUISITOS DE ACCESO A SISTEMAS
Se deberá poder acceder al sistema con todos los perfiles definidos para cada actor de los diferentes
flujos de proceso especificados.
6.3.11 PRUEBAS ESPECIALES
Pruebas Unitarias
Se realizaron al culminar el desarrollo de cada caso de uso.
Pruebas Integrales
No Aplica.
6.3.12 PRUEBAS DE ACEPTACIÓN
Las pruebas de aceptación serán realizadas por el Usuario Final del aplicativo. Producción se
compromete a sugerir el Set de Casos a probar, quedando finalmente a criterio del Gerente del Proyecto
y del Usuario el alcance de las mismas.
Al finalizar sus pruebas se deberá emitir un informe final, así como la conformidad de las pruebas
realizadas.
6.3.13 TIEMPOS Y RECURSOS REQUERIDOS
•
El tiempo estimado de las pruebas es 8 días
•
Participantes en las pruebas. - 2 recursos.
Pruebas y Control de calidad
16 días
21/10/2017 – 06/11/2017
Elaborar plan de pruebas
2 días
21/10/2017 – 23/10/2017
Erick Benites
Diseñar casos de pruebas
4 días
23/10/2017 – 27/10/2017
Erick Benites
Ejecución de pruebas
5 días
27/10/2017 – 01/11/2017
Pablo Guevara
Corrección de defectos en pruebas
3 días
01/11/2017 – 04/11/2017
Erick Benites
Revisión de correcciones
2 días
04/11/2017 – 06/11/2017
Pablo Guevara
Tabla 95: Pruebas y control de calidad (Fuente: propia)
Página 198
6.4
EJECUCIÓN DE LOS CASOS DE PRUEBAS
6.4.1 CASO DE PRUEBA AV_CP040_NOTIFICACIÓN DE SEGUIMIENTO AL LÍDER
A continuación, se describe el caso de prueba para el caso de uso “AV_CUS040_Notificación de
seguimiento al líder” con sus diferentes escenarios:
Caso de Prueba AV_CP_038_Notificación de seguimiento al líder.xlsx
6.4.2 CASO DE PRUEBA AV_CP024_IMPORTAR INFORMACIÓN DE CURSOS
LLEVADOS Y DISPONIBLES
A continuación, se describe el caso de prueba para el caso de uso “AV_CUS024_Importar información
de cursos llevados y disponibles” con sus diferentes escenarios:
Caso de Prueba AV_CP_024_Importar información de cursos llevados y disponibl
Página 199
6.5
CONCLUSIONES
En este capítulo se identificó que la organización no cuenta con una política de calidad, por lo tanto se
identificó cual serían los lineamientos de calidad que se proponen a seguir y basado en esos lineamientos
se definen los objetivos de calidad de la organización Comunidad Cristiana Agua Viva. Por lo tanto,
esto fue el punto de partida para definir los objetivos de calidad y determinar las métricas aplicadas al
software construido.
La política de calidad de la organización y los objetivos de la calidad permiten alinear las métricas y los
casos de prueba para garantizar la calidad basada en los requerimientos funcionales del sistema. Los
requerimientos del sistema son esenciales para medir la calidad del software a entregar, ya que al no
cumplir con los requerimientos de los usuarios señala que no se está brindando un producto de calidad.
Se revisó la normatividad aplicable para la organización, ya que no cuenta con una normatividad
específica para el desarrollo de software se eligió la norma ISO 25000 debido a que es la norma vigente
y remplazó a la norma 9126 y basado en esta norma se eligieron las métricas adecuadas para el software
considerando los requerimientos que se tuvo al inicio del desarrollo. Cada una de las normas y estándares
existentes sirven como guía para aplicar ingeniería de Software y ofrecer un producto de calidad.
El plan de pruebas permite planificar y tener un control de las pruebas a aplicar en el desarrollo del
software y basado en este plan de pruebas se definen un set de pruebas con escenarios que permitirán
probar y validar cada uno de los casos de uso del sistema, y cada escenario ha sido evidenciado con una
captura de pantalla de la aplicación y adjuntada a los documentos Excel presente en el documento.
Página 200
7
CAPITULO 7: CONSTRUCCIÓN
7.1
INTRODUCCIÓN
En el presente capítulo se detalla los patrones de diseño seleccionados para la construcción del software
junto a sus respectivos diagramas como la evidencia de su aplicación en el código fuente del sistema.
Además, se presenta el modelo de datos que da soporte al software como su respectivo diccionario de
datos.
7.2
PATRONES DE LA SOLUCIÓN PROPUESTA
En la siguiente sección se exponen los patrones de diseño utilizados en el desarrollo del sistema.
Asimismo, esto permite demostrar las buenas prácticas de desarrollos aplicadas en la construcción del
software que contribuyen en la entrega de un producto de calidad. Entre los patrones a mencionar
tenemos; el patrón MVC, Repository y Adapter.
7.2.1 PATRÓN MVC
Nombre del Patrón
Modelo Vista Controlador (MVC)
Problema que se
Situaciones en las que los bloques de código encargados de la lógica
busca resolver
de negocio y acceso a datos se encuentran entreverada con bloques de
código relacionado a mostrar la información. Esto complica la
mantenibilidad y el desarrollo de componentes especializados y
reutilizables.
Solución
Propone la división en tres capas, la capa modelo encargada del acceso
a datos y la lógica de negocio, la capa de vista encargada de la lógica
de presentación y el controlador se encarga de intermediar entre ambas
capas. Esto favorece a la administración correcta de los diferentes
componentes, facilita la funcionalidad, mantenibilidad y escalabilidad
del sistema.
Ventajas
•
•
•
•
•
•
Contribuye al desarrollo de un sistema modular y escalable.
Separa la lógica de presentación y la lógica de negocio.
Permite la reutilización de componentes especializados.
Permite el uso de plantillas de diseño.
Facilita el mantenimiento en caso de presentarse errores
dentro de alguna de las capas.
Mejor entendimiento y adopción por parte de los
programadores.
Página 201
Desventajas
Motivo
experiencia de uso
•
Dicho patrón está orientado a objetos, por lo cual su
implementación es compleja y costosa al inicio del proyecto,
más aún si no se cuenta con buenos frameworks que faciliten
su implementación.
y Actualmente es uno de los patrones de diseños más utilizados en un
desarrollo de aplicaciones web, existen muchos frameworks de
desarrollo que la implementan y ofrecen una puesta en marcha de las
aplicaciones en minutos. Permite, además, la reutilización de
componentes del modelo para las distintas interfaces de hoy en día
como web y mobile. Asimismo, permite que la aplicación sea escalable
y reduciendo los errores por parte de los desarrolladores, mejorando la
calidad del producto final.
Tabla 96: Patrón MVC (Fuente: propia)
Diagrama del Patrón MVC
Figura 71: Patrón MVC (Fuente propia)
Página 202
Evidencia del uso del Patrón MVC
Modelo
Vista
Controlador
Figura 72: Estructura MVC (Fuente: propia)
Página 203
Figura 73: Modelo Grupo.php (Fuente: propia)
Página 204
Figura 74: Vista grupos/view.blade.php (Fuente: propia)
Página 205
Figura 75: Controlador GruposController.php (Fuente: propia)
Página 206
7.2.2 PATRÓN REPOSITORY
Nombre del Patrón
Repositorio (Repository)
Problema que se
Los componentes que contienen la lógica de negocio se encuentra
busca resolver
fuertemente acoplada de la lógica de acceso a datos, en caso sea
necesario cambiar el tipo de fuente de datos de una base de datos
relacional a un archivo xml, json o servicio se presentará muchas
dificultades para dar mantenimiento a la aplicación.
Solución
El patrón Repositorio permite la separación de la lógica de negocio y
la lógica de acceso a una fuente de datos de forma que nos ofrece una
capa de abstracción que nos sirve de datos sin importar de dónde o de
qué maneras estas son obtenidas.
Ventajas
•
•
•
Desventajas
Motivo
experiencia de uso
•
Desacopla nuestra lógica de negocio de nuestra fuente de datos
a través de una nueva capa de abstracción que hace más simple,
reutilizable y mantenible la lógica de acceso y la persistencia
de la información.
Facilita la creación de componentes de acceso a datos
reutilizables e intercambiables.
Favorece el análisis y la determinación de causa de errores.
Incrementa el grado de complejidad al añadir una capa más de
abstracción en nuestro sistema.
y Esta buena práctica es muy difundida dentro del desarrollo de
aplicaciones web por la facilidad que nos ofrece para desarrollar
aplicaciones más complejas con solo reutilizar componentes, además
muchas de las operaciones habituales con los objetos de datos son
registrados en estos componentes como crear, leer, actualizar,
eliminar, etc y hace que el mantenimiento sea menos costoso.
Tabla 97: Patrón Repository (Fuente: propia)
Página 207
Diagrama del Patrón Repositorio
Figura 76: Patrón Repositorio (Fuente https://bosnadev.com/2015/03/07/using-repository-pattern-in-laravel-5/)
Figura 77: Evidencia del uso del Patrón Repositorio (Fuente: propia)
Página 208
7.2.3 PATRÓN ADAPTER
Nombre del Patrón
Adaptador (Adapter)
Problema que se
Situaciones en las cuales es necesario interactuar dos tipos
busca resolver
componentes cuyas interfaces son incompatibles.
Solución
El patrón Adapter define una clase adaptadora que se encarga de la
traducción de la interface de la clase adaptada a una interface
compatible con la clase cliente.
Ventajas
•
•
•
•
Desventajas
Motivo
experiencia de uso
•
Permite encapsular clases aún más complejas que no podemos
controlar en interfaces estándares para nuestra aplicación.
Si nuestros componentes adaptados cambiar o aparecen otros
simplemente podeos crear otro adaptador para este caso.
Permite que los componentes de vista fácilmente puedan
cambiar el tipo de fuente de datos con solo cambiar el
adaptador.
Se asemeja mucho en la realidad con el adaptador de enchufe
de corriente.
El diseño se hace aún más complejo, puesto que requiere de
otros componentes intermediarios.
y Dentro del desarrollo móvil en Android se suele utilizar este patrón de
diseño puesto que existen componentes de vista (RecyclerView) que
crear listas de datos pero estas vistas deben
interactuar con
componentes de fuentes de datos diversos (base de datos, archivos,
servicios, etc), para ello dichas vistas hacen uso de adaptadores que les
facilita el acceso a esta información sin importar su naturaleza.
Tabla 98: Patrón Adapter (Fuente: propia)
Página 209
Diagrama del Patrón Adapter
Figura 78: Patrón Adapter (Fuente propia)
Evidencia del uso del Patrón Repositorio
Figura 79: Cliente (Fuente: propia)
Página 210
Figura 80: Adaptador (Fuente: propia)
7.3
MODELO DE DATOS
En esta sección se describe el modelo de datos que da soporte al software, así como su diccionario de
datos. En esta parre se construye el diagrama de datos según el diseño del almacenamiento físico en la
base de datos de la solución que se desarrolla. El nombre de las tablas y las columnas debe coincidir con
la construcción física de los datos.
Página 211
7.3.1 MODELO DE DATOS FÍSICO DEL SISTEMA
Figura 81: Modelo de Datos Físico del Sistema (Fuente: propia)
Página 212
7.3.2 DICCIONARIO DE DATOS
Tabla: creyentes
Descripción: Almacena todos los creyentes que tiene la organización.
Nombre
Tipo
No Nulo
PK
FK
Default
id
INT
Si
Si
No
Identificador único del creyente.
nombres
VARCHAR(100)
Si
No
No
Nombres del creyente
apellidos
VARCHAR(100)
Si
No
No
Apellidos del creyente
username
VARCHAR(100)
No
No
No
Nombre de usuario del creyente
password
VARCHAR(100)
No
No
No
Contraseña del creyente
sexo
CHAR(1)
No
No
No
Sexo del creyente, M:
Masculino o F: Femenino
fecnacimiento
DATE
No
No
No
Fecha de nacimiento del
creyente
dni
VARCHAR(10)
No
No
No
Número del documento de
identidad del creyente
correo
VARCHAR(100)
No
No
No
Correo electrónico del creyente
telefono
VARCHAR(20)
No
No
No
Número de teléfono fijo del
creyente
celular
VARCHAR(20)
No
No
No
Número de celular del creyente
distritos_id
CHAR(6)
No
No
Si
Identificador del distrito de
domicilio del creyente
direccion
VARCHAR(100)
No
No
No
Dirección de domicilio del
creyente
referencia
VARCHAR(100)
No
No
No
Referencia de la dirección de
domicilio del creyente
picture
VARCHAR(100)
No
No
No
Nombre de archivo de la foto
del creyente
lpdp
CHAR(1)
No
No
No
Aceptación a la Ley de
protección de datos del creyente
facebook_id
VARCHAR(64)
No
No
No
Identificador de la cuenta de
Facebook del creyente
google_id
VARCHAR(64)
No
No
No
Identificador de la cuenta de
Google del creyente
remember_token
VARCHAR(100)
No
No
No
Token de sesión persistente
registrantes_id
INT
No
No
Si
Identificador del líder que
registra al creyente
created_at
TIMESTAMP
No
No
No
Fecha de registro de datos del
creyente
updated_at
TIMESTAMP
No
No
No
Fecha de actualización de datos
del creyente
estado
CHAR(1)
Si
No
No
'A'
Comentarios
Estado del registro del creyente
Tabla 99: Creyentes (Fuente: propia)
Página 213
Tabla: departamentos
Descripción: Almacena todos los departamentos del Perú.
Nombre
Tipo
No Nulo
PK
FK
Default
Comentarios
id
CHAR(2)
Si
Si
No
Identificador único
del departamento
nombre
VARCHAR(50)
Si
No
No
Nombre del
departamento
Tabla 100: Departamentos (Fuente: propia)
Tabla: provincias
Descripción: Almacena todas las provincias del Perú.
Nombre
Tipo
No Nulo
PK
FK
Default
Comentarios
id
CHAR(4)
Si
Si
No
Identificador único de la
provincia
nombre
VARCHAR(50)
Si
No
No
Nombre de la provincia
departamentos_id
CHAR(2)
Si
No
Si
Identificador del departamento
donde se encuentra la provincia
Tabla 101: Provincias (Fuente: propia)
Tabla: distritos
Descripción: Almacena todos los distritos del Perú.
Nombre
Tipo
No Nulo
PK
FK
Default
Comentarios
id
CHAR(6)
Si
Si
No
Identificador único del distrito
nombre
VARCHAR(50)
Si
No
No
Nombre del distrito
provincias_id
CHAR(4)
Si
No
Si
Identificador de la provincia
donde se encuentra el distrito
Tabla 102: Distritos (Fuente: propia)
Tabla: lideres
Descripción: Almacena a todos los líderes de la organización.
Nombre
Tipo
No Nulo
PK
FK
Default
Comentarios
id
INT
Si
Si
Si
Identificador único del líder
numcedula
VARCHAR(45)
No
No
No
Número de Grupo asignado al
líder
created_at
TIMESTAMP
No
No
No
Fecha de registro del líder
updated_at
TIMESTAMP
No
No
No
Fecha de actuación del líder
Tabla 103: Líderes (Fuente: propia)
Página 214
Tabla: cursos_programados
Descripción: Almacena todos los cursos programados para la escuela de líderes.
Nombre
Tipo
No Nulo
PK
FK
Default
id
INT
Si
Si
No
Identificador único del curso
programado
cursos_biblicos_id
INT
Si
No
Si
Identificador de curso bíblico
que se ha programado
periodo
VARCHAR(50)
No
No
No
Periodo académico en del año
fecinicio
DATE
No
No
No
Fecha de inicio del curso
programado
fecfin
DATE
No
No
No
Fecha final del curso
programado
horarios
VARCHAR(50)
No
No
No
Horario generado para el curso
programado
estado
CHAR(1)
Si
No
No
'A'
Comentarios
Estado del curso programado
Tabla 104: Cursos programados (Fuente: propia)
Tabla: consolidaciones
Descripción: Almacena todos los procesos de consolidación de los creyentes.
Nombre
Tipo
No Nulo
PK
FK
Default
Comentarios
id
INT
Si
Si
No
Identificador único de la consolidación de
un creyente
creyentes_id
INT
Si
No
Si
Identificador del creyente
lideres_id
INT
Si
No
Si
Identificador del líder asignado
fecasignacion
DATETIME
No
No
No
Fecha de asignación de líder a creyente
feccierre
DATETIME
No
No
No
Fecha de cierre de la consolidación del
creyente
estado
CHAR(1)
Si
No
No
'A'
Estado del creyente en su proceso de
consolidación (A: Activo, C: Cerrado, X:
No desea ser contactado, I:Inactivo)
Tabla 105: Consolidaciones (Fuente: propia)
Tabla: programaciones
Descripción: Almacena las programaciones de contacto de los nuevos creyentes.
Nombre
Tipo
No Nulo
PK
FK
Default
Comentarios
id
INT
Si
Si
No
Identificador único de la
programación de contacto
consolidaciones_id
INT
Si
No
Si
Identificador de la consolidación del
nuevo creyente
fechahora
DATETIME
No
No
No
Fecha y hora de la programación de
contacto
tipo
CHAR(2)
Si
No
No
Tipo de programación de contacto
(V1: Visita 1, V2: Visita 2)
Página 215
estado
CHAR(1)
Si
No
No
'P'
Estado de la programación de
contacto (P: Pendiente, C: Cerrado,
N: No ubicado)
Tabla 106: Programaciones (Fuente: propia)
Tabla: reportes
Descripción: Almacena los reportes del contacto con el creyente.
Nombre
Tipo
No
Nulo
PK
FK
Default
Comentarios
id
INT
Si
Si
No
Identificador único del reporte del
nuevo creyente
programaciones_id
INT
Si
No
Si
Identificador de la programación
de contacto al creyente
estados_id
INT
Si
No
Si
Identificador de estado del reporte
observaciones
VARCHAR(500)
No
No
No
Observaciones de la visita
sugerencias
VARCHAR(500)
No
No
No
Comentario o aporte sobre la
consolidación del creyente
Tabla 107: Reportes (Fuente: propia)
Tabla: cursos_bíblicos
Descripción: Almacena todos los cursos bíblicos de la escuela de líderes.
Nombre
Tipo
No Nulo
PK
FK
Default
Comentarios
id
INT
Si
Si
No
Identificador único del curso bíblico
nombre
VARCHAR(100)
Si
No
No
Nombre del curso bíblico
areas_id
INT
Si
No
Si
Identificador del área que pertenece el
curso bíblico
frecuencia
INT
Si
No
No
0
Número de frecuencia con que se dicta
del curso bíblico
estado
CHAR(1)
Si
No
No
'A'
Estado actual del curso bíblico
Tabla 108: Cursos bíblicos (Fuente: propia)
Tabla: areas
Descripción: Almacena las áreas donde se encuentran los cursos bíblicos.
Nombre
Tipo
No Nulo
PK
FK
Default
Comentarios
id
INT
Si
Si
No
Identificador único del área que pertenece
un curso bíblico
nombre
VARCHAR(45)
Si
No
No
Nombre del área
Tabla 109: Areas (Fuente: propia)
Página 216
Tabla: creyentes_areas
Descripción: Almacena las áreas donde se encuentran los cursos bíblicos.
Nombre
Tipo
No Nulo
PK
FK
Default
Comentarios
areas_id
INT
Si
Si
Si
Identificador del área de cursos
creyentes_id
INT
Si
Si
Si
Identificador del creyente que va un
curso
Tabla 110: Creyentes áreas (Fuente: propia)
Tabla: inscripciones
Descripción: Almacena las inscripciones de los creyentes a los cursos bíblicos.
Nombre
Tipo
No
Nulo
PK
FK
Default
id
INT
Si
Si
No
Identificador único de la inscripción
de un creyente
cursos_programados_id
INT
Si
No
Si
Identificador de un curso
programado
creyentes_id
INT
Si
No
Si
Identificador de un creyente
fecregistro
DATETIME
No
No
No
Fecha de la inscripción del creyente
estado
CHAR(1)
Si
No
No
'A'
Comentarios
Estado actual de la inscripción
Tabla 111: Inscripciones (Fuente: propia)
Tabla: cursos_relaciones
Descripción: Almacena las reglas de asociación de cursos.
Nombre
Tipo
No
Nulo
PK
FK
Default
Comentarios
id
INT
Si
Si
No
confianza
DECIMAL(6,2)
Si
No
No
0
Nivel de confianza de la
regla de asociación
fecha
TIMESTAMP
Si
No
No
CURRENT_TIMESTAMP
Fecha de registro de la
regla de asociación
Identificador de la regla de
asociación
Tabla 112: Cursos relaciones (Fuente: propia)
Tabla: migraciones
Descripción: Almacena todos los procesos de migración de cursos e inscripciones.
Nombre
Tipo
No Nulo
PK
FK
Default
Comentarios
id
INT
Si
Si
No
Identificador del proceso de
migración
creyentes_id
INT
Si
No
Si
Identificador del creyente
fecinicio
DATETIME
No
No
No
Fecha y hora de inicio del
proceso de migración
Página 217
fecfin
DATETIME
No
No
No
Fecha y hora de finalización
del proceso de migración
descripcion
VARCHAR(500)
No
No
No
Detalles del proceso de
migración
totalprogramaciones
INT(10)
No
No
No
Número total de
programaciones migradas
totalinscripciones
INT(10)
No
No
No
Número total de inscripciones
migradas
estado
CHAR(1)
Si
No
No
'C'
Estado del proceso de
migración
Tabla 113: Migraciones (Fuente: propia)
Tabla: grupos
Descripción: Almacena las migraciones de las inscripciones y programaciones de los cursos bíblicos
Nombre
Tipo
No
Nulo
PK
FK
Default
Comentarios
id
INT
Si
Si
No
Identificador único del grupo familiar
descripcion
VARCHAR(100)
No
No
No
Descripción del grupo familiar
latitud
DECIMAL(12,9)
No
No
No
Latitud de geolocalización
longitud
DECIMAL(12,9)
No
No
No
Longitud de geolocalización
direccion
VARCHAR(100)
No
No
No
Dirección del grupo familiar
distritos_id
CHAR(6)
Si
No
Si
Identificador del distrito del grupo
familiar
telefono
VARCHAR(20)
No
No
No
Teléfono del grupo familiar
dia
INT(1)
No
No
No
Día que se realiza el grupo familiar
hora
INT(2)
No
No
No
Hora del grupo familiar
lideres_id
INT
Si
No
Si
Identificador del líder del grupo familiar
codcelula
VARCHAR(10)
No
No
No
Código del grupo familiar
tipocelula
CHAR(1)
Si
No
No
0
Tipo del grupo familiar
tiporeunion
CHAR(1)
Si
No
No
0
Tipo de reunión del grupo familiar
estado
CHAR(1)
Si
No
No
'A'
Estado del grupo familiar
Tabla 114: Grupos (Fuente: propia)
Tabla: instancias
Descripción: Alamacena las instancias de la aplicación móvil en cada dispositivo del usuario.
Nombre
Tipo
No Nulo
PK
FK
Default
Comentarios
id
INT
Si
Si
No
identificador de la instancia
creyentes_id
INT
Si
No
Si
Identificador del creyente
instanceid
VARCHAR(152)
Si
No
No
Token único de la instancia
created_at
TIMESTAMP
No
No
No
Fecha de registro de la instancia
updated_at
TIMESTAMP
No
No
No
Fecha de actualización de la instancia
Página 218
estado
CHAR(1)
Si
No
No
'A'
Estado de la instancia
Tabla 115: Instancias (Fuente: propia)
Tabla: roles
Descripción: Almacena los roles del sistema.
Nombre
Tipo
No Nulo
PK
FK
Default
Comentarios
id
INT
Si
Si
No
Identificador del rol
nombre
VARCHAR(50)
Si
No
No
Nombre del rol
Tabla 116: Roles (Fuente: propia)
Tabla: creyentes_roles
Descripción: Almacena las relaciones de muchos a muchos entre los roles y los creyentes.
Nombre
Tipo
No Nulo
PK
FK
Default
Comentarios
creyentes_id
INT
Si
Si
Si
Identificador del creyente
roles_id
INT
Si
Si
Si
Identificador del rol
Tabla 117: Creyentes_roles (Fuente: propia)
Tabla: peticiones
Descripción: Almacena las peticiones de oración de cada creyente.
Nombre
Tipo
No
Nulo
PK
FK
Default
id
INT
Si
Si
No
Identificador de la
petición
creyentes_id
INT
Si
No
Si
Identificador del
creyente
tipopeticiones_id
INT
Si
No
Si
Identificador del
tipo de petición
contenido
VARCHAR(500)
Si
No
No
Contenido del
pedido de oración
created_at
TIMESTAMP
No
No
No
updated_at
TIMESTAMP
No
No
No
estado
CHAR(1)
Si
No
No
CURRENT_TIMESTAMP
Comentarios
Fecha de registro
de la petición
Fecha de
actualización de la
petición
'A'
Estado de registro
de la petición
Tabla 118: Peticiones (Fuente: propia)
Página 219
Tabla: tipopeticiones
Descripción: Almacena los tipos de pedido de oración.
Nombre
Tipo
No Nulo
PK
FK
Default
Comentarios
id
INT
Si
Si
No
Identificador del tipo de pedido de oración
detalle
VARCHAR(100)
Si
No
No
Nombre del tipo de pedido de oración
Tabla 119: Tipopeticiones (Fuente: propia)
Tabla: reportes_estados
Descripción: Almacena los estados disponibles para los reportes.
Nombre
Tipo
No
Nulo
PK
FK
Default
id
INT
Si
Si
No
Identificador único del estado de reporte
descripcion
VARCHAR(100)
Si
No
No
Descripción del estado de reporte
estado
CHAR(1)
Si
No
No
'A'
Comentarios
Estado del registro para el estado de
reporte
Tabla 120: Reportes_estados (Fuente: propia)
Tabla: cursos_adquiridos
Descripción: Almacena la lista de cursos adquiridos relacionados a una regla de asociación.
Nombre
Tipo
No Nulo
PK
FK
Default
Comentarios
cursos_relaciones_id
INT
Si
Si
Si
Identificador de la regla de asociación
cursos_biblicos_id
INT
Si
Si
Si
Identificador del curso bíblico adquirido
Tabla 121: Cursos_adquiridos (Fuente: propia)
Tabla: cursos_sugeridos
Descripción: Almacena la lista de cursos sugeridos relacionados a una regla de asociación.
Nombre
Tipo
No Nulo
PK
FK
Default
Comentarios
cursos_relaciones_id
INT
Si
Si
Si
Identificador de la regla de asociación
cursos_biblicos_id
INT
Si
Si
Si
Identificador del curso bíblico sugerido
Tabla 122: Cursos_sugeridos (Fuente: propia)
Página 220
7.4
CONCLUSIONES
En el presente capítulo se describió algunos de los patrones de diseño aplicados dentro del desarrollo del
software, los usos de estas buenas prácticas fueron argumentadas con las ventajas y experiencias de uso
que estas ofrecen para un tipo de sistema web y móvil. Estos patrones de diseño dan una solución
específica de acuerdo al desarrollo de software que se hace para la organización.
Finalmente se detalla el modelo de datos y su respectivo diccionario que dan el soporte necesario al
sistema que se está desarrollando porque es de acuerdo al modelo físico de los datos que se usan en la
organización.
Página 221
8
CAPITULO 8: GESTIÓN DEL PROYECTO
8.1
INTRODUCCIÓN
En este capítulo se presenta el cronograma de actividades correspondiente a esta primera etapa del
proyecto, así como el EDT del proyecto indicando lo que abarcará el proyecto. Finalmente se muestra
el acta de conformidad de parte del cliente.
8.2
REGISTRO DE INTERESADOS
#
1
Interesado
Pedro Hornung
Cargo
Presidente de la Junta de
Administración
2
Sergio Hornung
Pastor principal
3
José León
4
Raúl Cabrera
5
Raúl Llamoza
Vicepresidente Junta de
Administración
Secretario Junta de
Administración
Jefe de Sistemas
6
Pablo Guevara
Jefe del proyecto
7
Erick Benites
Jefe del Desarrollo de
Software del proyecto
8
Miguel García
9
Jimmy García
10
Erick arias
11
Giancarlo Cabrera
12
13
14
Luis Contreras
Sandra Perona
Lizardo Silva
Encargado del área de
Consolidación
Encargado del área de
comunicaciones
Encargado del área de
sonido
Encargado del grupo de
música
Asesor de tesis
Asesor de tesis
Asesor de tesis
Descripción
Persona responsable de la toma de
decisiones a nivel económico y
administrativo.
Responsable del proceso ministerial en la
organización.
Persona responsable de la gestión del
personal y otros procesos administrativos.
Responsable del proceso de manejo y buena
administración de las finanzas.
Persona responsable de la gestión de los
sistemas y proyectos de TI en la dirección.
Integrante del equipo del Proyecto
encargado de dirigir y supervisar el
proyecto.
Integrante del equipo del proyecto que se
encarga del desarrollo del Software en todas
sus etapas.
Miembro y líder de la iglesia que participa
en las reuniones de la iglesia los domingos.
Miembro y líder de la iglesia que participa
en las reuniones de la iglesia los domingos.
Miembro y líder de la iglesia que participa
en las reuniones de la iglesia los domingos.
Miembro y líder de la iglesia que participa
en las reuniones de la iglesia los domingos.
Asesor de tesis en proyecto informático I
Asesor de tesis en proyecto informático II
Asesor de tesis en proyecto informático III
Tabla 123: Registro de Interesados del Proyecto (Fuente: propia)
Página 222
8.3
EDT
Figura 82: Estructura de Desglose del Trabajo del Proyecto (Fuente: propia)
Página 223
8.4
CRONOGRAMA DE EJECUCIÓN
Nombre de tarea
PROYECTO SISTEMA DE GESTIÓN DE
DESARROLLO MINISTERIAL
Fundamentos Teóricos
Levantamiento observaciones de Perfil de Proyecto
Marco Teórico
Objeto de estudio
Campo de acción
Análisis crítico de los problemas de información
Propuestas de Solución
Objetivos del proyecto
Beneficios del proyecto
Antecedentes
Tendencias y tecnologías propuestas
Modelado del Negocio
Reglas del Negocio
Modelos de caso de uso del negocio
Modelo de análisis del negocio
Realización de casos de uso del negocio
Gestionar Iniciación
Consolidar Creyentes
Organizar Encuentros
Realizar Escuela De Líderes
Organizar Grupos Familiares
Lista de actividades a automatizar
Requerimientos
Requerimientos Funcionales
Requerimientos No Funcionales
Modelo de Casos de Uso de Sistema
Diagrama de paquetes de sistema
Diagrama de actores de sistema
Diagrama de casos de uso de sistema
Atributos de casos de uso de sistema
Especificación de alto nivel de casos de uso de sistema
Especificación Detallada de los Casos de Uso de Sistema
Modelo Conceptual
Definición de clases entidad
Definición de los atributos de las clases
Definición de las asociaciones entre clases
Definición de la multiplicidad y navegabilidad de las
clases
Elaboración del diccionario de datos
Arquitectura de Software
Diagrama de los CUS más significativos para la
arquitectura
Metas de la arquitectura de software
Duración Comienzo
Fin
119 días mar 19/03/19 vie 30/08/19
9 días
1 día
3 días
2 días
1 día
2 días
9 días
3 días
2 días
2 días
2 días
7 días
2 días
3 días
2 días
12 días
2 días
2 días
2 días
2 días
2 días
2 días
5 días
3 días
2 días
9 días
1 día
1 día
2 días
1 día
2 días
2 días
6 días
2 días
1 día
1 día
mar 19/03/19 vie 29/03/19
mar 19/03/19 mar 19/03/19
mie 20/03/19 vie 22/03/19
lun 25/03/19 mar 26/03/19
mie 27/03/19 mie 27/03/19
jue 28/03/19 vie 29/03/19
lun 01/04/19 jue 11/04/19
lun 01/04/19 mie 03/04/19
jue 04/05/19 vie 05/04/19
lun 08/04/19 mar 09/04/19
mie 10/04/19 jue 11/04/19
vie 12/04/19 lun 22/04/19
vie 12/04/19 lun 15/04/19
mar 16/04/19 jue 18/04/19
vie 19/04/19 lun 22/04/19
mar 23/04/19 mie 08/05/19
mar 23/04/19 mie 24/04/19
jue 25/04/19 vie 26/04/19
lun 29/04/19 mar 30/04/19
mie 01/05/19 jue 02/05/19
vie 03/05/19 lun 06/05/19
mar 07/05/19 mie 08/05/19
jue 09/05/19 mie 15/05/19
jue 09/05/19 lun 13/05/19
mar 14/05/19 mie 15/05/19
jue 16/05/19 mar 28/05/19
jue 16/05/19 jue 16/05/19
vie 17/05/19 vie 17/05/19
lun 20/05/19 mar 21/05/19
mie 22/05/19 mie 22/05/19
jue 23/05/19 vie 24/05/19
lun 27/05/19 mar 28/05/19
mie 29/05/19 mie 05/06/19
mie 29/05/19 jue 30/05/19
vie 31/05/19 vie 31/05/19
lun 03/06/19 lun 03/06/19
1 día
mar 04/06/19 mar 04/06/19
1 día
15 días
mie 05/06/19 mie 05/06/19
jue 06/06/19 mie 26/06/19
1 día
jue 06/06/19
jue 06/06/19
1 día
vie 07/10/17
vie 07/10/17
Página 224
Restricciones de la arquitectura de software
Mecanismos de la arquitectura de software
Vista lógica de la arquitectura de software
Vista de implementación de la arquitectura de software
Vista de despliegue de la arquitectura de software
Programación de los dos CUS del núcleo central
Prueba de concepto de la arquitectura de software
Calidad y Pruebas del Software
Plan de la calidad del software
Pruebas del software
Construcción
Patrones de diseño
Modelo de Datos
Construcción de los casos de uso más significativos
Gestión de Proyectos
Elaboración de informe y actas de reunión
Elaboración del cronograma del proyecto
Elaboración de la EDT
Elaboración bibliográfica, glosario y conclusiones
1 día
1 día
1 día
1 día
1 día
6 días
2 días
10 días
5 días
5 días
37 días
3 días
4 días
30 días
11 días
2 días
4 días
2 días
3 días
lun 10/06/19 lun 10/06/19
mar 11/06/19 mar 11/06/19
mie 12/06/19 mie 12/06/19
jue 13/06/19 jue 13/06/19
vie 14/06/19 vie 14/06/19
lun 17/06/19 lun 24/06/19
mar 25/06/19 mie 26/06/19
vie 27/06/19 mie 10/07/19
jue 27/06/19 mie 03/07/19
jue 04/07/19 mie 10/07/19
jue 11/07/19 vie 30/08/19
jue 11/07/19 lun 15/07/19
mar 16/07/19 vie 19/07/19
lun 22/07/19 vie 30/08/19
mar 19/03/19 mie 16/10/19
mar 19/03/19 mie 20/03/19
jue 21/13/19 mar 26/03/19
mie 27/03/19 jue 28/03/19
vie 29/03/19 mar 02/04/19
Tabla 124: Cronograma de Ejecución del Proyecto (Fuente: Propia)
Página 225
8.5
ACTAS DE ACEPTACIÓN
Acta de Aceptación PI1
Página 226
Acta de Aceptación PI2
Página 227
Acta de Aceptación PI3
Página 228
8.6
CONCLUSIONES
La correcta Gestión del Proyecto nos permite tener un mayor control sobre cada una de las
actividades y tareas desarrolladas de cada capítulo, haciendo una buena gestión del tiempo
en cada proceso a desplegar desde la planificación hasta el desarrollo de los casos de uso del
ciclo cero del software. Asimismo, nos ayuda a realizar las acciones correctivas en algunos
procesos y ajustar el tiempo con el fin de evitar retrasos y cumplir con los plazos
establecidos.
Finalmente, la gestión del proyecto permite desarrollar de manera eficiente el sistema, así
como también organizar, planificar y cumplir con la presentación de los entregables en los
tiempos establecidos.
Página 229
9
CONCLUSIONES GENERALES
El presente trabajo ha permitido detallar información relevante con respecto a la
organización y al proceso de desarrollo ministerial, esto ayudó a analizar su situación actual
y la problemática presente en las actividades de dicho proceso. Durante las reuniones que se
tuvo con el Jefe de área de Sistemas se observó la predisposición de este para mejorar el
proceso ministerial con el buen uso de la tecnología.
Los objetivos del proyecto se definieron en base a las necesidades que se encontraron en el
análisis del proceso de desarrollo Ministerial que tiene la organización. Luego se detalló los
beneficios tangibles e intangibles que el sistema tendrá y se hizo una comparación con
soluciones existentes que al evaluarlas no son las óptimas para la organización y la propuesta
de solución que se propone, busca generar un valor agregado en la buena gestión de la
información de las personas. Todo esto con el objetivo de llevar una mejor administración y
optimización en los procesos del negocio y de cumplir con las funcionalidades y necesidades
que la organización requiere.
También se identificó los casos de uso, los actores, los trabajadores y entidades del negocio,
así como la relación que existe entre ellos. También se elaboró los diagramas de actividades
que permitió aclarar a detalle el flujo de actividades en cada una de las áreas del proceso
ministerial. La herramienta utilizada fue el lenguaje de modelado unificado (UML), que nos
facilitó el modelado y la comprensión de las actividades para luego ser automatizadas en el
sistema que se está desarrollando.
La identificación de los requerimientos funcionales llevará a la proyección de las funciones
del sistema. La descripción de los requerimientos no funcionales ayudó en el desarrollo de
la plataforma del sistema. La construcción del Modelo de Casos de Uso del Sistema permitió
la definición de la arquitectura del sistema, estos ayudaron a definir los casos de uso del ciclo
cero que resolverán en gran parte con la problemática de la organización. Asimismo, se
definieron seis paquetes del sistema que representan a las áreas más importantes dentro del
campo de acción. Finalmente se elabora un modelo conceptual que describe las distintas
entidades y relación entre ellas que servirán de soporte para la persistencia de la información
del sistema.
Las principales metas de la arquitectura de software fueron identificadas, así como las
restricciones técnicas presentes en cada una de ellas, esto nos permite proponer mecanismos
que nos ayuden a cumplir estas metas que son parte de los requerimientos no funcionales.
Página 230
Además, se detalló el escenario en el cual se desplegará los componentes de software como
el diagrama de despliegue y el diagrama de implementación. También se tiene el diagrama
de vista lógica la cual nos permitirá conocer la distribución de capas dentro del software
propuesto.
Posteriormente se definió cual era la normativa de calidad aplicable para el software que se
está desarrollando y los casos de prueba a desarrollar sobre los casos de uso de la prueba de
concepto para que garantice la calidad del software según los estándares que los usuarios
esperan del aplicativo.
En la fase de construcción se describió los patrones de diseño aplicados en el desarrollo del
software que aseguran un producto escalable y bajo costo en mantenimiento, además de la
descripción del modelo de datos que sirve de soporte al sistema.
La correcta Gestión del Proyecto nos permite tener un mayor control sobre cada una de las
actividades y tareas desarrolladas de cada etapa del proyecto. Asimismo, nos ayuda a realizar
las acciones correctivas en algunos procesos y ajustar el tiempo con el fin de evitar retrasos
y cumplir con los plazos establecidos.
En conclusión, la gestión del proyecto permite desarrollar de manera eficiente el sistema, así
como también organizar, planificar y cumplir con la presentación de los entregables en los
tiempos establecidos.
Finalmente se ha desarrollado un software que cumple con el objetivo de gestionar de manera
eficiente la información del Proceso Ministerial, y permitirá contar con información en línea
vía web y móvil para uso de los líderes y los pastores principales de la organización. Este
software desarrollado cumple con los objetivos específicos de actualizar la información de
las personas manteniéndola centralizada y disponible oportunamente. El sistema permite el
control y seguimiento del desarrollo ministerial de las personas que llegan a la organización.
También permite la notificación de alertas cuando se produzcan una falta de comunicación
o contacto entre los líderes y las personas asignadas a él. Asimismo, permite la inscripción
en línea de los creyentes a los cursos disponibles en la escuela de líderes. También permite
el control y seguimiento de los cursos que llevan los estudiantes. El sistema permite el
control y seguimiento del desempeño del líder en base a métricas que controlen su
participación y crecimiento.
Página 231
10 GLOSARIO DE TÉRMINOS
[1] A1 Es el formato para llenar datos de las personas que llegan por primera vez a la iglesia
por su propia cuenta sin que nadie les haya invitado.
[2] Célula Es el nombre de un grupo familiar.
[3] Consolidación Es el cuidado de las personas nuevas que llegan a la iglesia. Se les llama
por teléfono para luego visitarlos, el objetivo es que asistan a un Grupo familiar y participen
en las actividades de la organización.
[4] Consolidador Representa al líder quien hace la función de realizar la llamada y visita al
nuevo creyente; lo anima a asistir al Grupo Familiar y demás actividades de la organización.
[5] Encuentro Es un retiro espiritual en donde se realiza una reunión donde van desde 60
hasta 250 personas en una casa de retiro al sur de Lima para recibir temas de familia y de
liderazgo.
[6] Escuela de Líderes es el área donde se desarrollan los cursos bíblicos para la preparación
y formación de nuevos líderes.
[7] Evento Evangelístico Son reuniones donde se comparte una actividad con las personas
invitadas y se le comparte el evangelio de Cristo.
[8] Grupo Familiar Es un grupo familiar de personas que se reúnen una vez por semana en
una casa, allí hay un líder que comparte un mensaje bíblico.
[9] Proceso Ministerial Es el proceso donde se desarrollan todas las áreas como son
consolidación, encuentros, escuela de líderes y grupos familiares.
[10] Trigger Es un lanzador de notificaciones.
[11] Radar es un porcentaje de logro o avance en algún área ministerial con respecto a su
meta cuatrimestral. Cada área tiene un indicador con su fórmula correspondiente.
[12] Escuela Es un grupo que puede estar conformado por más de cien personas, algunos
llegan hasta mil, dos mil, y más personas. Están representados por un Pastor que tiene un
código de escuela que lo distingue de las demás escuelas.
[13] Pastor Es un líder que dirige una escuela de multitudes de personas. Tiene una
representación a través de un código que lo diferencia de otros pastores. Hay pastores en
Lima, en los conos y en las provincias.
Página 232
[14] Código jerárquico del líder (Código de Grupo) Es un código que de acuerdo al nivel
jerárquico tiene una cantidad de caracteres. Por ejemplo, el A1 es del pastor principal
hombre. El A2 es de la pastora principal. Estos a su vez tienen doce pastores bajo su
dirección que tendrían el siguiente nivel de jerarquía de bajo. Por ejemplo, el A12, El
siguiente nivel de bajo del árbol sería el A124, y así sucesivamente hasta llegar a la última
jerarquía de bajo de los líderes que hay en la organización. Para los conos los códigos son
C11, C12, C13, etc.
[15] Métrica del radar Es una fórmula que se saca en base al número de grupos familiares
que tiene el líder. Cada área ministerial tiene su métrica. Ver Anexo N° 4.
[16] Tipo de Grupo Hay dos tipos de grupos: Evangelística es cuando los miembros del
grupo la conforman nuevos convertidos. De líderes cuando la conforman solo líderes.
[17] Tipo de Reunión Hay dos tipos de reunión: Adultos, son personas que participan desde
la edad de los 30 años. Jóvenes, son personas que participan desde la edad de los 15 a 30
años.
[18] TAB Es cuando el usuario presiona alguna opción de la app en su Smartphone.
[19] Nuevo creyente Es la persona que llega por primera vez a la organización y empieza a
involucrase en las actividades de la misma.
[20] Preencuentro Es el curso previo que los nuevos creyentes necesitan llevar para poder
asistir a un encuentro.
Página 233
11 SIGLARIO
AN Actor del Negocio
AS Actor del Sistema
AV Agua Viva
CCAV Comunidad Cristiana agua Viva
CP Casos de prueba
CRM (Customer Relationship Management) o gestión de relación con los clientes. Es una
estrategia para enfocar los objetivos de una organización en los clientes.
CUN Caso de Uso del negocio
CUS Caso de Uso del Sistema
DNI Documento nacional de identidad
EDT Estructura de Descomposición del Trabajo
EN Entidad del Negocio
ESADE Universidad Ramón LLULL de España
FODA Fortalezas, Oportunidades, Debilidades. Amenazas
IEC Comisión Electrotécnica Internacional
ISO Organismo Internacional de Estándares
RF Requerimiento Funcional
RNF Requerimiento no funcional
RN Regla de Negocio
SUNAT Superintendencia Nacional de Administración Tributaria
TR Trabajador del negocio
UML Lenguaje unificado de modelado
Página 234
12 REFERENCIAS
Agua Viva | Comunidad Cristiana - http://www.ccaguaviva.com/press/
Mckenna, R. (1993). Relationship Marketing: Successful strategies for the age of the
consumer, Boston: Harvard School Business Press.
Llamas Alonso, M. R., Jiménez Zarco, y Dawson, J. (2006). Medición de resultados en la
estrategia CRM. Boletín Económico de Ice Nº 2887
Mendoza Quijada, M. M., & Vilela Goicochea, Y. S. (2014). Impacto de un modelo de
sistema CRM en la fidelización de los clientes de la distribuidora ferretera Ronny L
S.A.C. dela ciudad de Trujillo en el año 2014.
Noboa Herrera, B. M. (2016). El CRM para mejorar la calidad de servicio al cliente en el
banco del litoral para el año 2016. Guayaquil.
Díaz Alba, E., & Pinzón Salgado, M. A. (2014). Sistema de información web para la
gestión de clientes (CRM) para la empresa Mi-Martinez. Bogota.
García, I. (2001). CRM: gestión de la relación con los clientes. FC Editorial.
Croxatto, H. (2005). Creando valor en la relación con sus clientes. Dunken.
Gartner, I. (28 de 04 de 2017). Gartner IT. Una estrategia de negocios diseñada para
optimizar la rentabilidad, las utilidades y la satisfacción del Cliente.
Brunetta, H. (2014). Del marketing relacional al CRM. Buenos Aires: Todo Management.
Business Software. (2014). Top 40 CRM Software. Consultado el 03 de abril 2017.
Disponible en: http://www.business-software.com/offer/top-40-CRM-software/
Cabello, M. (2007). Sistemas de Gestión integrados para la Empresa: CRM | Digital
Learning - Formación online en Nuevas Tecnologías. Consultado el 4 de abril 2017.
Disponible en: http://www.digitallearning.es/tutoriales/sistemas-de-gestion-integradospara-la-empresa-CRM.html
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Croxatto, H. (2005). Creando Valor En La Relacion Con Sus Clientes (1° ed.). Buenos
Aires, Dunken: Editorial Dunken. Consultado el 04 de abril 2017. Disponible en:
https://books.google.es/books?id=2Ce8Ud3qrw8C
Chen, I. J., y Popovich, K. (2003). Understanding customer relationship management
(CRM): People, process and technology. Consultado el 04 de abril 2017.
Todman, C. (2000). Designing a Data Warehouse: Supporting Customer Relationship
Management. Upper Saddle River, NJ, USA. Consultado el 04 de abril 20147
Greenberg, P. (2003). CRM gestión de relaciones con los clientes (2003rd ed.). Madrid,
España: Mcgraw-hill/interamericana de España, S.A.U.
INCHE, P. E. (sábado de diciembre de 2011). Iglesias Evangélicas o protestantes.
Obtenido de
http://iglesiasevangelicasperu.blogspot.pe/search/label/Iglesia%20Evang%C3%A9lica%20
o%20protestante
INCHE, P. E. (domingo de julio de 2015). IGLESIA EVANGELICA LAS ASAMBLEAS
DE DIOS DEL PERU. Obtenido de
http://iglesiasevangelicasperu.blogspot.pe/search/label/IGLESIA%20ASAMBLEAS%20D
E%20DIOS%20DEL%20PERU
Mendoza, L. E., Marius, A., Pérez, M., y Grimán, A. C. (2007). Critical success factors for
a customer relationship management strategy. Consultado el 03 de abril 2017.
Morales, E., & González, J. (23 de 01 de 2012). Instituto Nacional de Astrofísica, Óptica y
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T. Menzies, Y. Hu. Data Mining For Busy People. IEEE Computer, Outubro de 2003, pgs.
18-25
Berzal, F. (2013). Departamento de Ciencias de la Computación e I.A. Universidad de
Granada. Obtenido de http://elvex.ugr.es/
R. Agrawal; T. Imielinski; A. Swami: Mining Association Rules Between Sets of Items in
Large Databases", SIGMOD Conference 1993: 207-216
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Agrawal, R., & Srikant, R. (1994). Fast Algorithms for Mining Association Rules.
Proceedings of the 20th International Conference on Very Large Data Bases, (págs. 487499).
Omnigaea. (05 de Enero de 2017). Qué son las APPS. Recuperado el 15 de Febrero de
2017, de http://www.omnigaea.com/mundo-app/que-son-las-apps/
Android, X. (08 de Febrero de 2011). ¿Qué es Android? Recuperado el 15 de Febrero de
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Administrador Fiel | Sistema comparativo 2 - https://administradorfiel.com/
ISO. (7 de noviembre de 2017). ISO / IEC 25000. Obtenido de
https://www.iso.org/standard/64764.html
ISO. (2008). ISO/IEC 25010. En SQUARE, Software Product Quality Model (págs. 1421).
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http://iso25000.com/index.php/normas-iso-25000/iso-25010
ISO. (2008). ISO/IEC 25010. En SQUARE, System Quality in Use Model (págs. 21-24).
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Justicia, M. d. (13 de octubre de 2003). Resolución Ministerial. Obtenido de
http://sistemas3.minjus.gob.pe/transparencia2/Directivas-ReglamentosMINJUS/Implementaci%C3%B3n%20de%20la%20Direcci%C3%B3n%20de%20Asuntos
%20Interconfesionales%20de%20la%20DNJ.pdf
Página 237
13 ANEXOS
Anexo 1
Formato AMC / A1.
Anexo 2
Hoja de Registro de Participantes que asisten al curso de Pre encuentro para ir al
Encuentro
Página 238
Anexo 3
Diagnóstico actual del desarrollo ministerial
Anexo 4
Propuesta de indicadores de proyección de crecimiento (RADARES)
Página 239
Anexo 5
Encuesta de satisfacción de uso del sistema para la métrica de uso
https://goo.gl/forms/lTmnv2aZdgaGnFly2
Página 240
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Correction: Correlation factors of translanguaging practices on English as a Foreign Language contexts: the perception of teachers
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Muhammad Alrayes
malrayes@ksu.edu.sa Correction: Correlation factors of translanguaging practices
on English as a Foreign Language contexts: the perception
of teachers Muhammad Alrayes1 Published online: 17 November 2023
© The Author(s) 2023 Published online: 17 November 2023
© The Author(s) 2023 Education and Information Technologies (2024) 29:1247
https://doi.org/10.1007/s10639-023-12359-3 Education and Information Technologies (2024) 29:1247
https://doi.org/10.1007/s10639-023-12359-3 CORRECTION CORRECTION 1
English Language Department, College of Language Sciences, King Saud University,
Riyadh, Saudi Arabia The online version of the original article can be found at https://doi.org/10.1007/s10639-023-12137-1. Education and Information Technologies
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English Language Department, College of Language Sciences, King Saud University,
Riyadh, Saudi Arabia
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No commercial reuse without permission. See http://emh.ch/en/services/permissions.html. Errata | Published 04 November 2021 | doi:10.4414/SMW.2021.w30104
Cite this as: Swiss Med Wkly. 2021;151:w30104 The correct wording is: Treatment with tafamidis 61 mg
should be considered for patients with cardiac ATTRwt
amyloidosis and dyspnea NYHA I-III. [IIa, B]. Published under the copyright license “Attribution – Non-Commercial – No Derivatives 4.0”.
No commercial reuse without permission See http://emh ch/en/services/permissions html Swiss Medical Weekly · PDF of the online version · www.smw.ch Published under the copyright license “Attribution – Non-Commercial – No Derivatives 4.0”.
No commercial reuse without permission. See http://emh.ch/en/services/permissions.html. Published under the copyright license “Attribution – Non-Commercial – No Derivatives 4.0”.
No commercial reuse without permission. See http://emh.ch/en/services/permissions.html. Erratum to: Management of transthyretin
amyloidosis Erratum to Swiss Med Wkly. 2021;151:w30053 (DOI 10.4414/smw.2021.w30053 The correct wording is: Treatment with tafamidis 61 mg
should be considered for patients with cardiac ATTRwt
amyloidosis and dyspnea NYHA I-III. [IIa, B]. The published manuscript contained a typing error in the
chapter "Wild-type transthyretin amyloidosis (ATTR-
wt)", subchapter "Treatment" (page 13 of the PDF ver-
sion). It reads: Treatment with tafamidis 61 mg should
be considered for patients with cardiac ATTRwt
amyloidosis and dyspnea NYHA I-II. [IIa, B]. The error occured during manuscript editing and is not in
the authors responsability. It has now been corrected on-
line. We apologize to the authors and readers for this error. Swiss Medical Weekly · PDF of the online version · www.smw.ch Page 1 of 1
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LINGUISTIC VARIATION BETWEEN MALE AND FEMALE STUDENTS IN ACADEMIC WRITING
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Language literacy : journal of linguistics, literature, and language teaching
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| ERROR: type should be string, got "https://jurnal.uisu.ac.id/index.php/languageliteracy \nNationally Accredited SINTA 3, and indexed in DOAJ and Copernicus Language Literacy: Journal of Linguistics, Literature, and Language Teaching \nVolume 6, Number 2, pp: 423-436, December 2022 \ne-ISSN: 2580-9962 | p-ISSN: 2580-8672 \n \n \nDOI: 10.30743/ll.v6i2.5910 Language Literacy: Journal of Linguistics, Literature, and Language Teaching \nVolume 6, Number 2, pp: 423-436, December 2022 \ne-ISSN: 2580-9962 | p-ISSN: 2580-8672 \n \n \nDOI: 10.30743/ll.v6i2.5910 Accepted: 2022-09-26 Accepted: 2022-09-26 Abstract The variation of linguistic characteristics in academic writing between male \nand female is inevitable to happen. Therefore, this study aimed at finding \nout the differences of male and female students in linguistic characteristics \nof academic writing from perspective of linguistics, especially at the level of \ntext analysis. The quantitative research method with convergent parallel \ndesign was used to analyze sixty students’ essay writing chosen by using a \npurposive sampling technique. The result revealed that there were some \ndifferences of linguistic characteristics in academic writing between male \nand female. The female students outperformed male students in all \nlinguistic characteristics in text-level analysis. The female students were \nfound more capable in paragraphing competences, linking devices, text \noutput counts, topical organization, and ending. The findings generally \nsupport the notion that differences of linguistic characteristics between \nmale and female should be considered in teaching academic writing to \ncreate a friendly teaching-learning process for both genders. Received: 2022-09-08 Received: 2022-09-08 Received: 2022-09-08 Surya Asra\n1, Grace Josephine Tiwon Wiradisastra\n2 \n1Universitas Samudra, Langsa, Indonesia \n2Universitas Indonesia, Depok, Indonesia \nE-mail: suryaasra2019@unsam.ac.id Surya Asra\n1, Grace Josephine Tiwon Wiradisastra\n2 \n1Universitas Samudra, Langsa, Indonesia \n2Universitas Indonesia, Depok, Indonesia \nE-mail: suryaasra2019@unsam.ac.id Published: 2022-12-29 Published: 2022-12-29 1. Introduction The research on the influence of gender in language teaching has been carried out by \nmany experts (Andini & Prasetyowati, 2021; Cameron, 1997; Fattahi & Nushi, 2021; Gtowka, \n2014; Mahmud, 2018; Sunderland, 2000, 2006). The studies have been conducted since \ngender becomes one of factors that causes diversity in language (Holmes, 2013). However, \nthere are only a few of the studies that examine gender focused on writing skill (Reynolds et \nal., 2015; Sunderland, 2000; Waskita, 2008). This fact has an explanation that many people \nincluding some researchers consider speaking skill as a benchmark in assessing someone’s \nlanguage skill (Waskita, 2008). As a result, the other skills (listening, reading, and writing) are \nrarely used as research topics, especially in relation to gender influence. The above assumption is not fully wrong, yet the contribution of literacy skill, \nespecially writing skill cannot be ignored. Writing skill is one of the important factors to \nachieve a better future career. Tuan (2010) reveals that the success of EFL learners in English \nwriting skill brings benefits not only for the learning process but also for a better career in \nthe future. Many tasks in working life must be accomplished by writing, such as compiling \nreport, writing scientific journal, composing speech, and so on. Therefore, writing skill is an 423 Linguistic Variation between Male and Female Students in Academic Writing, Surya Asra, Grace Josephine \nTiwon Wiradisastra Linguistic Variation between Male and Female Students in Academic Writing, Surya Asra, Grace Josephine \nTiwon Wiradisastra important factor in academic life, especially in university. In the context of higher education, \nthe function of writing skill becomes very important. Students are required to write an \nacademic paper at the end of their study at university. Besides, they are also required to \ntake academic written tests such as TOEFL and IELTS. Therefore, gender studies in relation to \nwriting skill is very necessary to conduct. The influence of gender is manifested in language products produced by men and \nwomen, both in writing and speaking. Newman, Groom, Handelman, and Pennebaker (2008) \nstate that gender differences in language use between men and women are consistent. Furthermore, Hamdan and Hamdan (2013) state that from birth both genders have followed \ndifferent codes of conduct according to existing community rules and this has caused men \nand women to have different life experiences, ways of speaking, and ways of writing. https://jurnal.uisu.ac.id/index.php/languageliteracy \nNationally Accredited SINTA 3, and indexed in DOAJ and Copernicus 1. Introduction In \nsummary, writing skill as one of language skills cannot be separated from the influence of \ngender in it. Therefore, the study of gender influence in writing skill is the first step to \nunderstand the differences and then applies it to teaching-learning process of writing. There are three perspectives developed by Jones (2011) to explain the influence of \ngender in writing skill, namely cognitive psychology, socio-cultural, and linguistic \nperspectives. This study focused on linguistic perspective for analyzing written text (student \nwriting product). Linguistic analysis is in line with characteristics of academic writing in the \nclassroom context, especially for academic writing in higher education. The linguistic \nperspective analyzed the linguistic features of a text, such as text and lexical diversity. The \nanalysis carried out in this study was limited on analysis of linguistic characteristics in text \nlevel. 2. Literature Review The linguistic perspective is a comprehensive analysis that analyses written text \nbased on linguistic characteristics in text and sentence (Jones & Myhill, 2007). Linguistic \ncharacteristics in text-level analysis consist of topical organization, paragraphing \ncompetence, closing paragraph (endings), linking devices, and text output counts. Topical organization is a topic setting process of a paragraph to fit the topic of text \nwriting. The topic of text writing is usually determined by the organizer of writing test, and \nthis is an absolute requirement in writing text. In this study, there are four aspects of topical \norganization analyzed, consisting of: (1) paragraphs commencing with a topic sentence, (2) \nlogical order to paragraphs or following a systematic compilation of paragraphs into a logical \ntext, (3) paragraphs drifting off topic, and (4) paragraphs needing subdivision. Paragraphing competence is a process to arrange paragraphs into good text in a \nwriting. A good paragraph according to Oshima and Hogue (2006) and Johns (1997) must \nhave the following requirements: (1) topic sentence, (2) arranged logically, and (3) only one \nmain idea. In addition, Jones and Myhill (2007) and Johns (1997) state that a good paragraph \nmust use meta-discourse signs, references, and conclusion (it is not absolute). Whereas a \npoor paragraph can be seen from partial paragraphs (incomplete paragraphs), paragraphs \nwith one sentence, and the absence of paragraphs conditioned. Endings is a conclusion paragraph of an article. It consists of (1) thematic link with \nprevious paragraph, (2) thematic link with opening paragraph, (3) repetition of words or \nphrases in opening paragraph, (4) repetition of proper nouns in opening paragraph, and (5) \nsummaries, conclusions/ suggestions, or comments (Jones and Myhill, 2007). 424 Language Literacy: Journal of Linguistics, Literature, and Language Teaching \nVolume 6, Number 2, pp: 423-436, December 2022 \ne-ISSN: 2580-9962 | p-ISSN: 2580-8672 \n \n \nDOI: 10.30743/ll.v6i2.5910 Linking devices is hyphens that the author uses to connect words, sentences, and \nparagraphs into a complete article. The linking devices analyzed in this study consist of \n(Jones and Myhill, 2007): (1) temporal adverbials, (2) ordinal adverbials, (3) place adverbials, \n(4) manner adverbials, (5) additive adverbials, (6) adversative adverbials, (7) causal \nadverbials (8) repeated words; (9) repeated phrases; (10) repetition of proper nouns, (11) \nsynonyms, (12) hyponyms-hypernyms, (13) anaphoric pronouns, and (14) determiners. Text output counts is the total of characters, words, sentences, and paragraphs \nproduced by the author. https://jurnal.uisu.ac.id/index.php/languageliteracy \n \n425 \nNationally Accredited SINTA 3, and indexed in DOAJ and Copernicus 3. Research Method The method used in this research was a quantitative method. Quantitative data in \nthis study is in the form of numerical data obtained from coding linguistic characteristics of \nstudents’ essay writing and then this data is analyzed statistically (descriptive and \ninferential). Meanwhile, the convergent parallel design approach was used since the data \nfrom statistical analysis and data from coding linguistic characteristics in students writing \npaper were compared to find patterns and draw research conclusions (Creswell & Plano-\nClark, 2011; Nunan & Bailey, 2009). 3.1 Participants The population of this study were students of English Education Department who \nwere in the fourth semester 4 and they took Essay Writing course. There were 108 students \nwho participated in this study consisting of 48 male students and 60 female students. Meanwhile, the sample of this study was selected by purposive or judgmental sampling \ntechnique as the process of selecting a sample is based on the researcher's belief that the \nsample can be a representation of the population based on the researcher's knowledge of \nthe characteristics the group (Gay, Mills, & Airasian, 2006; Arikunto, 2013). As a result, 60 \nstudents were used as research samples consisting of 30 male students and 30 female \nstudents who were selected based on the characteristics that match the research objectives, \nnamely based on the average writing ability score. 2. Literature Review There are eight aspects of text output counts that are analyzed in \nthis study, including (Jones & Myhill, 2007): (1) number of characters (not including commas \nand spaces), (2) number of words, (3) number of sentences, (4) number of paragraphs, (5) \nnumber of sentences per paragraphs, (6) number of words per sentences, (7) number of \ncharacters per words, and (8) number of passive sentences. p\n,\n( )\np\n \nFor a brief description of text-level analysis, it can be seen in the following table. Category \nCriteria \nSub-criteria \nTopical \norganization \nParagraphs commencing with a \ntopic sentence \n \n \nLogical order to paragraphs \nParagraphs drifting off topic \nParagraphs needing subdivision \nParagraphing \ncompetence \nComplete paragraphs \nHaving a topic sentence, arranged \nlogically, explaining only one main idea \nUsing meta-discourse signs \nUsing references \nHaving a conclusion sentence \nIncomplete paragraphs \nPartial paragraphs \nParagraphs with one sentence \nNo paragraphs \n \nEndings \nThematic link with previous \nparagraph \n \nThematic link with opening \nparagraph \nRepetition of words or phrases in \nopening paragraph \nRepetition of proper nouns in \nopening paragraph \nSummaries, conclusions/ \nsuggestions, or comments \nLinking devices \nAdverbials \nTemporal \nOrdinal \nPlace \nManner \nAdditive \nAdversative \nCausal 425 Linguistic Variation between Male and Female Students in Academic Writing, Surya Asra, Grace Josephine \nTiwon Wiradisastra Repetition \nWords \nPhrases \nProper nouns \nSynonyms \n \nHyponym-hypernyms \nAnaphoric pronouns \nDeterminers \nText output counts \nNumber of characters \n \n \nNumber of words \nNumber of sentences \nNumber of paragraphs \nNumber of sentences per \nparagraphs \nNumber of words per sentences \nNumber of characters per words \nNumber of passive sentences \nAdapted from Jones and Myhill (2007) \nTable 1. Criterions for linguistic analysis in text-level analysis Repetition \nWords \nPhrases \nProper nouns \nSynonyms \n \nHyponym-hypernyms \nAnaphoric pronouns \nDeterminers \nText output counts \nNumber of characters \n \n \nNumber of words \nNumber of sentences \nNumber of paragraphs \nNumber of sentences per \nparagraphs \nNumber of words per sentences \nNumber of characters per words \nNumber of passive sentences \nAdapted from Jones and Myhill (2007) \nTable 1. Criterions for linguistic analysis in text-level analysis Table 1. Criterions for linguistic analysis in text-level analysis https://jurnal.uisu.ac.id/index.php/languageliteracy \nNationally Accredited SINTA 3, and indexed in DOAJ and Copernicus https://jurnal.uisu.ac.id/index.php/languageliteracy \nNationally Accredited SINTA 3, and indexed in DOAJ and Copernicus 3.3 Data analysis The data analysis procedure was by using SPSS v.22 and coding of the writing \nadapted from Jones and Myhill categories (2007), particularly analysis in text level (see table \n1). The statistical analysis was conducted to obtain an overview of the significance level of \ndifferences between male and female students. Descriptive and inferential statistical \nanalysis used included t-test to see the significance of the overall linguistic characteristics \nand two-way ANOVA test (two-way analysis of variance) to see the level of significance of \naspects of linguistics. The results of statistical analysis from the SPSS program were \npresented in the form of a table of mean and significance values with a comparative \nsignificance level of 5% (p=0.05). It means that the significance value was categorized to be \n“significant” if it was lower than p=0.05 and “insignificant” if it was higher than p=0.05. 3.2 Instruments Data for this study were collected through an academic written test of five-paragraph \nessay. The test was a paper-based test consisting of five paragraphs with one paragraph of \nintroduction, three paragraph of body, and one paragraph of conclusion. https://jurnal.uisu.ac.id/index.php/languageliteracy \nNationally Accredited SINTA 3, and indexed in DOAJ and Copernicus 426 Language Literacy: Journal of Linguistics, Literature, and Language Teaching \nVolume 6, Number 2, pp: 423-436, December 2022 \ne-ISSN: 2580-9962 | p-ISSN: 2580-8672 \n \n \nDOI: 10.30743/ll.v6i2.5910 Language Literacy: Journal of Linguistics, Literature, and Language Teaching \nVolume 6, Number 2, pp: 423-436, December 2022 \ne-ISSN: 2580-9962 | p-ISSN: 2580-8672 \n \n \nDOI: 10.30743/ll.v6i2.5910 4. Results and Discussion It was found that there were differences in linguistic characteristics produced \nbetween male and female students. The researchers found that female students \noutperformed male students in all linguistic characteristics in text-level analysis. 4.1. Topical organization The researchers found that female student ability to organize a topic in academic \nwriting were better than male student ability. This is due to the tendency of male students \nnot to follow the rules of good paragraph compilation, for example not having the same \ntopic among paragraphs and not arranging paragraphs in logical order. The following table \nshows a different frequency of topical organization aspects between male and female \nstudents. Aspects of topical organization \nFrequency of gender differences \nMale \nFemale \nParagraphs commencing with a topic sentence \n96 \n118 \nLogical order to paragraphs \n7 \n18 \nParagraphs drifting off topic \n54 \n32 \nParagraphs needing subdivision \n54 \n38 \nTable 3. Frequency of topical organization. Aspects of topical organization Firstly, for paragraphs commencing with a topic sentence, female students produced \n118 paragraphs, while male students produced 96 paragraphs. There are 22 paragraphs of \ndifference. Secondly, for logical order to paragraphs, there were only 7 male students who \narranged their paragraphs sequentially and logically, while there are 18 female students who \narranged their paragraphs sequentially and logically. The difference is 11 students. Thirdly, \nfor paragraphs drifting off topic, male students wrote 54 paragraphs that do not fit the topic, \nwhile female students only wrote 32 paragraphs. The difference is 22 paragraphs. Fourthly, \nfor paragraphs needing subdivision, male students wrote 54 paragraphs, while female \nstudents only wrote 38 paragraphs. The difference is 16 paragraphs. Generally, it was found that female students produce more paragraphs than male \nstudents in aspects of paragraphs commencing with a topic sentence and logical order to \nparagraphs. While male students produce more paragraphs than female students in aspects 427 Linguistic Variation between Male and Female Students in Academic Writing, Surya Asra, Grace Josephine \nTiwon Wiradisastra of paragraphs drifting off topic and paragraphs needing subdivision. The statistical analysis \non each aspect of topical organization can be seen in the table below. graphs drifting off topic and paragraphs needing subdivision. The statistical analysis \nh aspect of topical organization can be seen in the table below. Aspects of topical organization \nMean \nSig. of \nANOVA Test \nMale \nFemale Difference \nParagraphs commencing with a topic sentence \n3.2 \n3.9 \n.7 \n.006 \nLogical order to paragraphs \n.233 \n.6 \n.367 \n.003 \nParagraphs drifting off topic \n1.8 \n1.067 \n.733 \n.006 \nParagraphs needing subdivision \n1.8 \n1.267 \n.533 \n.031 \nTable 4. Statistical test of topical organization All aspects of topical organization have a significant difference value. 4.1. Topical organization Their values are \nlower than significance level p=0.05. It means that the difference is statistically significant. In \nother words, female students’ ability on topical organization is better than male students. Therefore, writing instructors must emphasize to their students that they need to write \nparagraphs according to the topic required by test organizer are important in writing \nacademic. In addition, they also need to state to their students that they must be logical and \navoid writing two different ideas in one paragraph. Nationally Accredited SINTA 3, and indexed in DOAJ and Copernicus Nationally Accredited SINTA 3, and indexed in DOAJ and Copernicus https://jurnal.uisu.ac.id/index.php/languageliteracy \n \n428 \nNationally Accredited SINTA 3, and indexed in DOAJ and Copernicus //jurnal.uisu.ac.id/index.php/languageliteracy \n \n42 https://jurnal.uisu.ac.id/index.php/languageliteracy https://jurnal.uisu.ac.id/index.php/languageliteracy \n \n428 \nNationally Accredited SINTA 3, and indexed in DOAJ and Copernicus 4.2. Paragraphing competence It was found that female students were better than male students to form \nparagraphs. They tend to follow some rules in writing a good paragraph, such as having a \ntopic sentence, writing in logical order, and explaining only one main idea. On the contrary, \nmale students tend to violate the rules of good paragraph compilation. In addition, male \nstudents also tend not to pay attention to the conditions requested by the test organizer, \nincluding number of paragraphs needed. The following table presents a different frequency \nof paragraphing competence aspects between male and female students. p\ng\np\ng\np\np\nAspects of \nparagraphing \ncompetence \n \nFrequency of gender differences \nMale \nFemale \nComplete paragraphs \nHaving a topic sentence, \narranged logically, explaining \nonly one main idea \n111 \n126 \nUsing meta-discourse signs \n36 \n75 \nUsing references \n0 \n2 \nHaving a conclusion sentence \n16 \n38 \nTotal \n163 \n241 \nIncomplete \nparagraphs \nPartial paragraphs \n73 \n23 \nParagraphs with one sentence \n8 \n4 \nTotal \n81 \n27 \nNo paragraphs \n \n2 \n0 \nTotal \n2 \n0 \nTable 5. Frequency of paragraphing competence. Frequency of gender differences Frequency of gender differences Aspects of \nparagraphing \ncompetence Aspects of \nparagraphing \ncompetence Firstly, for complete paragraphs with a topic sentence, logical arrangement, only one \nmain idea, female students produced 126 paragraphs, while male students produced 111 \nparagraphs. The difference is 15 paragraphs. For complete paragraphs using meta-discourse 428 Language Literacy: Journal of Linguistics, Literature, and Language Teaching \nVolume 6, Number 2, pp: 423-436, December 2022 \ne-ISSN: 2580-9962 | p-ISSN: 2580-8672 \n \n \n \nDOI: 10.30743/ll.v6i2.5910 \n \nsigns, female students wrote 75 paragraphs, while male students wrote 36 paragraphs. There are 39 paragraphs of difference. For complete paragraphs using references, female \nstudents compiled 2 paragraphs, while there were no male students compiling paragraphs \nwith references. Then, for complete paragraphs with a conclusion sentence, female students \nproduced 38 paragraphs, while male students produced 16 paragraphs. The difference is 22 \nparagraphs. If all those aspects are summed, female students produced 241 paragraphs for \naspect of complete paragraphs, while male students produced 163 paragraphs. There are 78 \nparagraphs of difference. Language Literacy: Journal of Linguistics, Literature, and Language Teaching \nVolume 6, Number 2, pp: 423-436, December 2022 \ne-ISSN: 2580-9962 | p-ISSN: 2580-8672 \n \n \nDOI: 10.30743/ll.v6i2.5910 signs, female students wrote 75 paragraphs, while male students wrote 36 paragraphs. There are 39 paragraphs of difference. For complete paragraphs using references, female \nstudents compiled 2 paragraphs, while there were no male students compiling paragraphs \nwith references. p\nj\np p\ng\ng\ny\nNationally Accredited SINTA 3, and indexed in DOAJ and Copernicus //jurnal.uisu.ac.id/index.php/languageliteracy \n \n42 Nationally Accredited SINTA 3, and indexed in DOAJ and Copernicus nally Accredited SINTA 3, and indexed in DOAJ and Copernicus https://jurnal.uisu.ac.id/index.php/languageliteracy \n \n429 \nNationally Accredited SINTA 3, and indexed in DOAJ and Copernicus 4.2. Paragraphing competence Then, for complete paragraphs with a conclusion sentence, female students \nproduced 38 paragraphs, while male students produced 16 paragraphs. The difference is 22 \nparagraphs. If all those aspects are summed, female students produced 241 paragraphs for \naspect of complete paragraphs, while male students produced 163 paragraphs. There are 78 \nparagraphs of difference. Secondly, for incomplete/partial paragraphs, female students only produced 23 \nparagraphs, while male students produced 73 paragraphs. The difference is 50 paragraphs. Then, for incomplete paragraphs with one sentence, female students wrote only 4 \nparagraphs, while male students wrote 8 paragraphs. If both of them are summed, female \nstudents only produced 27 paragraphs, while male students produced 81 paragraphs. There \nare 54 paragraphs of difference. Thirdly, for no paragraphs, there is no mistake made by \nfemale students, while male students had the lack number of paragraphs required by the \ntest organizer (no two paragraphs). It can be concluded that the number of good paragraphs with a topic sentence, \nlogical arrangement, only one main idea, meta-discourse signs, references, and conclusion \nsentence produced by female students is more than male students. While male students \nproduced more bad paragraphs, such as partial paragraphs and paragraphs with one \nsentence than female students. Even, they made the absence of 2 paragraphs (no \nparagraphs) out of the total 150 paragraphs that should have been produced in the test. The \nstatistical analysis on each aspect of paragraphing competence can be seen in the table \nbelow. be o\nAspects of \nparagraphing \ncompetence \n \nMean \nSig. of \nANOVA \nTest \n \nMale \nFemale \nDifference \nComplete \nparagraphs \nHaving a topic \nsentence, arranged \nlogically, explaining \nonly one main idea \n3.7 \n4.2 \n.5 \n.028 \n \nUsing meta-discourse \nsigns \n1.2 \n2.5 \n1.3 \n.001 \n \nUsing references \n.000 \n.067 \n.065 \n.155 \n \nHaving a conclusion \nsentence \n.533 \n1.267 \n.734 \n.009 \nIncomplete \nparagraphs \nPartial paragraphs \n2.433 \n.767 \n1.666 \n.000 \n \nParagraphs with one \nsentence \n.233 \n.133 \n.1 \n.374 \nNo paragraphs \n \n.067 \n.000 \n.067 \n.155 \nTable 6. Statistical test of paragraphing competence Aspects of \nparagraphing \ncompetence It appears that there are four aspects of paragraphing competence that have a \nstatistically significant difference between male and female students (their values are lower 429 Linguistic Variation between Male and Female Students in Academic Writing, Surya Asra, Grace Josephine \nTiwon Wiradisastra Linguistic Variation between Male and Female Students in Academic Writing, Surya Asra, Grace Josephine \nTiwon Wiradisastra than significance level p=0.05). 4.2. Paragraphing competence There are (1) complete paragraphs with a topic sentence, \nlogical arrangement, and only one main idea, (2) complete paragraphs using meta-discourse, \n(3) complete paragraphs with a conclusion sentence, and (4) partial paragraphs. Related to \ncomplete paragraphs and partial paragraphs, writing instructors must emphasize to their \nstudent that there are three absolute requirements in compiling a good paragraph, \nespecially on a condition that there should be only one main idea in one paragraph. https://jurnal.uisu.ac.id/index.php/languageliteracy \n \n430 \nNationally Accredited SINTA 3, and indexed in DOAJ and Copernicus y Accredited SINTA 3, and indexed in DOAJ and Copernicus Nationally Accredited SINTA 3, and indexed in DOAJ and Copernicus 4.3. Endings Statistical test of endings 430 Language Literacy: Journal of Linguistics, Literature, and Language Teaching \nVolume 6, Number 2, pp: 423-436, December 2022 \ne-ISSN: 2580-9962 | p-ISSN: 2580-8672 \n \n \nDOI: 10.30743/ll.v6i2.5910 Language Literacy: Journal of Linguistics, Literature, an\nVolume 6, Number 2, pp: 423-436, December 2022 \ne-ISSN: 2580-9962 | p-ISSN: 2580-8672 \nDOI: 10.30743/ll.v6i2.5910 It can be seen that there are three aspects of endings that have statistically \nsignificant differences between male and female students, namely thematic link with \nprevious paragraph, thematic link with opening paragraph, and summaries, conclusions/ \nsuggestions, or comments. Their values are lower than significance level p= 0.05. Therefore, \nwriting instructors must pay attention to their student ability in compiling a closing \nparagraph, especially on significantly different aspects. https://jurnal.uisu.ac.id/index.php/languageliteracy \nNationally Accredited SINTA 3, and indexed in DOAJ and Copernicus 4.3. Endings It was found that the closing paragraph quality of female students was better than \nmale students. Yet, the difference is not so prominent between them because it only focuses \non the last paragraph. To see the difference on aspects of endings (closing paragraph) \nbetween male and female students, the following table presents their different frequency. Aspects of endings \nFrequency of gender \ndifferences \nMale \nFemale \nThematic link with previous paragraph \n21 \n28 \nThematic link with opening paragraph \n20 \n28 \nRepetition of words or phrases in opening paragraph \n7 \n11 \nRepetition of proper nouns in opening paragraph \n0 \n0 \nSummaries, conclusions/ suggestions, or comments \n20 \n29 \nTable 7. Frequency of endings Firstly, there were 28 female students who had closing paragraph related to previous \nparagraph, while there were 21 male students who had it. The difference is 7 students. Secondly, there were also 28 female students who had closing paragraph related to opening \nparagraph, while there were 20 male students who had it. There are 8 people of difference. Thirdly, for repetition of words or phrases in opening paragraph, there were 11 female \nstudents who repeated words or phrases in opening paragraph, while there were 7 female \nstudents who did it. The difference are 4 students. Fourthly, for repetition of proper nouns \nin opening paragraph, there were no students who did it, both male and female students. Fifthly, there were 29 female students who had closing paragraph containing summaries, \nconclusions/ suggestions, or comments, while there were only 20 male students who had it. The difference were 9 students. From those points, it can be concluded that closing \nparagraph aspects of female students are better than male students. The statistical analysis \non each aspect of endings (closing paragraph) can be seen in the table below. Aspects of endings \nMean \nSig. of \nANOVA Test \nMale Female \nDifference \nThematic link with previous paragraph \n.7 \n.933 \n.233 \n.019 \nThematic link with opening paragraph \n.667 \n.933 \n.266 \n.009 \nRepetition of words or phrases in opening \nparagraph \n.233 \n.367 \n.134 \n.267 \nRepetition of proper nouns in opening \nparagraph \n.000 \n.000 \n. . Summaries, conclusions/ suggestions, or \ncomments \n.667 \n.967 \n.3 \n.002 \nTable 8. Statistical test of endings Table 8. 4.4 Linking devices Female students wrote 806 items, while male students \nwrote 686 items with 120 items of difference. Secondly, for repeated words, female students produced 5.548 words, while male \nstudents produced 4.444 words. The difference is 1.104 items. For repeated phrases, female \nstudents wrote 383 phrases, while male students wrote 381 phrases. There are only 2 \nphrases. Then for repetition of proper nouns, it was not found in all writings, both male and \nfemale students. If those repetitions are summed, there are 5931 repetitions made by \nfemale students, while there are 4825 repetitions made by male students. There are 1105 \nrepetitions of words, phrases, and proper nouns. However, the small number of repetitions \ndoes not make writing of male students better than female students because the small \nrepetition also shows a small amount of text output (number of words and phrases \nproduced). Thirdly, female students used 241 synonyms, while male students used 231 \nsynonyms. The difference is 10 synonyms. With that slight difference, it can be concluded \nthat the ability to use synonyms between genders is no different. An article is categorized to \nbe good if the repetition of words/phrases is few and the use of synonyms increases. Yet, \nthis study found that the use of synonyms between male and female students is almost the \nsame. Fourthly, female students produced 87 hyponyms-hypernyms, while male students \nproduced 68 hyponyms-hypernyms. The difference is 19 hyponyms-hypernyms. It means \nthat the ability of female students to use hyponyms-hypernyms is better than female \nstudents. Therefore, the writing of female students becomes more detailed using more \nspecific terms. Fifthly, female students produced 608 anaphoric pronouns, while male \nstudents used 404 anaphoric pronouns. The difference is 204 anaphoric pronouns. Sixthly, \nfemale students used 1280 determiners, while male students used 1093 determiners. There \nare 187 determiners of difference. It means that the writing of female students has more \nwords than male students. Generally, it can be concluded that for the use of adverbials, repetitions, \nsynonyms, hyponyms-hypernyms, anaphoric pronouns, and determiners, female students \noutperformed male students. The statistical analysis on each aspect of linking devices can be \nseen in the table below. https://jurnal.uisu.ac.id/index.php/languageliteracy \n \n432 \nNationally Accredited SINTA 3, and indexed in DOAJ and Copernicus \n \n \n \n \nAspects of linking \ndevices \nMean \nSig. 4.4 Linking devices The researchers found that female students were better in using linking devices than \nmale students, both in terms of usage and number. However, female students did more \nword repetition. This is because the number of words produced by female students is also \nmore than male students. Besides, female students were also better at using hyponym-\nhypernym. Therefore, female student's writing became more detailed, and their argument \nbecame stronger due to adding examples. To see each aspect of linking devices, the \nfollowing table presents a different frequency between male and female students. Aspects of linking \ndevices \n \nFrequency of gender \ndifferences \nMale \nFemale \nAdverbials \nTemporal \n174 \n228 \n \nOrdinal \n26 \n61 \n \nPlace \n2 \n3 \n \nManner \n77 \n90 \n \nAdditive \n141 \n158 \n \nAdversative \n126 \n131 \n \nCausal \n140 \n135 \nTotal \n686 \n806 \nRepetition \nWords \n4444 \n5548 \n \nPhrases \n381 \n383 \n \nProper nouns \n0 \n0 \nTotal \n4825 \n5931 \nSynonyms \n \n231 \n241 \nHyponym-hypernyms \n \n68 \n87 \nAnaphoric pronouns \n \n404 \n608 \nDeterminers \n \n1093 \n1280 \nTable 9. Frequency of linking devices Firstly, for temporal adverbials, female students produced 228 items, while male \nstudents produced 174 items. There are 54 items of difference. For ordinal adverbials, \nfemale student made 61 items, while male students made 26. The difference is 35 items. For \nplace adverbials, female students wrote 3 items, while male students wrote 2 items. There \nwas only one item for difference. For manner adverbials, female students made 90 items, \nwhile male students made 77 items. The difference was 13 items. For additive adverbials, \nfemale students produced 158 items, while male students produced 141 items. the \ndifference is 17 items. For adversative adverbials, female students wrote 131 items, while \nmale students wrote 126. There are 5 items of difference. Then, for causal adverbials, female 431 https://jurnal.uisu.ac.id/index.php/languageliteracy \nNationally Accredited SINTA 3, and indexed in DOAJ and Copernicus Linguistic Variation between Male and Female Students in Academic Writing, Surya Asra, Grace Josephine \nTiwon Wiradisastra Linguistic Variation between Male and Female Students in Academic Writing, Surya Asra, Grace Josephine \nTiwon Wiradisastra Linguistic Variation between Male and Female Students in Academic Writing, Surya Asra, Grace Josephine \nTiwon Wiradisastra students produced 135 items, while male students produced 140 items. There are also 5 \nitems of difference, but male students produced more causal adverbials than female \nstudents. However, if all aspects of adverbials are summed, female students still produced \nmore adverbials than male students. y Accredited SINTA 3, and indexed in DOAJ and Copernicus https://jurnal.uisu.ac.id/index.php/languageliteracy \n \n432 \nNationally Accredited SINTA 3, and indexed in DOAJ and Copernicus Nationally Accredited SINTA 3, and indexed in DOAJ and Copernicus al.uisu.ac.id/index.php/languageliteracy \n \n432 4.4 Linking devices of \nANOV\nA Test \nMale \nFemale \nDifference \nTemporal adverbials \n5.8 \n7.6 \n1.8 \n.046 \nOrdinal adverbials \n.867 \n2.033 \n1.166 \n.014 \nPlace adverbials \n.067 \n.1 \n.033 \n.703 \nManner adverbials \n2.567 \n3 \n.433 \n.438 \nAdditive adverbials \n4.7 \n5.267 \n.567 \n.392 \nAdversative \nadverbials \n4.2 \n4.367 \n.167 \n.833 \nCausal adverbials \n4.667 \n4.5 \n.167 \n.796 \nRepeated words \n148.133 \n184.933 \n36.8 \n.001 nal uisu ac id/index php/languageliteracy\nAspects of linking \ndevices \nMean \nSig. of \nANOV\nA Test \nMale \nFemale \nDifference \nTemporal adverbials \n5.8 \n7.6 \n1.8 \n.046 \nOrdinal adverbials \n.867 \n2.033 \n1.166 \n.014 \nPlace adverbials \n.067 \n.1 \n.033 \n.703 \nManner adverbials \n2.567 \n3 \n.433 \n.438 \nAdditive adverbials \n4.7 \n5.267 \n.567 \n.392 \nAdversative \nadverbials \n4.2 \n4.367 \n.167 \n.833 \nCausal adverbials \n4.667 \n4.5 \n.167 \n.796 \nRepeated words \n148.133 \n184.933 \n36.8 \n.001 432 https://jurnal.uisu.ac.id/index.php/languageliteracy \n \n432 \nNationally Accredited SINTA 3, and indexed in DOAJ and Copernicus Language Literacy: Journal of Linguistics, Literature, and Language Teaching \nVolume 6, Number 2, pp: 423-436, December 2022 \ne-ISSN: 2580-9962 | p-ISSN: 2580-8672 \n \n \n \nDOI: 10.30743/ll.v6i2.5910 \n \nRepeated phrases \n12.7 \n12.767 \n.067 \n.962 \nRepetition of proper \nnouns \n.000 \n.000 \n. . Synonyms \n7.7 \n8.033 \n.333 \n.612 \nHyponym-hypernyms \n2.267 \n2.9 \n.633 \n.094 \nAnaphoric pronouns \n13.467 \n20.267 \n6.8 \n.009 \nDeterminers \n36.433 \n42.667 \n6.234 \n.028 \nTable 10. Statistical test of linking devices It can be concluded that there are five aspects of linking devices that have significant \ndifferences between male and female students for significance level of p=0.05, namely \ntemporal adverbials, ordinal adverbials, repeated words, anaphoric pronouns, and \ndeterminers. Therefore, writing instructors must pay attention to all significant aspects of \nlinking devices when teaching writing skill. https://jurnal.uisu.ac.id/index.php/languageliteracy \nNationally Accredited SINTA 3, and indexed in DOAJ and Copernicus https://jurnal.uisu.ac.id/index.php/languageliteracy \n \n433 \nNationally Accredited SINTA 3, and indexed in DOAJ and Copernicus 4.5 Text output counts From the analysis of student writing using Markin 4, it was found that number of text \noutput counts of female students was better than male students. It means that female \nstudents produced a greater number of text output count than male students. It makes the \nwriting of female students is better and more detail than female students. To see the \ndifferent aspects of the number of text output counts, the following table presents the \ndifferent frequency between male and female students. Aspects of text output \ncounts \nFrequency of gender differences \nMale \nFemale \nNumber of characters \n34049 \n40529 \nNumber of words \n7601 \n9081 \nNumber of sentences \n475 \n601 \nNumber of paragraphs \n148 \n150 \nNumber of sentences per \nparagraphs \n96 \n120.2 \nNumber of words per \nsentences \n494.24 \n452.96 \nNumber of characters per \nwords \n133.29 \n133.73 \nNumber of passive \nsentences \n31 \n38 \nTable 11. Frequency of text output counts Firstly, for number of characters, female students produced 40529 characters, while \nmale students produced 34049 characters. The difference is 6480 characters. Secondly, for \nnumber of words, female students produced 9081 words, while male students only \nproduced 7601 words. The difference is 1480 words. Thirdly, for number of sentences, \nfemale students produced 601 sentences, while male students produced 475 sentences. The \ndifference is 126 sentences. Fourthly, for number of paragraphs, female students produced \n150 paragraphs. while male students produced 148 paragraphs. It has only 2 paragraphs of 433 Linguistic Variation between Male and Female Students in Academic Writing, Surya Asra, Grace Josephine \nTiwon Wiradisastra Linguistic Variation between Male and Female Students in Academic Writing, Surya Asra, Grace Josephine \nTiwon Wiradisastra difference because there are 2 male students who only write 4 paragraphs. It should be the \nsame if male students followed the requirements stated by the test organizer. It has been \ndiscussed in the aspect of no paragraphs. Fifthly, for number of sentences per paragraph, \nfemale students got 120.2 sentences, while male students got 96 sentences. The difference \nis 24.2 sentences. It means that female students have more sentences for each paragraph \nthan male students. difference because there are 2 male students who only write 4 paragraphs. It should be the \nsame if male students followed the requirements stated by the test organizer. It has been \ndiscussed in the aspect of no paragraphs. Fifthly, for number of sentences per paragraph, \nfemale students got 120.2 sentences, while male students got 96 sentences. https://jurnal.uisu.ac.id/index.php/languageliteracy \nNationally Accredited SINTA 3, and indexed in DOAJ and Copernicus 5. Conclusion The highest significant difference of linguistic characteristics in text-level analysis is \nparagraphing competences. Paragraphing competences consist of complete paragraphs \n(topic sentence, one main idea, logical order, paragraphs with meta-discourse, paragraphs \nwith references, and paragraphs with conclusion sentences), incomplete paragraphs (partial \nparagraphs and paragraphs with one sentence), and no paragraphs. For this linguistic \ncharacteristic, it is found that female students produced more complete paragraphs. In other \nwords, female students outperformed male students. Otherwise, male students produced \nmore partial paragraphs, paragraphs with only one sentence, and no paragraphs. The most \ndominant cause that makes female students are superior to male students in paragraphing \ncompetences is meta-discourse. Female students in writings tend to use meta-discourse \nsigns, such as first (ly), second (ly), third (ly), last (ly), in conclusion, therefore, and so in their \nwriting. The use of meta-discourse signs also influences other linguistic characteristics, such \nas topical organization and endings. Meta-discourse signs make topical organization easier, \nand endings (closing paragraphs) has a good quality. There are several notions that need to be considered by writing instructors and \nfurther researcher. Writing instructors (teachers, lecturers, tutors, etc.) should consider \ngender differences in teaching English, especially in teaching academic writing skill and pay \nmore attention when teaching students with different gender in one class. This attention can \nbe focused on aspects of linguistic characteristics of academic writing in order to specifically \nhelp their students in teaching-learning process. Besides, further researchers can do more \nwritten tests as a research instrument, not just one written test in order to validate research \ndata. Further researchers can also use different types (genres) of text, such as analyzing \nstudent daily journals, both in text and sentence levels. 4.5 Text output counts They can excel almost in all of these linguistic characteristics in text-level \nanalysis (topical organization, paragraphing competences, ending, linking devices, and text 434 Language Literacy: Journal of Linguistics, Literature, and Language Teaching \nVolume 6, Number 2, pp: 423-436, December 2022 \ne-ISSN: 2580-9962 | p-ISSN: 2580-8672 \n \n \n \nDOI: 10.30743/ll.v6i2.5910 \n \noutput counts. Besides, it was also found that all of these linguistic characteristics obtained \nsignificant differences at a significance level of p=0.05. However, the differences are not \nstatistically significant. From previous research conducted by Jones and Myhill (2007), it was \nalso found that men tend to write long paragraphs. On the contrary, this research proves \nthat female students tend to write longer paragraphs with a higher number of sentences. Language Literacy: Journal of Linguistics, Literature, and Language Teaching \nVolume 6, Number 2, pp: 423-436, December 2022 \ne-ISSN: 2580-9962 | p-ISSN: 2580-8672 \n \n \nDOI: 10.30743/ll.v6i2.5910 output counts. Besides, it was also found that all of these linguistic characteristics obtained \nsignificant differences at a significance level of p=0.05. However, the differences are not \nstatistically significant. From previous research conducted by Jones and Myhill (2007), it was \nalso found that men tend to write long paragraphs. On the contrary, this research proves \nthat female students tend to write longer paragraphs with a higher number of sentences. 4.5 Text output counts The difference \nis 24.2 sentences. It means that female students have more sentences for each paragraph \nthan male students. Sixthly, for number of words per sentence, female students got 452.96 words, while \nmale students got 494.24 words. The difference is 41.28 words. Male students have more \nwords for each sentence than female students in this aspect. This is due to the smaller \nnumber of sentences of male students than female students. Seventhly, for number of \ncharacters per word, female students got 133.73 characters, while male students got 133.29 \ncharacters. There is only 0.44 characters of difference. It means that both male and female \nstudents have the same number of characters for each word. Eighthly, for number of passive \nsentences, female students produced 38 sentences, while male students produced 31 \nsentences. The difference is 7 sentences. Although the difference of text output counts is not too many, it can still be inferred \nthat the writing of female students is better than female students. The statistical analysis on \neach aspect of text output counts can be seen in the table below. Aspects of text output \ncounts \nMean \nSig. of \nANOVA \nTest \nMale \n \nFemale \n \nDifference \n \nNumber of characters \n1.134.967 1.350.967 \n216 \n.003 \nNumber of words \n253.367 \n302.7 \n49.333 \n.002 \nNumber of sentences \n15.833 \n20 \n4.167 \n.000 \nNumber of paragraphs \n4.933 \n5 \n0.067 \n.155 \nNumber of sentences \nper paragraphs \n3.2 \n4 \n0.8 \n.001 \nNumber of words per \nsentences \n16.475 \n15.56 \n0.915 \n.218 \nNumber of characters \nper words \n4.443 \n4.458 \n0.015 \n.817 \nNumber of passive \nsentences \n1.033 \n1.267 \n0.234 \n.500 \nTable 12. Statistical test of text output counts It can be concluded that there are four aspects of text output counts that have \nsignificant difference values for significance level of p=0.05, namely number of characters, \nnumber of words, number of sentences, and number of sentences per paragraph. These four \naspects, especially number of words sentences must be writing instructors’ attention in \nteaching writing skill. In addition, although number of passive sentences does not experience \na significant difference, writing instructors should also emphasize to their student about the \nimportance of using passive sentences in academic writing to produce formal writing and \nobjective writing styles (Jordan, 2003). Female students had better abilities than male students in all linguistic characteristics \nof text analysis. Acknowledgements This paper was funded by the Indonesia Endowment Fund for Education (LPDP). Therefore, we would like to thank the Indonesia LPDP for funding this research. https://jurnal.uisu.ac.id/index.php/languageliteracy \nNationally Accredited SINTA 3, and indexed in DOAJ and Copernicus References Andini, T., M. & Prasetyowati, S. (2021). Gender differences learning strategy at English \nlanguage education department students university of muhammadiyah Malang. Jurnal \nInovasi \nPembelajaran, \n7 \n(2), \n217-226. Retrieved \nfrom \nhttp://ejournal.umm.ac.id/index.php/jinop Andini, T., M. & Prasetyowati, S. (2021). Gender differences learning strategy at English \nlanguage education department students university of muhammadiyah Malang. Jurnal \nInovasi \nPembelajaran, \n7 \n(2), \n217-226. Retrieved \nfrom \nhttp://ejournal.umm.ac.id/index.php/jinop Andini, T., M. & Prasetyowati, S. (2021). Gender differences learning strategy at English \nlanguage education department students university of muhammadiyah Malang. Jurnal \nInovasi \nPembelajaran, \n7 \n(2), \n217-226. Retrieved \nfrom \nhttp://ejournal.umm.ac.id/index.php/jinop Arikunto, S. (2013). Prosedur Penelitian: Suatu Pendekatan Praktik. Jakarta: Rineka Cip Cameron, D. (1997). Performing gender Identity: Young men’s talk and the Construction of \nheterosexual masculinity. In Sally, J. & Meinhof, U. (Eds.), Language and masculinity. Oxford: Blackwell. https://jurnal.uisu.ac.id/index.php/languageliteracy \nNationally Accredited SINTA 3, and indexed in DOAJ and Copernicus 435 Linguistic Variation between Male and Female Students in Academic Writing, Surya Asra, Grace Josephine \nTiwon Wiradisastra Linguistic Variation between Male and Female Students in Academic Writing, Surya Asra, Grace Josephine \nTiwon Wiradisastra Linguistic Variation between Male and Female Students in Academic Writing, Surya Asra, Grace Josephine \nTiwon Wiradisastra Creswell, J. W. & Plano-Clark, V. L. (2011). Designing and Conducting Mixed Methods \nResearch (2nd ed.). Thousand Oaks, CA: Sage. Fattahi, N. & Nushi, M. (2021). The effect of gender and language proficiency on the \nmetaphor use in the writing of TEFL students. Asian. J. Second. Foreign. Lang. Educ. 6, 19. https://doi.org/10.1186/s40862-021-00126-1 Gay, L. R., Mills, G. E. & Airasian, P. (2012). Educational research: Competencies for analysis \nand application. (8th ed.). New Jersey: Pearson Prentices Hall. Gtowka, D. (2014). The impact of gender on attainment in learning english as a foreign \nlanguage. Studies in Second Language Learning and Teaching (SSLLT), 4 (4), 617-635. doi: 10.14746/ssllt.2014.4.4.3 Hamdan, S. M. & Hamdan, J. M. (2013). Authors’ perceptions of author’s gender: A myth or a \ntruth?. International Journal of English and Literature, 4 (10), 523-528. Retrieved from \nhttps://academicjournals.org/article/article1383241609_Hamdan%20and%20Hamda\nn.pdf Holmes, J. (2013). An introduction to sociolinguistics (4th ed.). New York: Routledge. Johns, A. M. (1997). Text, role, and context: Developing academic literacies. Cambridge: \nCambridge University Press. Jones, S. & Myhill, D. (2007). Discourses of differences? Examining gender differences in \nlinguistic characteristics of writing. Canadian Journal of Education,30 (2), 456-482. doi: https://doi.org/10.2307/20466646 Jones, S. (2011). Mapping the landscape: gender and the writing classroom. Journal of \nWriting Research, 3 (3), 161-179. doi: 10.17239/jowr-2012.03.03.2 Jordan, R. R. (2003). https://jurnal.uisu.ac.id/index.php/languageliteracy \nNationally Accredited SINTA 3, and indexed in DOAJ and Copernicus References Academic writing course: Study skills in English. London: Pearson \nEducation Limited. Mahmud, M. (2018). Gender differences in English language teaching. Confrence TESOL \nIndonesia \n– \nAsian \nEFL \nJournal, \n20 \n(5). Retrieved \nfrom \nhttps://www.researchgate.net/publication/321722800_Gender_Difference_in_Engli\nsh_Language_Teaching Newman, M. L., Groom. C. J., Handelman, L. D. & Pennebaker, J. W. (2008). Gender \ndifferences in language use: An analysis of 14.000 text samples. Discourse Processes, \n45, 211-236. doi: 10.1080/01638530802073712 Nunan, D. & Bailey, K. M. (2009). Exploring second language classroom research: A \ncomprehensive guide. Boston: Heinle, Cengage Learning. Oshima, A. S. & Hogue, A. (2006). Writing academic English. (4th ed.). New York: Longman. Reynolds, M.R., Scheiber, C., Hajovsky, D. B., Schwartz, B., & Kaufman, A. S. (2015). Gender \ndifferences in academic achievement: Is writing an exception to the gender \nsimilarities hypothesis?. The Journal of Genetic Psychology, 176 (4), 211-234. doi: \nhttps://doi.org/10.1080/00221325.2015.1036833 Sunderland, J. (2000). Issues of language and gender in second and foreign language \neducation. Language Teaching, 33, 203-223. doi: 10.1017/S0261444800015688 Sunderland, J. (2006). Language and gender. New York: Routledge Applied Linguistics. Tuan, L. T. (2010). Enhancing efl learners’ wrriting skill via journal writing. English language \nTeaching, 3 (3), 81-88. doi: https://doi.org/10.31538/alsuna.v2i2.397 Waskita, D. (2008). Differences in men’s and women’s ESL academic writing at the University \nof Melbourne. Jounal of Sociotechnology, 14 (7), 448-463. Retrieved from \nhttp://journals.itb.ac.id/index.php/sostek/article/view/1005 Waskita, D. (2008). Differences in men’s and women’s ESL academic writing at the University \nof Melbourne. Jounal of Sociotechnology, 14 (7), 448-463. Retrieved from \nhttp://journals.itb.ac.id/index.php/sostek/article/view/1005 436"
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Influência do teor de umidade na determinação do módulo de elasticidade de vigas de Pinus sp.
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Influência do teor de umidade na determinação
do módulo de elasticidade de vigas de Pinus sp. Influence of moisture content on the determination of
the modulus of elasticity of Pinus sp. beams SEGUNDINHO, P. G. de A.; CARREIRA, M. R.; REGAZZI, A. J.; DIAS, A. A. Influência do teor de umidade
na determinação do módulo de elasticidade de vigas de Pinus sp. Ambiente Construído, Porto Alegre,
v. 17, n. 3, p. 319-329, jul./set. 2017.
ISSN 1678-8621 Associação Nacional de Tecnologia do Ambiente Construído.
http://dx.doi.org/10.1590/s1678-86212017000300179 Resumo o processo de secagem da madeira o módulo de elasticidade estático
não sofre alteração até o ponto de saturação das fibras (PSF); para
teores de umidade abaixo do PSF, contudo, o módulo de elasticidade
estático é afetado. O objetivo deste artigo foi investigar a influência do
teor de umidade na obtenção do módulo de elasticidade de vigas de madeira
serrada de Pinus sp. pelos ensaios de vibração transversal livre e flexão estática. Foram utilizadas 20 vigas com dimensões nominais de 5 cm x 10 cm x 200 cm
originadas de plantios de reflorestamento do estado de São Paulo. Tais vigas foram
extraídas de toras recém-abatidas e condicionadas em ambiente fechado para
secagem com temperatura e umidade controladas. O módulo de elasticidade
longitudinal aparente obtido pelo método de vibração transversal livre estabilizou-
se com umidade na faixa de variação do PSF, tal como ocorre no ensaio de flexão
estática. Constatou-se que o ensaio de vibração transversal livre pode ser utilizado
para obter o módulo de elasticidade longitudinal aparente em peças de madeira
tanto com teor de umidade acima quanto abaixo do PSF. N N Palavras-chave: Modelo de regressão segmentada. Madeira de Pinus sp. Ensaio não
destrutivo. Ponto de saturação das fibras. Pedro Gutemberg de Alcântara
Segundinho
Universidade Federal do Espírito
Santo
Jerônimo Monteiro - ES - Brasil Keywords: Segmented regression model. Pinus sp. wood. Non-destructive testing. Fiber
saturation point. Introdução Barret e Hong (2009) desenvolveram uma
investigação experimental sobre o efeito do teor de
umidade na medição do módulo de elasticidade
dinâmico de vigas de madeira. Nesse estudo a água
livre
foi
simulada
com
massas
(moedas)
distribuídas ao longo do comprimento. Os autores
observaram que, adicionando-se massas para
simular teores de umidade acima de 30%, o
módulo de elasticidade dinâmico permanece
constante. Barret e Hong (2009) concluíram
também que o procedimento da norma ASTM D
1990 (ASTM, 1997) – Standard Practice for
Establishing Allowable Properties for Visually-
Graded Dimension Lumber from In-Grade Tests of
Full-Size Specimens – para correção do módulo de
elasticidade em função do teor de umidade pode
ser usado para calcular o módulo de elasticidade
dinâmico das vigas no teor de umidade de 15%. No processo de secagem da madeira até o ponto de
saturação
das
fibras
(PSF)
o
módulo
de
elasticidade estático não sofre alteração e é
admitido como constante. Para teores de umidade
abaixo do PSF o módulo de elasticidade estático é
afetado pelo teor de umidade (BODIG; JAYNE,
1982; LOGSDON, 1998). O módulo de elasticidade na flexão pode ser
avaliado por meio de ensaio estático ou ensaios
dinâmicos, como ultrassom, ondas de tensão e
vibração transversal livre. Entre esses métodos de
avaliação não destrutiva, o ensaio de vibração
transversal livre apresenta algumas vantagens em
relação aos demais, como menor tempo para
execução, repetitividade e possibilidade de obter o
módulo de elasticidade levando-se em conta as
deformações devidas ao cisalhamento. Deve-se
destacar ainda que a técnica de vibração
transversal livre tem demonstrado boa exatidão na
estimativa da rigidez à flexão de peças estruturais
de madeira serrada em razão da boa aderência
entre o modelo matemático e o modelo físico do
ensaio (BALLARIN; TARGA; PALMA, 2002). Tal aderência entre modelos também foi verificada
por Carreira et al. (2003, 2012), Carreira,
Segundinho e Dias (2012) e Candian e Sales
(2009). Vale ressaltar que tais trabalhos realizados
no Brasil por meio de vibração transversal foram
em peças de madeira com teor de umidade abaixo
do PSF. Unterwieser e Schickhofer (2010) estudaram a
influência do teor de umidade no módulo de
elasticidade dinâmico de vigas de madeira serrada
e propuseram equações para correção do módulo
de elasticidade da espécie Spruce para teores de
umidade abaixo e acima do PSF. Abstract In the wood drying process, the static modulus of elasticity does not change until
the fibre saturation point (FSP) is reached; however, for lower than FSP moisture
content, the static modulus of elasticity changes. The aim of this paper is to
investigate the influence of moisture content on obtaining the modulus of elasticity
of sawn wooden beams of Pinus sp. through free transverse vibration and static
bending tests. We used 20 beams with nominal dimensions of 5 cm x 10 cm x 200
cm from reforestation forests in the State of São Paulo. The beams were taken
from newly harvested logs that were kept indoors for drying under controlled
temperature and humidity. The apparent longitudinal modulus of elasticity,
obtained through the free transverse vibration method, stabilized with humidity in
the FSP variation range, as it occurs in the static bending test. The study showed
that the free transverse vibration test can be used to obtain the apparent
longitudinal modulus of elasticity from pieces of wood with a moisture content that
is either above or below the FSP. Marcelo Rodrigo Carreira
Universidade Tecnológica Federal do
Paraná
Campo Mourão - PR - Brasil Adair José Regazzi
Universidade Federal do Espírito
Santo
Jerônimo Monteiro - ES - Brasil Adair José Regazzi
Universidade Federal do Espírito
Santo
Jerônimo Monteiro - ES - Brasil Antonio Alves Dias
Universidade de São Paulo
São Carlos – SP – Brasil Antonio Alves Dias
Universidade de São Paulo
São Carlos – SP – Brasil Keywords: Segmented regression model. Pinus sp. wood. Non-destructive testing. Fiber
saturation point. Recebido em 10/10/15
Aceito em 26/11/16 319 Ambiente Construído, Porto Alegre, v. 17, n. 3, p. 319-329, jul./set. 2017. Introdução A norma NBR 7190 (ABNT, 1997) recomenda a
correção do módulo de elasticidade obtido por
meio de ensaios estáticos para o teor de umidade
de 12% e indica que não sejam utilizadas madeiras
em aplicação estrutural com teores de umidade
superiores a 20%, pois a resistência e a rigidez da
madeira sofrem variações durante o intervalo
higroscópico até atingir o PSF, teor de umidade
próximo de 30%, a partir do qual se considera que
o módulo de elasticidade permanece constante. A norma ASTM D 6874 (ASTM, 2003a) –
Standard
Test
Methods
for
Nondestructive
Evaluation of Wood-Based Flexural Members
Using Transverse Vibration – padroniza os
procedimentos do ensaio de vibração transversal
livre em vigas de madeira serrada. Segundo essa
norma, o módulo de elasticidade dinâmico de vigas
de madeira serrada com teor de umidade não
superior a 22% pode ser corrigido empregando-se
os procedimentos da norma ASTM D 2915
(ASTM, 2003b) – Practice for Evaluating
Allowable Properties for Grades of Structural
Lumber –, e para teores de umidade acima de 22%
a norma ASTM D 6874 (ASTM, 2003a) não
recomenda o emprego da norma ASTM D 2915
(ASTM, 2003b), deixando claro que qualquer
ajuste do módulo de elasticidade acima desse teor
de umidade deve ser documentado. A norma
ASTM D 6874 (ASTM, 2003a) recomenda ainda
que o emprego dos procedimentos da norma
ASTM D 2915 (ASTM, 2003b) deve ser feito com
certa cautela, uma vez que a aplicabilidade de tais
procedimentos a resultados de ensaios dinâmicos
não foi bem determinada. O objetivo do trabalho foi investigar a influência
do teor de umidade na obtenção do módulo de
elasticidade de vigas de madeira serrada de Pinus
sp. pelos ensaios de vibração transversal livre e
flexão estática simplesmente apoiada. Neste estudo
ajustou-se um modelo de regressão segmentada
para identificar o teor de umidade a partir do qual
o módulo de elasticidade tende a ser constante. Segundinho, P. G. de A.; Carreira, M. R.; Regazzi, A. J.; Dias, A. A.
320 Métodos Foi aplicada uma força crescente, tendo sido
anotadas as forças necessárias para provocar
deslocamentos verticais de 4 mm e 8 mm. O
módulo de elasticidade foi calculado a partir
desses valores utilizando a Equação 1. Inicialmente foram estimados o teor de umidade e
as densidades básica e aparente das vigas, a partir
de corpos de prova extraídos delas, e medida a
massa inicial de cada viga com o intuito de estimar
o teor de umidade em cada observação. 3
MOE
48
st
F L
v I
Eq. 1 Eq. 1 A secagem das vigas foi feita de forma natural, e a
cada variação de aproximadamente 5% no teor de
umidade foram obtidas suas dimensões e massas e
realizados os ensaios para a obtenção do módulo
de elasticidade estático e das frequências naturais
do primeiro modo de vibração por flexão das
vigas. Tal procedimento foi realizado até as vigas
atingirem a umidade de equilíbrio com o ambiente. Então foram extraídos 15 corpos de prova de cada
viga para a determinação do teor de umidade
médio de acordo com a norma NBR 7190 (ABNT,
1997) e obteve-se a massa seca das vigas a partir
da determinação do teor de umidade final e
correção das umidades dos ensaios, possibilitando
calcular os teores de umidade efetivos em cada
ensaio. Em que: MOEst é o módulo de elasticidade a flexão (Pa); ΔF é o incremento de força (N); L é o vão livre (m); Δv é o incremento no deslocamento vertical (4·10-3
m); e I é o momento de inércia da seção transversal (m4). Ensaio de vibração transversal livre O ensaio de vibração transversal livre foi realizado
com excitação no plano vertical. As vigas foram
suspensas por duas linhas de nylon, com diâmetro
de 0,7 mm, posicionadas nas coordenadas 0,224l e
0,776l, que correspondem aos pontos nodais do
primeiro modo de flexão de uma viga em
suspensão livre-livre, sendo l o comprimento da
viga. A Figura 1a ilustra uma visão geral do
ensaio. Material Foram utilizadas 20 vigas de madeira serrada de
Pinus sp. com dimensões nominais de 5 cm x 10
cm x 200 cm originadas de plantios de
reflorestamento do estado de São Paulo. As vigas
foram extraídas de toras recém-abatidas e foram
condicionadas
em
uma
sala
fechada,
cuja
temperatura e umidade relativa do ar variaram de
21,4 a 24,6 oC e de 55% a 65% respectivamente. Segundinho, P. G. de A.; Carreira, M. R.; Regazzi, A. J.; Dias, A. A. 320 Ambiente Construído, Porto Alegre, v. 17, n. 3, p. 319-329, jul./set. 2017. Acrescenta-se que questões como tipos de lenhos,
juvenil e adulto, proporção de lenho inicial e
tardio, número de anéis de crescimento, que
interferem no módulo de elasticidade, foram
desconsiderados neste estudo. Salienta-se que para
trabalhos
com
madeira
estrutural
deve-se
selecionar madeira de lenho adulto. 37, e por essa razão o efeito do esforço cortante foi
desprezado no cálculo dos módulos de elasticidade
estático e dinâmico. 37, e por essa razão o efeito do esforço cortante foi
desprezado no cálculo dos módulos de elasticidade
estático e dinâmico. A força aplicada foi medida com um anel
dinamométrico com capacidade de 4,7 kN, e os
deslocamentos
verticais
na
metade
do
comprimento das vigas foram medidos por meio
de relógio comparador com resolução de 0,01 mm. Influência do teor de umidade na determinação do módulo de elasticidade de vigas de pinus sp.
321 Análise dos dados Em que SQR é a soma de quadrados do resíduo e
SQTC é a soma de quadrados total corrigida pela
média; e (iii) coeficiente de variação (CV),
definido por (Equação 6): A análise dos dados foi realizada considerando-se
o modelo estatístico polinomial quadrático com
resposta constante (MPQC). É o mesmo modelo
polinomial quadrático com resposta em platô, mas
no estudo em questão a equação terá ponto de
mínimo, não de máximo. O ajuste do modelo foi
realizado
a
partir
dos
dados
obtidos
experimentalmente para as 20 vigas estudadas por
meio do procedimento NLIN (PROC NLIN) do
software estatístico SAS (Statistical Analysis
Software) (STATISTICAL..., 2004).
%
100
yx
S
CV
= y
Eq. 6 Eq. 6 Em que
yx
S
=
QMResíduo é o desvio padrão Em que
yx
S
=
QMResíduo é o desvio padrão
residual obtido da análise de variância da regressão
(MPQC). O coeficiente de variação mede a
dispersão relativa das observações, porque, por
definição, é o cociente entre a medida da dispersão
dos pontos em torno da curva ajustada (Syx) e o
valor médio da variável dependente (ӯ). O
resultado é tanto melhor quanto menor for o
coeficiente de variação. Em que
yx
S
=
QMResíduo é o desvio padrão
residual obtido da análise de variância da regressão
(MPQC). O coeficiente de variação mede a
dispersão relativa das observações, porque, por
definição, é o cociente entre a medida da dispersão
dos pontos em torno da curva ajustada (Syx) e o
valor médio da variável dependente (ӯ). O
resultado é tanto melhor quanto menor for o
coeficiente de variação. Em que: Em que: y é o módulo de elasticidade longitudinal aparente
(Ea); e x é o teor de umidade. A justaposição dos dois submodelos é concordante
no ponto de interseção x0. Dessa forma, para
valores de x menores que x0, o modelo que
descreve a resposta y é uma função quadrática, e
para valores de x maiores ou iguais a x0 o modelo é
uma constante. Para estimação dos parâmetros, o
modelo deve apresentar propriedades matemáticas
adequadas, isto é, deve ser uma função contínua e
diferençável em x0. Essa condição implica que x0=-
b/2c e p=a-(b2/4c), em que x0 é ponto de mínimo, o
teor de umidade x para um mínimo módulo de
elasticidade longitudinal aparente e o ponto de
interseção das duas linhas, e p é a constante, sendo
a, b e c os parâmetros do modelo a serem
estimados. Assim, para valores de umidade
maiores que x0 o módulo de elasticidade estimado
pelo modelo será a constante p. O módulo de elasticidade dinâmico foi calculado
pela Equação 2, obtida pela solução da equação de
movimento de uma viga de Bernoulli para uma
viga
em
suspensão
livre-livre
(CLOUGH;
PENZIEN, 1995). 2
3
1
1
2
MOE
vt
f
m l
I
Eq. 2 Eq. 2 Em que: MOEvt é o módulo de elasticidade longitudinal
obtido pelo método de vibração transversal livre
(MPa); Ensaio de flexão estática O ensaio de flexão estática foi realizado com o
esquema de viga simplesmente apoiada com força
concentrada na metade do vão da viga. Elas foram
fletidas em relação ao eixo de menor inércia, com
um vão de 185 cm. A relação vão-altura foi igual a Figura 1- Ensaio de vibração transversal livre nas vigas serradas de Pinus sp. (a) Vista geral do ensaio
(b) Acelerômetro fixado na
extremidade da viga
(c) Mola empregada
para suspensão ivre nas vigas serradas de Pinus sp. (b) Acelerômetro fixado na
extremidade da viga
(c) Mola empregada
para suspensão Figura 1- Ensaio de vibração transversal livre nas vigas serradas de Pinus sp. (a) Vista geral do ensaio (a) Vista geral do ensaio (a) Vista geral do ensaio (b) Acelerômetro fixado na
extremidade da viga (c) Mola empregada
para suspensão Influência do teor de umidade na determinação do módulo de elasticidade de vigas de pinus sp. 321 Ambiente Construído, Porto Alegre, v. 17, n. 3, p. 319-329, jul./set. 2017. Os sinais do martelo e do acelerômetro foram
enviados
a
um
condicionador
de
sinais
desenvolvido pelo primeiro autor e então foram
convertidos em sinais digitais por uma placa USB
6009 da National Instruments Corporation. Os
sinais foram adquiridos e analisados pelo programa
Impact, também desenvolvido pelo primeiro autor,
sendo a função de resposta em frequência (FRF)
calculada com a média de 10 espectros. A
identificação dos parâmetros modais foi feita com
o programa Modal-Id, que faz a identificação
multimodal pelo método da razão de polinômios. Avaliação do modelo Foi ajustado um modelo para cada uma das 20
vigas. A qualidade do ajustamento do modelo
polinomial quadrático com resposta constante
(MPQC) foi avaliada por meio dos seguintes
critérios: (i) teste F para regressão; (ii) coeficiente
de determinação (R2), obtido da seguinte forma
(uma vez que o modelo inclui o intercepto)
(Equação 5): f1 é a primeira frequência de vibração transversal
livre (Hz); e f1 é a primeira frequência de vibração transversal
livre (Hz); e γ1 é obtido por meio da equação (n + 0,5)2, onde n
é igual a 1 (número do modo harmônico); m é a massa do corpo de prova (kg); m é a massa do corpo de prova (kg); l é o comprimento da peça (m); e
2 %
100
SQR
R
= 1- SQTC
Eq. 5 Eq. 5 I é o momento de inércia da seção transversal (m4). Modelo Polinomial Quadrático com
Resposta Constante (MPQC) Consideraram-se os seguintes modelos (Equações
3 e 4): Para os testes estatísticos o nível de significância
adotado foi de até 5% de probabilidade. Assim,
para valor-P > 0,05, o resultado é declarado não
significativo estatisticamente. Eq. 4 y= p+e se
0
x
x (constante)
Eq. 4 Segundinho, P. G. de A.; Carreira, M. R.; Regazzi, A. J.; Dias, A. A. 322 Ambiente Construído, Porto Alegre, v. 17, n. 3, p. 319-329, jul./set. 2017. Nas Figuras 2 e 3 são plotados os dados
experimentais (módulo de elasticidade em função
da umidade), valores estimados e pontos de
interseção obtidos nos ensaios de flexão estática
simplesmente apoiada e vibração transversal livre
respectivamente. Resultados e discussão Os valores médios da densidade básica, densidade
aparente e teor de umidade da madeira de Pinus sp. obtidos após a secagem natural são apresentados
na Tabela 1. Tabela 1 - Valores médios da densidade básica, densidade aparente e teor de umidade da madeira de
Pinus sp. obtidos após a secagem natural
Densidade básica (g/m3)
0,49 (0,06)*
Densidade aparente (g/m3)
0,56 (0,07)*
Teor de umidade (%)
13,07 (0,76)*
Nota: *o valor entre parênteses é o desvio padrão. ela 1 - Valores médios da densidade básica, densidade aparente e teor de umidade da madeira de
us sp. obtidos após a secagem natural Nota: *o valor entre parênteses é o desvio padrão. Figura 2 - Dados experimentais (módulo de elasticidade em função da umidade), valores estimados e
pontos de interseção obtidos no ensaio de flexão estática simplesmente apoiada
Figura 3 - Dados experimentais (módulo de elasticidade em função da umidade), valores estimados e
pontos de interseção obtidos no ensaio de vibração transversal livre Figura 2 - Dados experimentais (módulo de elasticidade em função da umidade), valores estimados e
pontos de interseção obtidos no ensaio de flexão estática simplesmente apoiada Influência do teor de umidade na determinação do módulo de elasticidade de vigas de pinus sp. pontos de interseção obtidos no ensaio de flexão estática simplesmente apoiada
Figura 3 - Dados experimentais (módulo de elasticidade em função da umidade), valores estimados e
pontos de interseção obtidos no ensaio de vibração transversal livre Figura 3 - Dados experimentais (módulo de elasticidade em função da umidade), valores estimados e
pontos de interseção obtidos no ensaio de vibração transversal livre Influência do teor de umidade na determinação do módulo de elasticidade de vigas de pinus sp. 323
pontos de interseção obtidos no ensaio de vibração transversal livre Influência do teor de umidade na determinação do módulo de elasticidade de vigas de pinus sp. 323 Ambiente Construído, Porto Alegre, v. 17, n. 3, p. 319-329, jul./set. 2017. Na Tabela 2 está a análise de variância e teste F
para regressão considerando o modelo polinomial
quadrático com resposta constante para madeira de
Pinus sp. com a carga aplicada formando ângulos
quaisquer com as direções tangencial e radial no
ensaio de flexão estática. para madeira de Pinus sp. no ensaio de flexão
estática, com os coeficientes de determinação R2
(%), coeficiente de variação CV (%), teor de
umidade mínimo x0 (%) e a constante p. Segundinho, P. G. de A.; Carreira, M. R.; Regazzi, A. J.; Dias, A. A.
324 Nota: **significativo em nível de 1% de probabilidade (P < 0,01) e n o número de observações em cada viga. a: **significativo em nível de 1% de probabilidade (P < 0,01) e n o número de observações em cada viga. Resultados e discussão O teor de
umidade a partir do qual não houve alteração
significativa no módulo de elasticidade das vigas
variou entre 25,42% e 36,95%. Na Tabela 3, por sua vez, estão as equações
ajustadas para o modelo de regressão utilizado Tabela 2 - Análise de variância e teste F para regressão considerando o modelo polinomial quadrático
com resposta constante para madeira de Pinus sp. com a carga aplicada formando ângulos quaisquer
com as direções tangencial e radial no ensaio de flexão estática resposta constante para madeira de Pinus sp. com a carga aplicada formando ângulos quaisque
as direções tangencial e radial no ensaio de flexão estática
Vigas
n
Fonte de variação
G. L. Q. M. F
1
18
Regressão
2
4,9035
125,09**
Resíduo
15
0,0392
2
18
Regressão
2
3,0196
46,31**
Resíduo
15
0,0652
3
19
Regressão
2
4,5203
162,60**
Resíduo
16
0,0278
4
19
Regressão
2
5,2534
56,49**
Resíduo
16
0,0930
5
19
Regressão
2
4,2254
53,96**
Resíduo
16
0,0783
6
20
Regressão
2
6,0642
153,52**
Resíduo
17
0,0395
7
18
Regressão
2
8,9438
153,41**
Resíduo
15
0,0583
8
18
Regressão
2
10,5378
200,72**
Resíduo
15
0,0525
9
19
Regressão
2
5,1881
220,77**
Resíduo
16
0,0235
10
18
Regressão
2
3,2821
119,78**
Resíduo
15
0,0274
11
20
Regressão
2
7,9446
99,43**
Resíduo
17
0,0799
12
18
Regressão
2
1,8129
88,43**
Resíduo
15
0,0205
13
18
Regressão
2
2,1358
133,49**
Resíduo
15
0,0160
14
18
Regressão
2
2,9753
89,08**
Resíduo
15
0,0334
15
19
Regressão
2
3,8809
102,13**
Resíduo
16
0,0380
16
20
Regressão
2
7,0327
193,74**
Resíduo
17
0,0363
17
21
Regressão
2
4,4031
128,75**
Resíduo
18
0,0342
18
21
Regressão
2
5,5653
205,36**
Resíduo
18
0,0271
19
18
Regressão
2
3,7467
37,69**
Resíduo
15
0,0994
20
20
Regressão
2
5,7293
116,45**
Resíduo
17
0,0492
**significativo em nível de 1% de probabilidade (P < 0,01) e n o número de observações em cada viga. Ambiente Construído, Porto Alegre, v. 17, n. 3, p. 319-329, jul./set. 2017. Tabela 3 - Equações ajustadas para o modelo polinomial quadrático com resposta constante (p),
coeficiente de determinação (R2), coeficiente de variação (CV), teor de umidade mínimo (X0) e a
constante (p) para madeira de Pinus sp. Resultados e discussão com a carga aplicada formando ângulos quaisquer com as
direções tangencial e radial no ensaio de flexão estática Vigas
Equações ajustadas*
R2 (%)
CV (%)
X0 (%)
p (Gpa)
1
2
ˆy=10,2561-0,2335x+0,00328x
94,34
2,79
35,58
6,10
2
2
ˆy= 9,2624 -0,2189x+0,00344x
86,06
4,05
31,80
5,78
3
2
ˆy=11,3465-0,2679x+0,00426x
95,32
2,15
31,43
7,14
4
2
ˆy=16,0390 -0,2905x+0,00452x
87,59
2,48
32,12
11,37
5
2
ˆy=11,7616 -0,2300x+0,00340x
87,08
3,24
33,81
7,87
6
2
ˆy =11,4574 -0,2643x+0,00393x
94,75
2,55
33,62
7,01
7
2
ˆy=13,2954 -0,3318x+0,00497x
95,34
2,73
33,37
7,76
8
2
ˆy =14,2565-0,3219x+0,00436x
96,39
2,40
36,95
8,31
9
2
ˆy=11,8368 -0,3641x+0,00681x
96,50
2,00
26,73
6,97
10
2
ˆy =12,2603-0,4029x+0,00793x
94,11
2,14
25,42
7,14
11
2
ˆy =15,5763-0,5230x+0,01010x
92,12
2,95
25,82
8,83
12
2
ˆy =6,5202-0,1432x+0,00215x
92,19
3,10
33,25
4,14
13
2
ˆy= 9,7409 -0,1938x+0,00292x
94,68
1,80
33,14
6,53
14
2
ˆy =11,0754 -0,1860x+0,00260x
92,23
2,18
35,78
7,75
15
2
ˆy = 9,6130 -0,2148x+0,00309x
92,74
2,99
34,79
5,88
16
2
ˆy =14,9831-0,3752x+0,00610x
95,80
1,90
30,73
9,22
17
2
ˆy = 8,4174 -0,2425x+0,00366x
93,46
3,62
33,11
4,40
18
2
ˆy = 9,8605-0,2196x+0,00298x
95,81
2,49
36,80
5,82
19
2
ˆy =10,6202-0,2098x+0,00292x
83,41
4,17
35,99
6,84
20
2
ˆy =14,6632-0,3166x+0,00523x
93,20
2,10
30,28
9,87
Nota: *y é o módulo de elasticidade e x é o teor de umidade (%). polinomial quadrático com resposta constante
adotado no ajuste dos dados experimentais obtidos
nos dois ensaios é adequado. Analisando os resultados da Tabela 3, pode-se
notar que os coeficientes de determinação para o
modelo mostraram-se altos, com valores superiores
a 83,41%, além de coeficiente de variação com
valores inferiores a 4,17%, indicando que o
modelo proporcionou bom ajuste dos dados. Analisando os resultados da Tabela 3, pode-se
notar que os coeficientes de determinação para o
modelo mostraram-se altos, com valores superiores
a 83,41%, além de coeficiente de variação com
valores inferiores a 4,17%, indicando que o
modelo proporcionou bom ajuste dos dados. A partir dos dados das Tabelas 3 e 5, e
considerando dados pareados, constatou-se que na
comparação das médias de teor de umidade
mínimo (média de x0 no ensaio de flexão estática é
igual a 32,5% e média de x0 no ensaio de vibração
transversal livre é igual a 31,6%), pelo teste t de
Student, elas não diferiram estatisticamente (valor-
P = 0,3109). Nota-se que os valores apresentaram
variação entre 23,48% e 39,90%. Resultados e discussão Na Tabela 4 está a análise de variância e teste F
para regressão considerando o modelo polinomial
quadrático com resposta constante para madeira de
Pinus sp. no ensaio de vibração transversal livre. Na Tabela 5 estão as equações ajustadas para o
modelo de regressão utilizado para madeira de
Pinus sp. no ensaio de vibração transversal livre,
com os coeficientes de determinação (R2),
coeficiente de variação (CV), teor de umidade
mínimo (x0) e a constante p. O teor de umidade a
partir do qual não houve alteração significativa no
módulo de elasticidade das vigas variou entre
23,48% e 39,90%. Tal como Oliveira e Sales (2005), utilizando
aparelho de velocidade ultrassônica, observou-se
uma inflexão em torno do PSF para várias
espécies,
entre
essas
pínus
e
eucaliptos,
demonstrando que o efeito do teor de umidade
abaixo do PSF é mais significativo do que acima
desse ponto. Segundo Calegari et al. (2007), a
grande influência do teor de umidade sobre o
MOE, na faixa que corresponde à madeira
completamente seca até o PSF, seria responsável
pela variação da velocidade ultrassônica. Analisando os resultados da Tabela 5, pode-se
notar que os coeficientes de determinação para o
modelo mostraram-se altos, com valores superiores
a 77,58%, e coeficiente de variação com valores
inferiores a 2,71%. Logo, verifica-se que o modelo 325 Influência do teor de umidade na determinação do módulo de elasticidade de vigas de pinus sp. 325 Ambiente Construído, Porto Alegre, v. 17, n. 3, p. 319-329, jul./set. 2017. Tabela 4 - Análise de variância e teste F para regressão considerando o modelo polinomial quadrático
com resposta constante para madeira de Pinus sp. no ensaio de vibração transversal livre Tabela 4 - Análise de variância e teste F para regressão considerando o modelo polinomial quadrátic
com resposta constante para madeira de Pinus sp. no ensaio de vibração transversal livre p
p
ç
Vigas
n
Fonte de variação
G. L. Q. M. Segundinho, P. G. de A.; Carreira, M. R.; Regazzi, A. J.; Dias, A. A.
326 Nota: *significativo em nível de 1% de probabilidade (P < 0,01) e n o número de observações em cada viga. Nota: *significativo em nível de 1% de probabilidade (P < 0,01) e n o número de observações em cada viga. Resultados e discussão F
1
18
Regressão
2
5,7578
329,02*
Resíduo
15
0,0175
2
18
Regressão
2
1,3673
52,39*
Resíduo
15
0,0261
3
19
Regressão
2
3,2296
188,87*
Resíduo
16
0,0171
4
19
Regressão
2
2,7410
44,57*
Resíduo
16
0,0615
5
19
Regressão
2
2,8679
52,53*
Resíduo
16
0,0546
6
20
Regressão
2
3,6625
237,82*
Resíduo
17
0,0154
7
18
Regressão
2
3,9739
82,45*
Resíduo
15
0,0482
8
18
Regressão
2
3,8576
105,98*
Resíduo
15
0,0364
9
19
Regressão
2
3,7240
135,91*
Resíduo
16
0,0274
10
18
Regressão
2
2,1029
83,78*
Resíduo
15
0,0251
11
20
Regressão
2
4,7126
59,20*
Resíduo
17
0,0796
12
18
Regressão
2
1,0528
152,58*
Resíduo
15
0,0069
13
18
Regressão
2
1,4955
271,91*
Resíduo
15
0,0055
14
18
Regressão
2
1,6693
56,78*
Resíduo
15
0,0294
15
19
Regressão
2
1,9418
106,69*
Resíduo
16
0,0182
16
20
Regressão
2
2,4304
29,42*
Resíduo
17
0,0826
17
21
Regressão
2
7,5247
917,65*
Resíduo
18
0,0082
18
21
Regressão
2
3,6588
292,70*
Resíduo
18
0,0125
19
18
Regressão
2
2,6342
51,65*
Resíduo
15
0,0510
20
20
Regressão
2
5,7195
101,59*
Resíduo
17
0,0563
í
l d
1% d
b bilid d
(P
0 01)
ú
d
b
õ
d Ambiente Construído, Porto Alegre, v. 17, n. 3, p. 319-329, jul./set. 2017. Tabela 5 - Equações ajustadas para o modelo polinomial quadrático com resposta constante,
coeficiente de determinação (R2), coeficiente de variação (CV), teor de umidade mínimo (X0) e a
constante (p) para madeira de Pinus sp. no ensaio de vibração transversal livre Tabela 5 - Equações ajustadas para o modelo polinomial quadrático com resposta constante,
coeficiente de determinação (R2), coeficiente de variação (CV), teor de umidade mínimo (X0) e a
constante (p) para madeira de Pinus sp. no ensaio de vibração transversal livre coeficiente de determinação (R2), coeficiente de variação (CV), teor de umidade mínimo (X0) e a
constante (p) para madeira de Pinus sp. Resultados e discussão no ensaio de vibração transversal livre
Vigas
Equações ajustadas*
R2 (%)
CV (%)
x0 (%)
p (GPa)
1
2
ˆy=11,6187 -0,3158x+0,00505x
97,77
1,74
31,29
6,68
2
2
ˆy= 8,8567 -0,1175x+0,00165x
87,48
2,26
35,52
6,77
3
2
ˆy=11,3837 -0,2335x+0,00377x
95,94
1,58
30,98
7,77
4
2
ˆy=15,6518 -0,1756x+0,00247x
84,78
1,87
35,53
12,53
5
2
ˆy=12,1194 -0,2106x+0,00329x
86,77
2,50
31,97
8,75
6
2
ˆy=11,8371-0,2284x+0,00357x
96,54
1,42
31,98
8,18
7
2
ˆy=12,7506 -0,2660x+0,00437x
91,67
2,34
30,45
8,70
8
2
ˆy=14,0312-0,3030x+0,00508x
93,40
1,88
29,81
9,52
9
2
ˆy=13,0362-0,4146x+0,00883x
94,44
1,90
23,48
8,17
10
2
ˆy=11,7025-0,2938x+0,00555x
91,77
1,91
26,45
7,82
11
2
ˆy=15,2873-0,4370x+0,00876x
87,45
2,71
24,94
9,84
12
2
ˆy=6,3069-0,0805x+0,00101x
95,30
1,62
39,90
4,70
13
2
ˆy= 9,3756 -0,1209x+0,00159x
97,33
0,99
37,97
7,08
14
2
ˆy=11,1439 -0,1547x+0,00228x
88,34
1,91
33,96
8,52
15
2
ˆy= 9,9393-0,2281x+0,00393x
93,02
1,92
29,01
6,63
16
2
ˆy=14,1099 -0,2764x+0,00494x
77,58
2,69
27,98
10,24
17
2
ˆy=11,7578 -0,4809x+0,00878x
99,02
1,52
27,38
5,17
18
2
ˆy= 9,8687 -0,1818x+0,00250x
97,02
1,55
36,42
6,56
19
2
ˆy=11,2564 -0,2130x+0,00326x
87,33
2,71
32,62
7,78
20
2
ˆy=14,7031-0,2358x+0,00338x
92,28
2,09
34,90
10,59
Nota: *y é o módulo de elasticidade e x é o teor de umidade (%). Nota: *y é o módulo de elasticidade e x é o teor de umidade (%). No estudo realizado por Gerhards (1982), a partir
de diversos trabalhos, é confirmada a redução da
resistência e do módulo de elasticidade da madeira
com o acréscimo da umidade, até valores de teor
de umidade próximos de 30%, o que comprova a
influência da umidade e da temperatura em várias
propriedades mecânicas da madeira e, dessa forma,
constata-se que os acréscimos de teores de
umidade acima desse ponto não mais influenciam
os parâmetros mecânicos. simplesmente apoiada quanto no ensaio de
vibração transversal livre. O teor de umidade a partir do qual não há alteração
significativa no módulo de elasticidade das vigas
variou entre 25,42% e 36,95% no caso da flexão
estática simplesmente apoiada e entre 23,48% e
39,90% no caso da vibração transversal livre. A
análise estatística efetuada para o teor de umidade
mínimo, considerando dados pareados, permite
concluir pela equivalência entre as médias obtidas
nos dois tipos de ensaio. O módulo de elasticidade
longitudinal aparente obtido pelo método de
vibração transversal livre estabilizou-se com
umidade na faixa de variação do PSF. Resultados e discussão Para efeito
de classificação estrutural comparativa entre peças
de madeira, pode-se concluir que a obtenção do
módulo de elasticidade longitudinal aparente com
valores altos do teor de umidade, acima do PSF,
não tem influência. Segundo Lima, Della Lucia e Vital (1986), o ponto
de interseção encontrado, que relaciona as
propriedades mecânicas da madeira, foi de 31,0%
de umidade quando se estudou a influência da
variação no teor de umidade da madeira de
Eucalyptus saligna com 40 anos de idade. Influência do teor de umidade na determinação do módulo de elasticidade de vigas de pinus sp.
327 Referências AMERICAN SOCIETY OF TESTING AND
MATERIAL. ASTM D 1990: standard practice for
establishing allowable properties for visually-
graded dimension lumber from in-grade tests of
full-size specimens. Philadelphia, 1997. CARREIRA, M. R.; SEGUNDINHO, P. G. A.;
DIAS, A. A. Determination of the E/G Ratio of
Wood Logs Using Transverse Vibration. Wood
Research, Bratislava, v. 57, p. 207-220, 2012. AMERICAN SOCIETY OF TESTING AND
MATERIAL. ASTM D 6874: standard test
method for nondestructive evaluation of wood-
based flexural members using transverse vibration. Philadelphia, 2003a. CLOUGH, R. W.; PENZIEN, J. Dynamics of
structures. 3. ed. Berkeley: Computers &
Structures, 1995. AMERICAN SOCIETY OF TESTING AND
MATERIAL. ASTM D 2915: practice for
evaluating allowable properties for grades of
structural lumber. Philadelphia, 2003b. GERHARDS, C. C. Effect of the Moisture Content
and Temperature on the Mechanical Properties of
Wood and Analysis of Immediate Effects. Wood
and Fiber, v. 14, n. 1, p. 4-36, 1982. ASSOCIAÇÃO BRASILEIRA DE NORMAS
TÉCNICAS. NBR 7190: projeto de estruturas de
madeira. Rio de Janeiro, 1997. ASSOCIAÇÃO BRASILEIRA DE NORMAS
TÉCNICAS. NBR 7190: projeto de estruturas de
madeira. Rio de Janeiro, 1997. LIMA, J. T.; DELLA LUCIA, R. M.; VITAL, B. R. Influência do teor de umidade nas propriedades
mecânicas da madeira de Eucalyptus saligna
Smith. Revista Árvore, v. 10, n. 1, p. 27-43, 1986. LOGSDON, N. B. Influência da Umidade nas
Propriedades de Resistência e Rigidez da Madeira. São Paulo, 1998. 174 f. Tese (Doutorado em
Engenharia Civil) – Escola de Engenharia,
Universidade de São Paulo, 1998. LIMA, J. T.; DELLA LUCIA, R. M.; VITAL, B. R. Influência do teor de umidade nas propriedades
mecânicas da madeira de Eucalyptus saligna
Smith. Revista Árvore, v. 10, n. 1, p. 27-43, 1986. BALLARIN, A. W.; TARGA, L. A.; PALMA, H. A. L. Ensaios Não-Destrutivos de Vibração
Transversal na Avaliação do Módulo de
Elasticidade de Madeiras de Reflorestamento. In:
ENCONTRO BRASILEIRO EM MADEIRAS E
ESTRUTURAS DE MADEIRA, 8., Uberlândia,
2002. Anais… Uberlândia, 2002. BALLARIN, A. W.; TARGA, L. A.; PALMA, H. A. L. Ensaios Não-Destrutivos de Vibração
Transversal na Avaliação do Módulo de
Elasticidade de Madeiras de Reflorestamento. In: Elasticidade de Madeiras de Reflorestamento. In: ENCONTRO BRASILEIRO EM MADEIRAS E
ESTRUTURAS DE MADEIRA, 8., Uberlândia,
2002. Anais… Uberlândia, 2002. OLIVEIRA, F. G. R.; SALES, A. Efeito da
Densidade e do Teor de Umidade na Velocidade
Ultra-Sônica da Madeira. Revista Minerva, v. 2,
n. 1, p. 25-31, 2005. BARRET, J. D.; HONG, J. P. Referências Moisture Content
Adjustments for Dynamic Modulus of Elasticity of
Wood Members. Wood Science and Technology,
v. 44, n. 3, p. 485-495, 2009. STATISTICAL ANALYSIS SOFTWARE. SAS/STAT, Version 9.1, User’s Guide, USA:
Cary, North Carolina, 2004. BODIG, J.; JAYNE, B. A. Mechanics of Wood
and Wood Composites. New York: Van Nostran-
Reinhold, 1982. UNTERWIESER, H.; SCHICKHOFER, G. Influence of Moisture Content of Wood on Sound
Velocity and Dynamic MOE of Natural
Frequency- and Ultrasonic Runtime Measurement. European Journal of Wood and Wood
Products, v. 69, n. 2, p. 171-181, 2010. CALEGARI, L. et al. Monitoramento do Teor de
Umidade de Madeiras de Pinus elliottii Engelm e CALEGARI, L. et al. Monitoramento do Teor de
Umidade de Madeiras de Pinus elliottii Engelm e
Eucalyptus grandis W. HILL ex Maiden, Sob
Diferentes Temperaturas de Secagem, Através do
Ultra-Som. Ciência Florestal, Santa Maria, v. 17,
n. 4, p. 399-408, 2007. Diferentes Temperaturas de Secagem, Através do Ultra-Som. Ciência Florestal, Santa Maria, v. 17,
n. 4, p. 399-408, 2007. CANDIAN, M.; SALES, A. Aplicação das
Técnicas Não-Destrutivas de Ultra-Som, Vibração
Transversal e Ondas de Tensão Para Avaliação de
Madeira. Ambiente Construído, Porto Alegre, v. 9, n. 4, p. 83-98, out./dez. 2009. CANDIAN, M.; SALES, A. Aplicação das
Técnicas Não-Destrutivas de Ultra-Som, Vibração Conclusões O modelo polinomial quadrático com resposta
constante mostrou-se adequado no ajuste dos
dados. Logo, comprovou-se que esse modelo pode
ser usado para descrever a variação do módulo de
elasticidade aparente em função do teor de
umidade tanto no ensaio de flexão estática Ambiente Construído, Porto Alegre, v. 17, n. 3, p. 319-329, jul./set. 2017. CARREIRA, M. R. et al. Bending Stiffness
Evaluation of Teca and Guajará Lumber Through
Tests of Transverse and Longitudinal Vibration. Acta Scientiarum. Technology, v. 34, p. 27-32,
2012. CARREIRA, M. R. et al. Bending Stiffness
Evaluation of Teca and Guajará Lumber Through
Tests of Transverse and Longitudinal Vibration. Acta Scientiarum. Technology, v. 34, p. 27-32,
2012. Segundinho, P. G. de A.; Carreira, M. R.; Regazzi, A. J.; Dias, A. A.
328 Ambiente Construído, Porto Alegre, v. 17, n. 3, p. 319-329, jul./set. 2017. Agradecimentos Os autores agradecem ao Conselho Nacional de
Desenvolvimento
Científico
e
Tecnológico
(CNPq) e à Fundação de Amparo à Pesquisa do
Espírito Santo (FAPES). Transversal e Ondas de Tensão Para Avaliação de
Madeira. Ambiente Construído, Porto Alegre, v. 9, n. 4, p. 83-98, out./dez. 2009. CARREIRA, M. R. et al. Avaliação de Técnicas
Não Destrutivas Aplicadas à Madeira de Pinus sp. In: CONFERÊNCIA PAN-AMERICANA DE
ENSAIOS NÃO DESTRUTIVOS, 3., Rio de
Janeiro, 2003. Anais... Rio de Janeiro, 2003. Ambiente Construído, Porto Alegre, v. 17, n. 3, p. 319-329, jul./set. 2017. Pedro Gutemberg de Alcântara Segundinho
Departamento de Ciências Florestais e da Madeira | Universidade Federal do Espírito Santo | Av. Gov. Lindemberg, 316, Centro |
Jerônimo Monteiro - ES – Brasil | CEP 29550-000 | Tel.: (28) 3558-2515 | E-mail: p_gutemberg2001@yahoo.com.br Pedro Gutemberg de Alcântara Segundinho Marcelo Rodrigo Carreira
Departamento Acadêmico de Construção Civil | Universidade Tecnológica Federal do Paraná | Via Marginal Rosalina Maria dos Santos |
Campus Campo Mourão, Área Urbanizada I | Campo Mourão - PR – Brasil | Caixa Postal 271 | CEP 87301-899 | Tel.: (44) 3518-1400 |
E-mail: carreira@utfpr.edu.br Marcelo Rodrigo Carreira
Departamento Acadêmico de Construção Civil | Universidade Tecnológica Federal do Paraná | Via Marginal Rosalina Maria dos Santos |
Campus Campo Mourão, Área Urbanizada I | Campo Mourão - PR – Brasil | Caixa Postal 271 | CEP 87301-899 | Tel.: (44) 3518-1400 |
E mail: carreira@utfpr edu br Adair José Regazzi
Departamento de Ciências Florestais e da Madeira | Universidade Federal do Espírito Santo | Tel.: (31) 9617-1458 |
E-mail: adairreg@ufv.br Adair José Regazzi
Departamento de Ciências Florestais e da Madeira | Universidade Federal do Espírito Santo | Tel.: (31) 9617-1458 |
E-mail: adairreg@ufv.br Antonio Alves Dias
Departamento de Engenharia de Estruturas, Escola de Engenharia | Universidade de São Paulo | Av. Trabalhador Sãocarlense, 400, Parque
Arnold Schimidt | São Carlos – SP – Brasil | CEP 13566-590 | Tel.: (16) 3373-8207 | E-mail: dias@sc.usp.br Antonio Alves Dias
Departamento de Engenharia de Estruturas, Escola de Engenharia | Universidade de São Paulo | Av. Trabalhador Sãocarlense, 400
Arnold Schimidt | São Carlos – SP – Brasil | CEP 13566-590 | Tel.: (16) 3373-8207 | E-mail: dias@sc.usp.br artamento de Engenharia de Estruturas, Escola de Engenharia | Universidade de São Paulo | Av. Agradecimentos Trabalhador Sãocarlense, 400, Parque
ld Schimidt | São Carlos – SP – Brasil | CEP 13566-590 | Tel.: (16) 3373-8207 | E-mail: dias@sc.usp.br Revista Ambiente Construído
Associação Nacional de Tecnologia do Ambiente Construído
Av. Osvaldo Aranha, 99 - 3º andar, Centro
Porto Alegre – RS - Brasil
CEP 90035-190
Telefone: +55 (51) 3308-4084
Fax: +55 (51) 3308-4054
www.seer.ufrgs.br/ambienteconstruido
E-mail: ambienteconstruido@ufrgs.br Influência do teor de umidade na determinação do módulo de elasticidade de vigas de pinus sp. 329
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https://openalex.org/W3160204816
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https://www.researchsquare.com/article/rs-520476/latest.pdf
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English
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A Note on Neutrosophic Soft Set With Rough Set
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Research Square (Research Square)
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Research Article Keywords: Rough set, Soft set, Neutrosophic set and Neutrosophic soft lower approx- imation and
Neutrosophic upper approximation operators, Neutrosophic soft rough set Posted Date: May 18th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-520476/v1 License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Read Full License Page 1/2 Abstract Rough set is a very powerful invention to the whole world for dealing with uncertain, incomplete and
imprecise problems. Also soft set theory and neutrosophic set theory both are advance mathematical
tools to handle these uncertain, incomplete, inconsistent information in a better way. The purpose of this
article is to expand the scope of rough set, soft set and neutrosophic set theory. We have introduced the
concept of neutrosophic soft set with roughness without using full soft set. Some definition, properties
and examples have been established on neutrosophic soft rough set. Moreover, dispensable and
equalities are written on roughness with neutrosophic soft set. Full-text Due to technical limitations, full-text HTML conversion of this manuscript could not be completed. However, the manuscript can be downloaded and accessed as a PDF. Page 2/2
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https://www.medienpaed.com/article/download/579/538
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de
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Evaluation von E-Learning: Checklisten, Kriterienkataloge oder Evaluationskonzepte? Zum Stand der Bewertungsverfahren für E-Learning-Arrangements
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ISSN 1424-3636
Jahrbuch Medienpädagogik 5. Evaluation und Analyse
Zweitveröffentlichung aus: Jahrbuch Medienpädagogik 5. (2005) Wiesbaden: VS Verlag für
Sozialwissenschaften. Hrsg. v. Ben Bachmair, Peter Diepold und Claudia de Witt.
Evaluation von E-Learning: Checklisten,
Kriterienkataloge oder Evaluationskonzepte?
Zum Stand der Bewertungsverfahren für E-Learning-Arrangements
Ulf-Daniel Ehlers
1.
Qualität als Ziel
Qualität in Bildungsmaßnahmen – als Legitimation oder aus wissenschaftlichem Interesse – ist ein Thema von hoher Bedeutung, auch – und gerade – im E-Learning.1 ELearning, als eine noch relativ junge, nicht völlig etablierte, aber – so stellt sich immer
häufiger heraus – kostenintensive Bildungsform, muss sich immer wieder der kritischen
Frage nach der Qualität stellen, zumeist assoziiert mit Lernerfolg.2 Vielfältige Instrumente, um die Qualität zu überprüfen oder zu entwickeln, begleitend oder im Nachhinein, sind bislang konzipiert worden (vgl. Ehlers/Pawlowski/Goertz 2003).
Der Artikel gibt einen Überblick zum derzeitigen Stand der Dinge in Bezug auf
zwei dieser Verfahrensweisen: zum einen geht es dabei um die Beurteilungen anhand
von Kriterienkatalogen bzw. Checklisten und zum anderen um Evaluationsverfahren.
Obwohl vielfach bereits umfassendere Managementsysteme zum Einsatz kommen, die
die Qualität bei Anbietern kontinuierlich verbessern sollen, haben beide Verfahren eine
steigende Bedeutung – da auch Managementverfahren immer wieder auf Kriterien oder
Evaluationsverfahren zurückgreifen.
Betrachtet man die Forschungsaktivitäten im Bereich der Qualitätsforschung beim
E-Learning, so wird deutlich, dass unterschiedliche „konjunkturelle Moden“ auszumachen sind. 3 Zum einen wurden seit Mitte der 1960er Jahre mit
1 E-Learning ist kein wissenschaftlicher Begriff. Der Begriff kam in den letzten Jahren (etwa seit 1999) als ein
Neologismus der Werbeindustrie auf. Er umfasst alle Formen des Lernens mit Hilfe elektronischer Medien,
sowohl online als auch offline. Es ist eine Lernform, bei der die neuen Informations- und Kommunikationsmedien (Computer und Internet) in Lernarrangements eingebunden werden, entweder zur Unterstützung des
Lernprozesses (als sogenannte „hybride“ Lernarrangements) oder als ausschließliche Form der Vermittlung
(vgl. Ehlers 2003).
2 Zur Bedeutung von Qualität im E-Learning vgl. auch Ehlers/Holmer/Gerteis/Jung (2003).
3 Dabei bedeutet das Aufkommen neuer Ansätze nicht zwangsläufig das Verwerfen von bereits bestehenden
Methoden. So standen zwar zunächst kriteriengestützte Expertenbeurteilungsverfahren im Vordergrund, dann
spezifische prozessorientierte Qualitätssicherungsverfahren, wie zum Beispiel spezielle Evaluationsverfahren
oder Benchmarks für E-Learning. Die schließlich an Bedeutung gewinnenden generischen Qualitätsmanagementansätze und Standardisierungsversuche greifen aber teilweise Elemente der anderen Qualitätsansätze
wieder auf beziehungsweise integrieren sie.
Ehlers, Ulf-Daniel. 2017. «Evaluation von E-Learning: Checklisten, Kriterienkataloge oder Evaluationskonzepte? Zum Stand der Bewertungsverfahren für E-Learning-Arrangements». MedienPädagogik, MedienPäd.Retro: Jahrbuch Medienpädagogik 5 (2005), 13–36. doi:10.21240/
mpaed/retro/2017.09.02.X.
dem Aufkommen des computerunterstützten Unterrichts (CUU) Qualitätskriterien für
diesen Unterrichtsansatz entwickelt. Diese wurden im Laufe der Zeit weiterentwickelt
und stellen heute sogenannte produktbezogene, normative Bewertungs-, Entwicklungsund Auswahlinstrumente für Lernplattformen, Lernsoftware oder E-Learning-Angebote dar. Zum anderen spielt das Entstehen von speziellen empirischen Evaluationsverfahren für die Beurteilung von Lernsituationen, in denen E-Learning eingesetzt wird,
eine wichtige Rolle. Nicht das Produkt alleine steht hier zur Disposition, sondern der
Lernprozess insgesamt. Damit rückt auch der Lerner in den Fokus der Analyse. Streng
genommen gilt die Bewertung von E-Learning-Angeboten mit Hilfe von Qualitätskriterienkatalogen auch als eine Form der Qualitätsevaluation, beispielsweise im Rahmen
einer sogenannten Expertenbeurteilung. Da sie sich aber im konzeptuellen Ansatz und
der Durchführung deutlich von eher prozessbezogenen Evaluationsansätzen unterscheidet, werden beide Arten gesondert voneinander aufgeführt.
Eine weitere Entwicklung ist in prozessorientierten Ansätzen zu sehen, die die
beiden vorherigen Konzepte jedoch in Teilen integriert und im Sinne übergreifender
Managementansätze versucht, sowohl Standards als auch Evaluationsverfahren einzusetzen, um die bestehenden Lehr- und Lernprozesse (zusätzlich auch die Administrationsprozesse u.a.) begleitend zu überwachen und ggf. zu optimieren. Die beschriebenen
Entwicklungsstränge stehen jedoch nicht unverbunden nebeneinander oder stellen eine
aufeinander aufbauende chronologische Entwicklung dar, sondern sind vielmehr miteinander verzahnt. Der folgende Artikel hat das Ziel, einerseits einen Überblick über die
derzeit bestehenden Instrumente im Bereich der Kriterienkataloge und Evaluationsverfahren zu geben und andererseits die Möglichkeiten, aber auch die Grenzen der jeweiligen Verfahren aufzeigen.
2.
Beurteilung von E-Learning anhand von Qualitätskriterien
Bewertungsinstrumente auf Basis von Qualitätskriterienkatalogen sind deswegen so
populär, weil sie Einschätzungen über die Lernwirksamkeit, die eigentlich nur über
aufwändige empirische Verfahren erhoben werden können, relativ einfach zu ermöglichen scheinen. Dadurch bieten sie auch Laien die Möglichkeit, ohne vorhergehende
empirische Studien Einschätzungen über die Qualität eines Lernarrangements oder einer Lernsoftware vorzunehmen. Als Qualitätskriterium wird dabei ein Merkmal einer
Lernsoftware bezeichnet, dessen Lernwirksamkeit in einer Validitätsstudie nachgewiesen wurde. Kohrt (1995) fasst diesen Sachverhalt folgendermaßen:
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„Ein potentielles Qualitätskriterium (das also in einer Checkliste auftauchen könnte) kann jedes
isolierte Merkmal eines Lehrprogramms oder seiner Begleitmaterialien sein, das sich messbar
positiv bzw. negativ auf den Lernerfolg auswirkt“ (ebenda, S. 6).
Viele Qualitätskriterienkataloge enthalten jedoch Kriterien, zu denen keine expliziten
Validitätsstudien vorliegen, sondern über die lediglich vermutet wird, sie seien lernwirksam. Die Entstehung von Qualitätskriterienkatalogen kann mit Gräber (1996) in
insgesamt vier Generationen von Bewertungsinstrumenten eingeteilt werden (siehe Abbildung 1). Sie unterscheiden sich sowohl hinsichtlich ihrer zeitlichen Entstehung als
auch bezüglich ihres Funktionsumfanges. Im Hinblick auf den Funktionsumfang ist zu
differenzieren zwischen einfachen Zusammenstellungen von Qualitätskriterien, die als
Anhaltspunkt für die Auswahl von Lernsoftware für Laien gedacht sind, nach computergestützten Bewertungssystemen, die Einschätzungen zu unterschiedlichen Bereichen
(bei-
Abb. 1: Vier Generationen von Kriterienkatalogen (nach Gräber 1996).
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spielsweise didaktische Gestaltung, Bildschirmpräsentation u.ä.) abfragen und am
Schluss einen Punktewert liefern, mit dem die bewertete Software gegenüber anderen
verglichen werden kann4, bis hin zu vollständig datenbankgestützten Kriteriensammlungen. Letztere stellen unter Zuhilfenahme unterschiedlicher Filter Kriterien entweder
für die Auswahl, für die Entwicklung oder die Beurteilung von Lernsoftware bereit.5
Gräber (1996, S. 17ff.) ordnet einfache Checklisten, aber auch strukturiertere, komplexere Instrumente der ersten Generation von Bewertungsinstrumenten zu. Er führt
aus, dass diese charakterisiert sind durch eine unzureichende Berücksichtung des Softwaretyps, der Lehr-Lernstrategie, der Aufgabenfelder beziehungsweise Inhaltsbereiche,
der Zielgruppen der Lerner sowie der Beurteiler und des Zeitpunktes der Bewertung.
Winship (1988, S. 371) weist darauf hin, dass es für unterschiedliche Verwendungszwecke von Lernsoftware auch unterschiedliche Beurteilungskriterien geben sollte.
Auf diese Forderung wurde in der zweiten Generation von Bewertungsinstrumenten
reagiert, indem Instrumente von vornherein für bestimmte Softwaretypen entwickelt
wurden. Insgesamt wird seit Anfang der 1990er Jahre verstärkt darauf hingewiesen,
dass „verschiedene Lernformen [...] unterschiedliche Computerprogramme [benötigen,
d. Autor], deren Verwendungszwecke demzufolge differenziert betrachtet werden müssen“ (Mandl et al. 1992, S. 15ff.). Mandl et al. (ebenda) unterscheiden nach Kanselaar
(1992) vier Lernformen, denen sie vier Computerprogrammtypen beiordnen (siehe Abbildung 2). Diesen können wiederum spezielle Bewertungsinstrumente zugeordnet werden, die in die sogenannte zweite Generation von Bewertungsinstrumenten fallen.
Neben Bewertungsinstrumenten für verschiedene Softwaretypen und Lernstrategien wurden auch spezifischere Bewertungsinstrumente entwickelt, unter anderem für
spezielle Aufgabenfelder (zum Beispiel Instrument zur Bewertung von Software zum
Lesen lernen von Squires/McDougall 1994, S. 127), für verschiedene Zielgruppen von
Lernern (etwa im Grundschulbereich: Schwarz/Lewis 1989 oder in der Erwachsenenbildung: Dejoy/Mills 1989), für verschiedene Zielgruppen von Bewertenden (beispielsweise computerunerfahrene Lehrer: EPASoft von Gräber et al. 1992; für computererfahrene
Lehrer: Reeves/Harmon 1994 oder für Lerner: Akademie des Deutschen Beamtenbundes
1986).
Neben solchen Bewertungsinstrumenten für spezifische Zwecke entstanden weitere,
die spezifische und allgemeine Teile verbinden sollten. Sie sind insgesamt allgemein
gehalten, setzen aber für bestimmte Bereiche spezifische Schwerpunkte. Hierunter fallen zum Beispiel Kriterienkataloge, die zum einen aus einem allgemeinen Teil bestehen
und zum anderen weitere Teilbereiche enthalten, die sich spezifischen Bereichen des
Lernens mit Lernsoftware wid4 Ein Beispiel für ein solches Bewertungsinstrument ist der vom Arbeitskreis der deutschen Automobilindustrie
entwickelte Kriterienkatalog AKAB.
5 Ein Beispiel für ein solches umfassendes Bewertungsinstrument stellt der datenbankgestützte Kriterienkatalog MEDA dar.
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Lernform
Computerprogramm
Bewertungsinstrumente (Beispiel)
Lernen als Wiederholen
und Memorieren
Übungsprogramm (etwa Drill& Practice-Programme zum
Vokabellernen)
„Drill and Practice Software Evaluation“, G. Bitter, A. Camuse
(1994), Arizona State University
Lernen als interaktiver und
konstruktiver Prozess
Tutorielle Programme
„Tutorial Software Evaluation“,
G. Bitter/A. Camuse (1994), Arizona State University
Lernen als explorativer
und entdeckender Prozess
Simulationsprogramme
„Simulation Software Evaluation“, G. Bitter/A. Camuse (1994),
Arizona State University
Lernen als Rekonstruktionsprozess
Cognitive Tools (wie etwa
Textverarbeitungswerkzeuge
oder Autorenwerkzeuge, sogenannte Teachware [Mandl et
al. 1992, S. 498])
„Word processing Software Evaluation“ G. Bitter/A. Camuse
(1994), Arizona State University
oder „Leistungsmerkmalskatalog
für Autorenwerkzeuge“, Siemens
AG
Abb. 2: Lernformen, Computerprogrammtypen und dazu kongruente Bewertungsinstrumente der
2. Generation (Lernformen und Lernprogramme vgl. Mandl et al. 1992, S. 16; Zuordnung
der Bewertungsinstrumente vgl. Gräber 1996, S. 19)
men. Dies können zum Beispiel der Bereich affektiver Lernprozesse (vgl. etwa Martin
1998) oder spezielle Fragen zu einzelnen Lernsoftwaretypen (vgl. etwa Blease 1986)
sein. Reay (1985, S. 79) entwickelt einen Kriterienkatalog für einen zweistufigen Bewertungsprozess. Zunächst bewertet ein Experte (in diesem Fall ein Lehrer) das Lernprogramm alleine und dann wird ein Schüler beim Lernen mit der Software beobachtet.
Zusammenfassend wird deutlich, dass die zweite Generation von Bewertungsinstrumenten aus sehr komplexen Instrumenten bestand, die für spezifische Fragestellungen
ausgelegt waren. Eine differenziertere Bewertungsmöglichkeit war so zwar möglich,
andererseits führte diese Entwicklung auch zu einer unüberschaubaren Menge an Bewertungsinstrumenten, da teilweise sogar für einzelne Programme eigene Kriterienkataloge zur Bewertung entwickelt wurden (vgl. Gräber 1996, S. 20). Eine intersubjektive
Vergleichbarkeit war daher teilweise nicht mehr gegeben.
Die dritte Generation von Bewertungsinstrumenten versuchte dieser Instrumentenvielfalt entgegenzuwirken, aber trotzdem die Differenzierungskraft beizubehalten, die
zuvor erzielt worden war. So zählt Gräber (ebenda, S. 22) zur dritten Generation von
Bewertungsinstrumenten solche, die versuchen, in einem Instrument alle Ansprüche und
Gegebenheiten zu berücksichtigen. Darunter fallen Instrumente wie die „Große Prüfliste von Lernsoftware“ (GPL), die Thomè (1988) im Rahmen einer Dissertation aus einer
Synopse von zwölf Kriterienkatalogen erstellte. Dieser Katalog enthält 221 Bewertungsaspekte, die in
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unterschiedliche Bewertungskategorien geordnet werden (vgl. Gräber 1996). Ein anderes Instrument, MEDA (Gräber 1991), stellt in Form einer Datenbank einen Katalog von
über 300 Fragen zur Verfügung, die bei der Beschreibung, Analyse und Bewertung didaktischer Software eingesetzt werden können. Der Beurteiler muss in einer Dreistufenhierarchie seine eigene Bewertungsintention und Zielvorstellungen wählen. Am Schluss
bekommt er eine Liste, die für seine jeweiligen Zwecke relevante Fragen präsentiert.
Die vierte Generation von Bewertungsinstrumenten benennt Gräber als prozessorientierte Instrumente (ebenda). Sie fokussieren nicht allein auf eine punktuelle Überprüfung von Softwaremerkmalen, sondern sollen eine Orientierungshilfe für Bildungsmöglichkeiten in unterschiedlichen Kontexten sein. Die Bewertungskriterien beziehen
sich hier darauf, den gesamten Prozess der Entwicklung, der Auswahl und des Einsatzes eines Programmes begleitend zu evaluieren. Als umfangreichste Arbeit in diesem
Zusammenhang kann die sogenannte „Evaluator’s Toolbox“ (Reeves 1992) angesehen
werden. Sie stellt ein sogenanntes Electronic Performance Support System (EPSS 6) dar.
Die Toolbox enthält elektronische Informationen, Beispiele, Checklisten und Fragebögen zur Evaluation von allen Schritten der Entwicklung interaktiver multimedialer
Programme. Alle Schritte werden evaluiert und erst auf Basis der Ergebnisse wird der
jeweils nächste Entwicklungsschritt geplant. Auch das Projekt Evaluationsnetz verfolgt
das Konzept, für unterschiedliche Phasen der Konzeption, des Designs und der Durchführung angemessene Kriterien zu spezifizieren. [www.evanetz.de].
Insgesamt kann der Schluss gezogen werden, dass sich die Bewertungsinstrumente
in zweierlei Hinsicht weiterentwickelt haben: Während die erste Generation an Bewertungsinstrumenten noch hauptsächlich technische Aspekte fokussierte, wurden nach
und nach immer stärker pädagogisch-didaktische Elemente berücksichtigt. Der zweite Entwicklungsstrang ist in Richtung einer zunehmend stärkeren Differenzierung von
Kontexten zu beobachten. Eine fundierte, theoriegeleitete und empirisch validierte Bewertungsgrundlage bieten die wenigsten derzeit verfügbaren Instrumente. Unter anderem daran schließt sich auch die Kritik an, die zur Bewertung mittels Katalogen von
Qualitätskriterien oder Checklisten geäußert wird.
Zunächst weist Meier (1995) darauf hin, dass in vielen Qualitätskriterienkatalogen
überwiegend Kriterien aus dem Bereich „Gestaltung der Bildschirmoberflächen“ oder
„Technik des Programmablaufs“ enthalten sind, didaktische Kriterien also oftmals unterrepräsentiert sind. Aus Metastudien zur Lernwirksamkeit von multimedialen Lernarrangements geht jedoch hervor, dass gerade
6 Gerry (1991) definiert die Funktionen eines EPSS folgendermaßen: „A system that provides electronic task
guidance and support to the user at the moment of need. EPSS can provide application help, reference information, guided instructions and/or tutorials, subject matter expert advice and hints on how to perform a task
more efficiently. An EPSS can combine various technologies to present the desired information. The information can be in the form of text, graphical displays, sound, and video presentations“ (ebenda).
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die didaktischen Konzepte, die im E-Learning umgesetzt sind und die zu Grunde liegenden Lernarrangements und -situationen günstigere Einflüsse auf den Lernprozess haben
als die verwendete „delivery technology“ (vgl. Weidenmann 1997; Kulik/Kulik 1991,
1994).
In der Literatur werden darüber hinaus vier Argumente gegen die Verwendung von
Kriterienkatalogen zur Beurteilung der Qualität von Lernsoftware genannt, die wie folgt
zusammengefasst werden können:
1) Mangelnde Beurteilerübereinstimmung bei der Quantifizierung von Qualitätskriterien: Fricke (2000, S. 76) weist darauf hin, dass ein Merkmal eines Lernprogrammes
dann als valide – und damit als lernwirksam – angesehen wird, wenn eine (zumindest
geringe) Korrelation zwischen Ausprägungsgrad des Merkmals und dem Lernergebnis zu erkennen ist. Reduziert sich die Messgenauigkeit des Merkmals, so reduziert
sich auch seine Validität. Bei der Quantifizierung von Merkmalen in Bezug auf eine
Lernsoftware kommt es oftmals zu großen Diskrepanzen zwischen den Beurteilern.
In diesem Fall wird von einer mangelnden Beurteilerübereinstimmung gesprochen.
Fricke (ebenda) berichtet von einem Versuch, bei dem sechs Experten ein und dasselbe
Lernprogramm anhand des oben beschriebenen Instruments MEDA unabhängig voneinander bewerten sollten. Weder war eine hinreichende Übereinstimmung in Bezug
auf die Bewertung der Relevanz der Kriterien zu beobachten noch in Bezug auf die
Einschätzung der Ausprägung der Kriterien bei der vorliegenden Lernsoftware.
2) Geringe praktische Signifikanz der Qualitätskriterien: Bei der Beurteilung von
Lernsoftware ist festzustellen, dass Kriterien, die objektiv gut operationalisierbar und
damit gut messbar sind, wie zum Beispiel Fragen nach dem Vorhandensein einer Hilfeschaltfläche in einem Lernprogramm, oftmals nur geringe Auswirkungen auf den
tatsächlichen Lernerfolg haben. Merkmale einer Lernsituation hingegen, die in der
Regel nur schwer operationalisiert werden und unter Gesichtspunkten der Beurteilerübereinstimmung auch nicht valide gemessen werden können, wie zum Beispiel der
Enthusiasmus eines Lerners oder Lehrers, scheinen einen sehr hohen Einfluss auf den
Lernerfolg zu haben (vgl. Fricke 2000). Das bedeutet, dass viele Merkmale, die eine
hohe Messgenauigkeit aufweisen, nur geringe Validitäten in Bezug auf das Lernergebnis haben. Umgekehrt konnten mit Merkmalen, die nur eine geringe Messgenauigkeit
bieten, bessere Voraussagen in Bezug auf das Lernergebnis getroffen werden (vgl. Rosenshine/Furst 1971). Fricke (ebenda) betont die Notwendigkeit, empirisch überprüfte
Lehr-Lerntheorien zur Erklärung dieser Paradoxien heranzuziehen, um eine unbefriedigende Zusammenstellung von Kriterien mit augenscheinlicher, aber unbewiesener
Validität („face validity“) zu vermeiden.
3) Differentielle Methodeneffekte bei Qualitätskriterien: Das Prinzip von differentiellen Methodeneffekten nach Cronbach und Snow (1971) lässt sich auch auf die Erforschung der Effektivität eines Lernarrangements anwenden. Legt man das von Fricke
(1995) vorgeschlagene Paradigma zur Konstruktion und
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Abb. 3: Konstituierende Faktoren von Lehr-Lernumgebungen (nach Fricke 1995).
Evaluation multimedialer Lehr-Lernumgebungen zu Grunde (vgl. Fricke 1995, S.
405), dann besteht ein Lernarrangement aus vier Hauptfaktoren: (a) die multimediale
Lernumgebung, (b) die Lernervariablen, (c) das Lerntbema und (d) das Lernergebnis
(siehe Abbildung 3).
Die Bewertung von Lernarrangements mit Kriterienkatalogen folgt in der Regel
dem Paradigma der Wirkungsforschung, das heißt, dass das Lernergebnis als abhängige
Variable und die anderen Variablen als unabhängige Variablen angesehen werden. Die
Ausprägungen der zu Grunde liegenden Kriterien bestimmen dann die Wirkung der unabhängigen Variablen auf die abhängige Variable, also das Lernergebnis.
Differentielle Methodeneffekte wirken dabei nun auf die Weise, dass die Zusammenhänge von unabhängigen und abhängigen Variablen geschwächt werden. Die Ursache hierfür liegt darin, dass die unabhängigen Variablen eines Lernarrangements nicht
nur auf die abhängigen Variablen Einfluss nehmen, sondern sich auch untereinander beeinflussen. So sind in Untersuchungen Wechselwirkungen zwischen Lernumgebung und
Lernstoff (vgl. Jung 1994), Lerner und Lehrstoff (vgl. Fricke 1989) und den Merkmalen
von Lernumgebungen untereinander (vgl. Fricke 1989; Meier/Baratelli 1991) nachgewiesen worden.
Fricke (2000) kommt zu dem Schluss, dass die Bewertung einer Bildungssoftware
nur aus ganzheitlicher Sicht erfolgen kann. Die aufgezeigten Wechselwirkungen führen
dazu, dass es so etwas wie die Effektivität eines Lernprogrammes nicht geben kann
– und auch nicht die Validität von Kriterien. Berücksichtig man ein Lernarrangement
ganzheitlich, so kann ein Kriterium einer Lernsoftware in einem Fall ein „positives“
Qualitätskriterium sein und in einem anderen Fall – etwa bei einer anderen Zielgruppe
– eher ein Nachteil.
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4) Nichtberücksichtung des Verwertungszusammenhangs einer Bildungssoftware:
Oftmals werden multimediale Lernprogramme als Substitut für konventionelle Fortbildungen eingesetzt. Dabei besteht die Hoffnung, Kosten zu sparen, da die Mitarbeiter am Arbeitsplatz lernen und keine – oder zumindest nur geringere – Ausfallzeiten
entstehen und keine Reisekosten anfallen (zu Kosten und Nutzen beim E-Learning
vgl. Gröhbiel 2003). Es ist unstrittig, dass E-Learning in der beruflichen Weiterbildung nur dann erfolgreich eingesetzt werden wird, wenn es Kostenvorteile mitbringt – oder zumindest bei gleichen Kosten eine viel versprechendem Alternative zu
konventionellen Qualifizierungsmaßnahmen darstellt. Genau diese Aspekte werden
in den meisten Kriterienkatalogen nicht mit berücksichtigt. Dazu kommt, dass in
Studien gezeigt werden konnte, dass der Einsatz von Lernprogrammen in Betrieben
oftmals zwar zu den gleichen Lerneffekten führt, aber insgesamt die Arbeitsorganisation und den Betriebsablauf so effektiviert, dass damit Kosteneinsparungen erzielt
werden konnten (vgl. Brinker 1991). Anders ausgedrückt scheint es von Bedeutung,
dass die Variable Lernergebnis in Frickes Modell (siehe Abbildung 3) noch um Merkmale der betriebswirtschaftlichen Kosten-Nutzen-Rechnung erweitert wird.
Insgesamt kann der Schluss gezogen werden, dass es gravierende Argumente gegen die
ausschließliche Verwendung von Kriterienkatalogen zur Bewertung der Qualität von
Lernsoftware gibt. Die Beurteilerübereinstimmung kann dabei zwar durch gezieltes
Training verbessert werden (vgl. dazu auch Flanders 1970) und auch Aspekte der betrieblichen Kosten-Nutzen-Rechnung können in Qualitätskriterien zumindest ansatzweise einfließen. Schwerer wiegen jedoch die Kritikpunkte der „geringen praktischen
Signifikanz“ von Kriterien und der „differentiellen Methodeneffekte“.
In Bezug auf einen diskursiven Qualitätsbegriff kann eine deutliche Abgrenzung
vorgenommen werden. Während ein am Lernenden orientierter Qualitätsbegriff sich auf
den ausgehandelten Lernprozess aus Sicht des lernenden Subjektes bezieht und Qualität damit an Bedingungen und Prozessen dieser Aushandlung beziehungsweise dieses
Erbringungsverhältnisses festmacht, liegt bei einer Bewertung durch Qualitätskriterien
ein von vornherein normativ fixierter Qualitätsbegriff vor.
3.
Evaluationsverfahren für E-Learning-Arrangements
Eine weitere Möglichkeit, Qualität beim E-Learning zu sichern beziehungsweise zu bewerten, besteht in der Anwendung von Evaluationsverfahren für E-Learning-Arrangements. Der Begriff Evaluation ist sehr vielschichtig und kann sehr unterschiedlich definiert werden. Die Definitionsvielfalt zeigt sich in der Literatur (Fricke [2002] führt eine
umfangreiche Sammlung von Literaturstellen mit unterschiedlichen Evaluationsbegriffe
auf: Stufflebeam 1969, 1972,
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1994; Wulf 1972; Stiefel 1974; Lange 1983; Prell 1981 und 1986; Gerl/Pehl 1983; Wittmann 1987; Fricke 1986; Wottawa 1986; Lösel/Nowack 1987; Will et al. 1987; Rossi
et al.1988; Wottawa/Thierau 1990; Berendt/Stary 1993; Götz 1993; Seidel/Park 1994).
E-Learning kann aus den verschiedensten Perspektiven evaluiert werden. Die Aufarbeitung des Forschungsstandes zu diesem Thema kann eingeteilt werden in verallgemeinerbare Ergebnisse, die vielfältige Evaluationsstudien bislang hervorgebracht haben
und Evaluationskonzepte, die speziell für das E-Learning entwickelt sind. Im folgenden Abschnitt werden vor allem Evaluationskonzepte berücksichtigt, da die bisherige
Evaluationsforschung nur wenige verallgemeinerbare Ergebnisse vorgebracht hat. Diesen Standpunkt vertritt auch Keil-Slavik (1999, S. 12), der beispielsweise die Situation im Bereich multimedialer Lehre in der Hochschule folgendermaßen einschätzt: „...
zu jedem Befund lässt sich mindestens eine Studie mit einem gegensätzlichen Befund
finden.“ Seiner Meinung nach könne das Ziel von Evaluation daher nicht sein, eine abschließende Bewertung vorzunehmend, sondern sie vielmehr evolutionär im Sinne ständiger Weiterentwicklung zu betreiben.
Der Begriff der Evaluation kann nicht allgemeingültig definiert werden, da er in
unterschiedlichsten Kontexten und theoretischen Zusammenhängen verwendet wird.
Evaluation geht aber über die reine Messung von einzelnen Variablen, zum Beispiel im
Sinne empirischer Lehr-Lernforschung (siehe dazu Kapitel 4.3), hinaus und stellt in der
Regel eine umfassende Bewertung von erhobenen Daten dar. Vier Elemente werden von
Will et al. (1987) besonders hervorgehoben:
„1. Evaluation ist ziel- und zweckorientiert. Sie hat primär das Ziel, praktische Maßnahmen zu
verbessern, zu legitimieren oder über sie zu entscheiden.
2. Grundlage der Evaluation ist eine systematisch gewonnene Datenbasis über Voraussetzungen, Kontext, Prozesse und Wirkungen einer praxisnahen Maßnahme.
3. Evaluation beinhaltet eine wertende Stellungsnahme, d.h. die methodologisch gewonnenen
Daten werden auf dem Hintergrund von Wertmaßstäben unter Anwendung bestimmter Regeln bewertet.
4. Evaluation bezieht sich im Gegensatz zur personenbezogenen Leistungsfeststellung oder
Testung auf einzelne Bereiche geplanter, durchgeführter oder abgeschlossener Bildungsmaßnahmen. Sie zielt also in der Regel nicht primär auf die Bewertung des Verhaltens (z.B.
von Leistungen) einzelner Personen, sondern ist Bestandteil der Entwicklung, Realisierung
und Kontrolle planvoller Bildungsarbeit“ (ebenda, S. 14).
Im Gegensatz zur Bewertung von E-Learning-Angeboten mit Qualitätskriterienkatalogen oder Checklisten steht bei der Evaluation nicht so sehr die Messung angebots-/
produktbezogener Merkmale im Vordergrund, sondern die Be-
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Wertung von Lernprozessen und das Erheben von Urteilen bzgl. Qualität, Wirkung (Akzeptanz, Lernerfolg) und wahrgenommenem Nutzen.
3.1
Grundlegende Evaluationsformen und -methoden
In der Evaluationsforschung wird allgemein unterschieden zwischen formativer und
summativer Evaluation. Formative Evaluation dient in der Regel eher der Qualitätssicherung. Ihr Ziel ist die Ermittlung von Schwachstellen. Die Forschungen verlaufen
entwicklungsbegleitend und dienen der Optimierung bereits während des laufenden
Prozesses. Summative Evaluation dient dahingegen eher der Kontrolle von Qualität,
Wirkung und Nutzen eines Bildungsangebotes. Der Evaluationszeitpunkt liegt daher zumeist am Ende eines Prozesses. Götz (1993, S. 105ff) stellt weitere Evaluationsmodelle
vor, beispielsweise Input-Outputevaluation, Fremd- vs. Selbstevaluation, Teil- und Gesamtevaluation, subjektive und objektive Evaluation, direkte und indirekte Evaluation,
intrinsische und extrinsische Evaluation, additive und integrierende Evaluation sowie
interne und externe Evaluation. Eine weitere Unterscheidung trifft Tergan (in Schenkel
2000) im Bereich von Evaluation, indem er zwischen Prozess- und Produktevaluation
differenziert. Es wird dabei zwischen solchen Evaluationen, bei denen der Evaluationsgegenstand eher die Prozesse einer Entwicklung sind, und solchen, die sich auf das
fertige Produkt am Ende einer Entwicklung konzentrieren, unterschieden.
Spezielle Evaluationskonzepte für E-Learning benötigen dahingehend eine theoretische Grundlegung, welche Prozesse beim Lernen mit Medienunterstützung sich in welcher Weise beeinflussen. Eine theoretische Basis bietet hier beispielsweise die Theorie
des Instruktionsdesigns von Reigeluth (1983). Nach Reigeluth gibt es drei Hauptkomponenten im Instruktionsprozess: Lernergebnisse („instructional outcomes“), Randbedingungen der Instruktion („instructional conditions“) und die Instruktionsmethode („instructional methods“). Fricke (vgl. 1995, 2000, 2002) entwickelt auf Basis dieses Modells
das bereits vorgestellte „Paradigma zur Konstruktion und Evaluation von Lehr-/Lernumgebungen“ (siehe auch Kapitel 2, Abbildung 3).
Das Modell kann deskriptiv oder präskriptiv gelesen werden (vgl. Fricke 1995,
2000, 2002). Im Sinne einer deskriptiven Theorie gibt das Modell Aufschluss darüber,
welche Effekte (Outcomes) verschiedene Lehrmethoden bei gegebenen Randbedingungen haben. Bei präskriptiver Lesart fungieren nicht die Lehrmethoden, sondern die Lernergebnisse als unabhängige Variable. Eine deskriptive Theorie soll also dazu dienen,
das Zustandekommen von Lernergebnissen bei bestimmten Lehrmethoden zu erklären,
während eine präskriptive Theorie geeignete Lehrmethoden zum Erreichen bestimmter
Lernergebnisse Vorschlägen soll.
Das Modell ermöglicht es auf diese Weise, zwischen unterschiedlichen Evaluationszielen und unterschiedlichen Komponenten eines Lernarrangements zu
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differenzieren. In Frickes Paradigma werden auch die Aspekte von Konstruktion einerseits und Evaluation andererseits zusammengeführt. Hier wird die Bedeutung von
prozessbegleitender Evaluation für die Qualität eines Lernarrangements deutlich. Ausdrücklich weisen auch Reigeluth (1983) und Reinmann-Rothmeier et al. (1994) auf die
Bedeutung einer solchen formativen Evaluation auf allen Stufen der Konstruktion und
Durchführung von E-Learning-Arrangements hin: „For the educational technology field,
evaluation was now being viewed as an integral and ongoing part of the instructional
development process“ (ebenda in Fricke 2002, S. 450).
3.2
Überblick über instruktionstheoretische Evaluationskonzepte für E-Learning
Kirkpatrick (1994) hat in einer Artikelserie „Techniken zur Evaluation von Trainingsprogrammen“ in den USA einen Evaluationsansatz formuliert, der zwischen vier Evaluationsebenen unterscheidet: Reaktionsebene, Lernebene, Verhaltensebene und Ergebnisebene. Kirkpatricks Modell erleichtert den Zugang zu Evaluationen und präzisen Ergebnissen. Zu häufig werden Aussagen über die Qualität von Weiterbildungsmaßnahmen
getroffen, ohne dass deutlich wird, ob sie sich auf die Reaktion der Lernenden, auf die
Handlungsfähigkeit der Absolventen, auf die Kosten oder auf den Erfolg der Weiterbildung beziehen.
Daneben existiert eine Reihe von Evaluationskonzepten speziell für multimediale
Lehr-Lernumgebungen. Reigeluths (1983, S. 77ff.) Instruktionsdesign kann als Grundlage für die Evaluation von multimedialen Lehr-Lernumgebungen genutzt werden. Zur
Konstruktion solcher Lernarrangements nach dem Paradigma des Instruktionsdesigns
unterscheidet er fünf Stufen:
1) Instructional Design: Auswahl geeigneter Ziele und entsprechender Instruktionsund Lehrmethoden für das Erreichen eines bestimmten Zieles bei einer vorgegebenen
Zielgruppe
2) Instructional Development: Entwicklung und Konstruktion konkreter Lehrmethoden (Reigeluth benutzt hier die Metapher des beziehfertigen Rohbaus eines Hauses)
3) Instructional Implementation: Genaue Anpassung der Lehrmethoden an die konkreten Rahmenbedingungen (Reigeluth benutzt hier die Metapher des „endgültigen
Ausbaus eines Hauses“ nach den Wünschen eines Mieters)
4) Instructional Management: Konkrete Einsatzplanung eines Qualifizierungsprogrammes, zum Beispiel in einem Unternehmen
5) Instructional Evaluation: Hier geht es um „[...] understanding, improving, and applying methods for assessing the effectiveness and efficiency of all [...] activities“
(Reigeluth 1983, S. 9)
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Im Evaluationsmodell von Ross und Morrison (vgl. Fricke 2002, S. 451) werden einige dieser Elemente aufgenommen. Formative und summative Ansätze, quantitative und
qualitative Methoden sowie Ansätze, die sowohl aus dem Instruktionsdesign als auch
aus der konstruktivistischen Lerntheorie stammen, sind hier kombiniert worden. Ihr
Evaluationsmodell enthält vier Stufen:
1) Needs Analysis: Festlegung der Ziele und Fragen der Evaluationsstudie
2) Methodology: Auswahl der Evaluationsmethoden und Implementierung der Methoden auf fünf Stufen
–– Programm Analysis: Analyse der Lehrziele und Präsentationsformen des Programms
–– Participant Analysis: Festlegung der Personenstichprobe
–– Evaluation design: Aufstellen des Versuchsplans und Festlegung der Messmethoden (sowohl formative als auch summative; qualitative als auch quantitative
Methoden)
–– Instrumentation: Zusammenstellung der Evaluationsinstrumente zu einem
kohärenten Gesamtevaluationsplan unter Berücksichtigung der gegebenen Beschränkungen
–– Implementation: Festlegen des zeitlichen Verlaufs der Evaluation
3) Data Analysis and Interpretation: Auswertung und Aufbereitung der Daten
4) Disseminating Results: Erstellung und Übergabe eines Abschlussberichts
Ross und Morrison betonen, dass die Evaluation als ein iterativer Prozess gesehen werden kann, der zu einer fortwährenden Verbesserung des Programms führen kann. Ihr
Vorschlag kann als Rahmenmodell für die Evaluation multimedialer Lehr-Lernarrangements angesehen werden und ist dementsprechend auch für E-Learning-Arrangements
anwendbar. Fricke (2002) bündelt beide zuvor vorgestellten Evaluationsbeziehungsweise Konstruktionsansätze in einem Modell (Fricke 2002, S. 455ff.). Dazu erweitert er das
von ihm nach Vorschlägen von Reigeluth entwickelte „Paradigma zur Konstruktion und
Evaluation multimedialer Lehr-Lernumgebungen“ (siehe Abbildung 3) um zwei weitere
Rahmenbedingungen des Lehrens und Lernens: zum einen handelt es sich dabei um die
Rahmenbedingung Lehr-Lerntheorien, da die Bewertung von Lernarrangements nicht
ohne die Berücksichtung der implizit oder explizit zu Grunde liegenden Lerntheorie
vorgenommen werden kann. Zum anderen ergänzt er das Modell um die Komponente
allgemeiner Rahmenbedingungen, wie zum Beispiel politische Vorgaben, finanzielle
Ressourcen, Produktionskosten, Vorgaben für Qualifikationen und Lehrziele, die seinem Verständnis nach ebenfalls eine wichtige Rahmenbedingung für die Bewertung von
Lernarrangements darstellen (siehe Abbildung 4).
Je nachdem, ob mit einem präskriptiven oder einem deskriptiven Ansatz evaluiert
wird, kommt er zu dem Schluss, dass sich insgesamt neun Evaluationsformen differenzieren lassen. Diese neun Evaluationsformen stellen sich als aufeinander aufbauende
Evaluationsschritte für jeweils einen Teilschritt bei der Kon-
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Abb. 4: Evaluationsformen auf Basis des Paradigmas zur Konstruktion und Evaluation multimedialer (Fricke 2002, S. 456).
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struktion einer multimedialen Lehr-Lernumgebung dar (zusammengefasst nach Fricke
2002, S. 457ff):
1) Die Ergebnisevaluation (Ergebnis als unabhängige Variable): Überprüfung der Lehrziele mit den Lehr-Lerntheorien, die Lernergebnisse sind dabei unabhängige Variablen.
2) Die Ergebnisevaluation (Ergebnis als abhängige Variable): Überprüfung der tatsächlich erreichten Lernergebnisse anhand von Lehr-Lerntheorien, die Lernergebnisse sind
hier abhängige Variablen.
Fricke (ebenda) unterscheidet im Weiteren zwischen Evaluation von Methodenkonstruktion, Evaluation von (differentiellen) Methodeneffekten (s. auch Kapitel 2), Methodenevaluation und Evaluation von Methodenwirkungen:
3) Evaluation der Methodenkonstruktion: Überprüfung der geplanten Lehrmethoden
mit den zu Grunde liegenden Lehr-Lerntheorien, zumeist geschieht dies als entwicklungsbegleitende, optimierende Evaluation.
4) Überprüfung differenzieller Methodeneffekte bezüglich unterschiedlicher Lernthemen: Während der Methodenkonstruktion müssen Theorien und Erkenntnisse über
differenzielle Methodeneffekte bezüglich unterschiedlicher Lernthemen berücksichtigt werden.
5) Überprüfung differenzieller Methodeneffekte bezüglich unterschiedlicher Lernervariablen: Während der Methodenkonstruktion müssen auch hierbei Theorien und Erkenntnisse über differenzielle Methodeneffekte bezüglich der Lernervariablen herangezogen werden.
6) Methodenevaluation 1 (Methoden als abhängige Variable): Bei dieser Evaluationsform wird das fertig konstruierte Lernarrangement auf Übereinstimmung mit den zu
Grunde liegenden Lehr-Lerntheorien überprüft, die Instruktionsmethoden fungieren
dabei als abhängige Variable.
7) Methodenevaluation 2 (Methoden als unabhängige Variable): Neue Instruktionsmethoden, die „ad-hoc“ in ein Lernarrangement eingebracht werden (beispielsweise vom
Anwender) und nicht das Ergebnis eines Konstruktionsprozesses darstellen, können
mit dieser Evaluationsform überprüft werden, indem sie als unabhängige Variable angesehen werden.
8) Evaluation der Methodenwirkungen: Hier werden die Medien- und Methodenwirkungen im Sinne der klassischen Medienwirkungsforschung evaluiert.
9) Metaevaluation als Kosten-Nutzen-Evaluation: Hier geht es nicht mehr um eine theoriegeleitete Konstruktion oder Überprüfung multimedialer Lehr-Lernumgebungen,
sondern um eine Analyse der eingesetzten Ressourcen im Vergleich zu den Wertigkeiten der erreichten Leistungen (zum Beispiel Wissensbestände, Kompetenzzuwächse).
Die einzelnen Evaluationsformen zeigen, dass Evaluation während des gesamten Konstruktions- und Durchführungsprozesses auf ein multimediales Lehr-Lernarrangements
angewendet werden kann. Fricke (2002) betont die Bedeutung der Orientierung und Überprüfung des Evaluationsprozesses an vorhande-
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Abb. 5: Evaluationsmodell Dresdner Ei (Schott 2000).
nen Lehr-Lerntheorien. Wie die oben aufgeführten Evaluationsformen zeigen, ist an
mindestens neun Stellen im Konstruktionsprozess eine solche Überprüfung sinnvoll.
Die neun Evaluationsformen können so der Qualitätssicherung im Sinne einer durchgehenden Orientierung an gleich bleibenden Bestimmungsgrößen dienen.
Ein weiterer Evaluationsansatz, der ebenfalls auf einer Instruktionstheorie, der sogenannten „Universal Constructive Instructional Theory“ (UCIT, vgl. Dijkstra et al. 1997;
Tennyson et al. 1997), aufbaut, liegt von Schott (2000) vor. Die sogenannte „Effiziente
Lern- und Informations-System-Evaluation“ (ELISE) besteht aus zehn Ablaufschritten
und stellt ein System an Bewertungsschritten dar, das eine strukturierte Evaluation ermöglicht. Es kombiniert Aspekte des Instruktionsdesigns und der Instruktionstheorie
UCIT in einem Modell, dem sogenannten „Dresdner Ei“ (siehe Abbildung 5).
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Dieses Modell stellt ebenfalls eine Verbindung von Konstruktionsprozessen und Evaluationsschritten dar. Der Konstruktionsprozess gemäß „Dresdner Ei“ geht von einer
Bedarfsanalyse aus, formuliert ein Gesamtziel, prüft analytisch den möglichen Gestaltungsraum anhand der Komponenten Lernaufgabe, mögliche Lernumgebung, Lerner und
Bezugsrahmen und realisiert dann ein Instruktionsvorhaben in Form einer spezifischen
situativen Lernlösung (siehe Abbildung 5).
Der Evaluationsprozess geht nun den umgekehrten Weg. Vom Endprodukt her beginnend, ist die Evaluation zunächst mit einem fertigen multimedialen Lern- beziehungsweise Informationssystem konfrontiert. (In der Terminologie der UCIT wird dies
als situativ rekonstruierte Lernaufgabe, eingebettet in eine situative rekonstruierte Lernumgebung [siehe Abbildung 5] bezeichnet.) Die Frage nach den Alternativen zu diesen
Aufgaben spannt nun zunächst ein analytisch-situatives Möglichkeiten-Grenzen-System auf.
Danach wird gefragt, ob mit der eingesetzten Software das angestrebte Gesamtziel
erreicht wird und die Probleme, derentwegen die Software entwickelt wurde, auch damit
gelöst werden. Die anfangs eingeforderte theoretische Grundlage des Evaluationsansatzes ist durch die enge Verknüpfung mit der UCIT gewährleistet, jedoch liegen bislang
nur wenig praktische Erfahrungen mit dem Modell vor (vgl. Schott et al. 2000).
Ein solches ganzheitliches Evaluationsverständnis, wie in den oben beschriebenen
Ansätzen, das sich nicht nur auf die Messung von erreichten Effekten bezieht, sondern während des ganzen Konstruktionsprozesses entwicklungsbegleitende Optimierung leisten kann, findet sich auch bei Tergan (in Schenkel 2000), Schenkel (2000) und
Mandl/Reinmann-Rothmeier (1995), von denen ebenfalls dokumentierte Evaluationsansätze vorliegen. In Bezug auf Qualität wird deutlich, dass Evaluation als Methode
der Qualitätssicherung fungieren kann. Sie liefert jedoch keine konkreten Dimensionen
subjektiver Qualität, sondern stellt in den vorgestellten Ansätzen vielmehr jeweils einen
methodischtheoretischen Rahmen dar, in dem Qualität bewertet werden kann.
3.3
Ein subjektorientierter Evaluationsansatz
Ein weiterer Evaluationsansatz von Zimmer und Psaralidis (2000) basiert explizit auf
einer Subjektperspektive. Er wendet sich gegen eine Evaluation im Sinne einer reinen
Medienwirkungsforschung und stellt vor allem das Ziel beruflichen Lernens in den Vordergrund, welches hier als Zuwachs beruflicher Handlungskompetenz definiert wird.
Zimmer und Psaralidis (2000) argumentieren in ihrem Beitrag, dass der Lernerfolg
die Qualität einer Lernsoftware bestimme und dieser durch den Erwerb von Handlungskompetenz definiert sei. Bislang werde jedoch in vielen Evaluationsmodellen beziehungsweise Evaluierungen davon ausgegangen, dass die Qualität einer Lernsoftware
den Lernerfolg bestimmt – und nicht umge-
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kehrt. Die Autoren kritisieren solche Evaluationsmodelle als zu weit angelehnt an „Maschinenmodelle der Wirkungsforschung“ (ebenda). Wird Lernen aber als ein Prozess begriffen, bei dem der Lernende den Lernerfolg (Handlungskompetenz) in Ko-Produktion
mit dem Lernangebot (Lehrender, Lernarrangement etc.) erst erstellt, so ist es schlicht
nicht möglich, die Qualität von Lernsoftware im Sinne eines Handlungskompetenzzuwachses mit Qualitätskriterien im vorhinein zu prognostizieren.7 Dieser Evaluationsansatz definiert die Lernsituation als eine ganzheitliche Situation, die nur in einer ganzheitlichen Betrachtung Aufschluss über die Wirkung der Lernsoftware liefern kann. Ziel
ist es dabei, subjektwissenschaftliche Erkenntnisse zu erlangen, also Erkenntnisse über
das Lernen vom Standpunkt des Subjekts aus (vgl. Holzkamp 1983, 1993; Zimmer 1987).
Beim instruktionstheoretischen Lernprozess wird der Lernerfolg als durch die
Lernsoftware bewirkt angesehen. Die Lernsoftware passt sich dabei durch die Aktionen
der Lerner an den Prozess der Erzeugung des Lernerfolges an und soll ihn individuell
optimieren. Die Lernumgebung sowie die individuellen und organisatorischen Kontexte
werden dabei als intervenierende Variablen berücksichtigt, denen gegenüber die Lerner
forschungsmethodologisch als „ausgeliefert“ betrachtet werden.
Beim handlungsbestimmten Lernprozess wird dagegen forschungsmethodologisch
von dem am eigenen Lernerfolg interessierten Subjekt ausgegangen, das mit dem Ziel
des Kompetenzgewinns die Lernsoftware nutzt. Die Lernumgebung sowie die individuellen und organisatorischen Situationen werden dabei als Bereiche betrachtet, die den
Lernerfolg fördern oder hemmen, aber durchaus auch vom Subjekt mitgestaltet oder
kontrolliert werden können.
Um die primäre Evaluationsfrage beantworten zu können – welcher Lernerfolg auf
das Lernen mit einer Lernsoftware zurückzuführen ist -, muss zunächst der Evaluationsgegenstand genau definiert werden. Der Gegenstand ist dabei nicht die Lernsoftware,
sondern zum einen der unmittelbare Lernerfolg mit der Lernsoftware und der daraus
folgende Zugewinn an subjektiver Handlungskompetenz, etwa in einer Arbeitssituation,
und zum anderen der Lernprozess, der zu Lernerfolg und Kompetenzgewinn führt.
Methodologisch schlagen Zimmer und Psaralidis (2000) dafür die Methode der logischen Rekonstruktion vor. Es geht dabei darum, den begründeten Zusammenhang der
Handlungskompetenzen und der zu ihnen führenden Handlungen in ihren Verbindungen, Brüchen, Gegensätzlichkeiten und Widersprüchlichkeiten zu rekonstruieren, und
zwar zu einem bestimmten Zeitpunkt und in einer bestimmten Situation hinsichtlich
konkreter Handlungsziele. Die logische Rekonstruktion unterstellt, dass alle Handlungen von Subjekten einen
7 Zimmer und Psaralidis (2000) lehnen sich dabei an die Definition des Lernprozesses von Holzkamp an: „Ein
Lernprozess vollzieht sich immer im Subjekt durch manuelle und geistige Handlungen an dinglichen oder
ideellen Gegenständen in kulturellen Zusammenhängen und Prozessen mit dem Ziel eines Gewinns an subjektiver Handlungskompetenz“ (vgl. Holzkamp 1983, S. 236).
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erkennbaren Grund haben (vgl. Holzkamp 1983, S. 342ff.). Unter dieser Voraussetzung
können die Dimensionen des Lernhandelns bestimmt und beschrieben werden.
Qualität ist demnach nicht eine per se vorhandene Eigenschaft einer Lernsoftware
(Wirkungsforschung), sondern wird erst im Lernprozess konstruiert. Darüber hinaus
wird der Lernprozess auch als situativ beeinflusst angesehen und als ganzheitliche
Handlungssituation wahrgenommen. Subjektive Qualität beim E-Learning zu bestimmen heißt also auch, die ganzheitliche Handlungssituation zu berücksichtigen.
3.4
Besonderheiten der Evaluation vernetzten Lernens
Abschließend soll noch auf einen Beitrag von Baumgartner (1997b) eingegangen werden, der einige Besonderheiten der Evaluation des netzgestützten Lernens aufzeigt. Er
geht von folgender These aus:
„Durch die besonderen Merkmale interaktiver Medien und der internationalen Netzwerke
(Internet) ergeben sich für den Lernprozess neue Möglichkeiten, aber auch neue Herausforderungen. Das muss sich notwendigerweise auch in den Inhalten und Formen von Evaluationen
widerspiegeln. Diese spezifischen Anforderungen wurden jedoch bisher beim Design von Evaluationen noch kaum berücksichtigt“ (Baumgartner 1997b, S. 10).
Baumgartner geht insbesondere auf fünf Besonderheiten interaktiver Lernarrangements
ein, die Einfluss auf die Evaluation nehmen und im Folgenden zusammengefasst werden
(ebenda).
1) Zugang: Durch die Möglichkeit des individuellen Zugangs zur Software gibt es keine einheitlichen Zeiten, keine gemeinsamen und öffentlich zugänglichen Örtlichkeiten für Lernprozesse mehr. Das Lernen findet potenziell in der Form von privatem
Studium – oft auch in den eigenen vier Wänden -, teilweise unabhängig von Lehrkräften oder anderen Lernern, statt. E-Learning ermöglicht einen individuellen und
freien Zugang zu den Lernmaterialien (anytime und anywhere). „Viele Methoden der
traditionellen Evaluation [...], die für gruppenbasierten Unterricht adäquat sein können, sind für die Evaluation interaktiver Lernsoftware entweder nicht stimmig oder
aus Gründen des Kosten- und/oder Zeitaufwands nicht brauchbar“ (ebenda, S. 6).
2) Ausgangs- und Bedürfnisstruktur: Beim E-Learning gibt es in der Gruppe der
Lerner eine potenziell heterogene Ausgangssituation. Dies gilt hinsichtlich demographischer Komponenten (berufliche Stellung, Bildungsstand etc.), inhaltlicher
Komponenten (Vorwissen, Kenntnisse etc.) und der Lernerfahrungen, die Lernende
haben. Auf einheitliche Voraussetzungen für eine Lerngruppe kann nicht ohne weiteres zurückgegriffen werden. Während der traditionelle Gruppenunterricht auf relativ
homogene Voraussetzungen angewiesen ist, ist
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E-Learning, als Form des selbstgesteuerten Lernens, nicht mehr dieser Restriktion
unterworfen. Zudem gilt: Was wann, wie lange, wie oft gelernt wird, liegt in der
Kontrolle des Lerners. Eine ähnlich heterogene Ausgangslage liegt zumeist auch
hinsichtlich der Bedürfnisstruktur vor. Potenziell ist medial gestütztes Lernen auch
in den Zielvorstellungen und den damit zusammenhängenden Motivationsstrukturen
völlig offen. Diese unterschiedlichen Bedürfnisse beeinflussen aber nicht nur den
Lernerfolg, sondern sind auch für seine Definition entscheidend. Die unterschiedlichen Zielstrukturen sind dabei vielfältig: eine private Fortbildung aus Interesse am
Thema ist ebenso denkbar wie ein weiterer Berufsabschluss oder eine im Berufsalltag notwendig gewordene Weiterqualifizierung. „Für Evaluationen heißt dies, dass
sie komplexer werden und sowohl die unterschiedlichen Ausgangssituationen als
auch die individuellen Lernstrategien und -ziele reflektieren müssen“ (ebenda, S. 7).
3) Wahlfreiheit des Angebotes: Beim E-Learning findet durch die eingesetzten Medien eine scheinbare Trennung von Inhalten und Lernprozess statt. Diese Trennung,
so argumentiert Baumgartner, sei jedoch nur scheinbar. Tatsächlich gehe es gerade nicht darum, eine inhaltliche, statische Qualität eines Materials zu evaluieren,
sondern vielmehr darum, zu analysieren, wie weit es in der Lage ist, Lernprozesse
durch Interaktionen anzustoßen und zu unterstützen. Dementsprechend seien auch
die Check- und Prüflisten für Softwareevaluationen nur mit äußerster Vorsicht und
eingeschränktem Wirkungsgrad anzuwenden (ebenda, S. 8).
4) Lernsituation: Baumgartner regt an, die besondere Situation des netzgestützten Lernens im Evaluationsdesign zu berücksichtigen. Insbesondere sollte eine Differenzierung der Möglichkeiten und Effekte der Infrastruktur (delivery technology) und der
didaktischen Potenziale (instruction technology) berücksichtigt werden.
5) Kosten: Baumgartner verweist darauf, dass bei den Kosten- und Nutzenanalysen
zwei Aspekte ins Kalkül zu ziehen sind, die seiner Meinung nach immer noch zu
wenig Beachtung finden. Zum einen argumentiert er mit Levin dahingehend, nicht
nur ökonomisch-finanzielle Kosten zu berücksichtigen, sondern auch soziale Kosten:
„In economic terms, a cost is a sacrifice of an option. If a resource is applied to one use, it is not
available for another use. In a purely economic sense, the cost of any decision is the value of
what is sacrificed with respect to the best alternative use of that resource“ (Levin 1981, S. 30).
Zum anderen sei zwischen Kosten-Nutzen-Analysen und Kosten-Wirksamkeits-Analysen zu unterscheiden. Während im ersten Fall inhaltlich unterschiedliche Teilgebiete
(beispielsweise Bildungs- vs. Transportwesen) miteinander verglichen werden könnten,
sei dies bei der Kosten-Wirksamkeits-Analyse nicht möglich: „So ist z.B. schon ein
Vergleich zwischen Lese- und Rechenfertigkeiten nicht mehr zulässig, weil sie verschiedene Testkriterien erforderlich
Ulf-Daniel Ehlers
www.medienpaed.com > 2.9.2017
32
machen, deren Punktwerte jedoch nicht mehr dasselbe aussagen“ (ebenda, S. 10). Baumgartner kommt zu dem Schluss, dass die großen individuellen Steuerungsmöglichkeiten des E-Learning es erfordern, vor allem qualitative Analysen bei der Evaluation als
grundlegende Methode zu verwenden.
Ein weiterer, bislang wenig beachteter Aspekt schließt Baumgartners Ausführen ab.
Hier rückt ins Blickfeld, dass nicht nur die bereits genannte Individualisierung des netzgestützten Lernens besondere Anforderungen bei der Evaluation bewirkt, sondern auch
und gerade die Möglichkeit zur sozialen Interaktion eine besondere und neue Art und
Weise erfordere, über Evaluation von gruppengestützten Lernprozessen nachzudenken.
Lernen ist demnach ein interaktiver sozialer Prozess, der zwischen den beteiligten Subjekten ausgehandelt wird und ist in diesem Sinne situiert (zu situiertem Lernen [situated
learning] vgl. Brown et al. 1988, Chaiklin/Lave 1993; Collins et al. 1989; Lave 1988;
Lave/ Wenger 1990; Suchman 1988). Ein Lernerfolg ist demnach nicht in der kognitiven
Leistung einer Einzelperson zu finden, sondern im gesamten Lernsystem.
„In die Sprache der Evaluationstheorie übersetzt heißt dies: Der Evaluand (d.h. die zu evaluierende Sache) ist die Gruppenperformance, die selbst wiederum situationsabhängig kontextgebunden, sozial verteilt ist und konstruiert (d.h. jeweils interpretativ ausgehandelt) wird“ (ebenda, S. 12).
Baumgartner lenkt mit seinem Beitrag das Augenmerk in der Debatte um die Evaluation
netzgestützten Lernens sehr deutlich auf neue Herausforderungen, für die es erst noch
Lösungen und Erfahrungen zu finden gilt.
4.
Fazit: Qualitätsevaluation auf allen Ebenen
Bislang beschränkt sich die Erfolgskontrolle bei Weiterbildungsmaßnahmen oft auf
die Befragung von Teilnehmern nach ihrer Zufriedenheit, bestenfalls vielleicht auf die
Überprüfung der unmittelbaren Lernergebnisse (vgl. Bliesener 1997). Wie bereits dargelegt, gibt es jedoch auch für den Bereich des E-Learnings umfassende Evaluationsmodelle. Insgesamt gilt hier: Qualitätssicherung ist mehr als die Kontrolle unmittelbarer
Lerneffekte. Es geht darum, alle Einflussfaktoren zu berücksichtigen: den Lerner, das
Lernthema, die gewünschten Ergebnisse und die Lernumgebung, sowohl die technologische als auch die soziale Lernumgebung (Arbeitsplatz, betriebliche Lernkultur, private
Lernsituation etc.). Eine effektive Qualitätssicherung bedeutet Evaluation in jeder Phase
des Qualifizierungsprozesses: von der Planung, über die Entwicklung, Durchführung
bis hin zur Transfersicherung.
Die Lernerposition und die den Lerner beeinflussenden Kontextfaktoren müssen in
Maßnahmen zur Qualitätssicherung verstärkt berücksichtigt werden. Ziel des Lernens
ist es, beim Lernenden einen Zuwachs an Handlungskompetenz zu erreichen. Das bedeutet, dass der primäre Qualitätsmaßstab der Zuwachs an Handlungskompetenz beim
Lerner ist und nicht – überspitzt formu-
Ulf-Daniel Ehlers
www.medienpaed.com > 2.9.2017
33
liert – die Güte der Lernsoftware, etwa prognostiv ermittelt anhand von Kriterienkatalogen.
Qualitätssicherung erschöpft sich daher nicht in guter Planung oder Vorbereitung,
sondern muss sich auf alle Phasen des Qualifikationsprozesses erstrecken und den Lerner mit einbeziehen. Qualitätssicherung beginnt beim Qualitätsbewusstsein und endet
bei der Sicherung der Transferleistungen, um die es ja letztlich geht. Entscheidend ist
demnach auch ein maßgeschneidertes Evaluationskonzept, um die Qualität zu sichern.
Das Evaluationskonzept muss dabei situationsgerecht angepasst werden.
Ein weit verbreiteter Irrtum ist es, zu glauben, dass E-Learning lediglich gut geplant
und vorbereitet sein müsse, um den gewünschten Erfolg zu erzielen. Ein detailliertes
Qualitätssicherungskonzept umfasst alle Phasen eines Qualifizierungsprozesses und
geht über Standardevaluationsverfahren für Weiterbildungsmaßnahmen hinaus, indem
es zusätzlich zu einer detaillierten Evaluation der Einsatzphase auch noch die Planungsund Entwicklungsphase und auch die Auswirkungen der Maßnahme (z.B. ROI in der betrieblichen Bildung) sowie Administrations- und Managementprozesse mit einbezieht.
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https://openalex.org/W4392845931
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https://dl.acm.org/doi/pdf/10.1145/3625007.3627486
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English
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Utilizing Textual Reviews for Visualizing and Understanding User Preferences
| null | 2,023
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cc-by
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2023 IEEE/ACM International Conference on Advances in Social Networks Analysis and M 2023 IEEE/ACM International Conference on Advances in Social Networks Analysis and M nternational Conference on Advances in Social Networks Analysis and Mining (ASONAM) Utilizing Textual Reviews for Visualizing and
Understanding User Preferences Tuan M. V. Le
Department of Computer Science
New Mexico State University, Las Cruces, USA
tuanle@nmsu.edu Dang Pham
Department of Computer Science
New Mexico State University, Las Cruces, USA
dangpnh@nmsu.edu For making sense of how users rate items, visualization is
an effective means to present user preferences and explore
item recommendations. Visualization approach has show its
effectiveness in different tasks such as presenting and manip-
ulating user preferences [7], exploring social recommendations
[8], visualizing explanations in recommender systems [9]. In
these methods, one possible way to display users and items
is by embedding their latent vectors to a 2- or 3-dimensional
space using a dimensionality reduction method such as Mul-
tidimensional Scaling [7]. However, most of the existing
visualization methods ignore the reviews when performing the
dimensionality reduction of user and item vectors. Therefore,
the explanations based on reviews may not be transferred to
and visualized in the visualization space for making sense of
the item recommendations. Although document visualization
methods can be utilized to visualize topics in reviews [10],
[11], they do not model users, items, and ratings. Therefore,
the output visualization may be not relevant and reflect well
the user rating behavior. Abstract—Latent factor models are widely used in recom-
mender systems. In these models, users and items are represented
as vectors in a joint latent factor space. The inner products of
user vectors and item vectors are used to model the user-item
interactions (e.g., ratings). A review is often posted by the user
to explain the given rating. Therefore, reviews can be used to
understand how users rate the items and to interpret the latent
dimensions of user and item vectors. In this paper, we propose
a probabilistic model that learns latent vectors of users and
items in a two- or three-dimensional space for visualization. Our
proposed model also extracts review topics and visualizes them
in the same visualization space for interpreting the ratings. We
model the user-item interactions by using the distances between
users and items in the visualization space. Extensive experiments
using several real-world datasets demonstrate the effectiveness of
our proposed model in recommendation and visualization tasks. Index Terms—latent factor models, visualization, topic models Index Terms—latent factor models, visualization, topic models I. INTRODUCTION In recommender systems, users often express their prefer-
ences by giving ratings and writing textual reviews to explain
the ratings they give. To understand user preferences, latent
factor models are widely used to analyze the ratings [1], [2]. In these models, users and items are represented as vectors
in a latent factor space. The ratings are modeled as the inner
products of user and item vectors. Given training data, the
latent vectors are typically learned by minimizing the mean
square error. While the learned latent vectors are good for
rating prediction, one shortcoming is that we may not have
a clear understanding of why users would give such ratings. In this regard, reviews can be a great source for extracting
discussed topics and aspects that could provide an explanation
on the given ratings. Therefore, there have been several works
that leverage reviews for understanding rating factors and
improving the performance of recommender systems [3]–[6]. To address the above limitations of the existing methods for
visualizing and understanding user preferences, we propose a
probabilistic semantic latent factor model, named SeeP, that
embeds users, items, and topics of reviews in a 2- or 3-
dimensional space for visualization and explanation. Different
from traditional latent factor models that needs to reduce the
dimension of high-dimensional latent vectors for visualization,
our proposed model directly learns for each user and each item
a latent vector in a 2- or 3-dimensional space. Since users
and items are in the same visualization space, we propose
using distances between users and items, instead of using dot
products, to model the ratings. Intuitively, a user close to an
item would give a high rating to that item. Moreover, since
we want to extract the topics from reviews and use those to
explain why a user prefers some items, we also embed review
topics into the visualization space. Topics next to a user and
an item can give an explanation on why that user gives such
a rating to that item. Since our proposed model optimizes and
learns the representations of users, items, and review topics
at the same time, the learned visualization ensures that both
rating behavior and semantic aspects of ratings are preserved
as much as possible. This could have important applications
for visual analysis of user preferences and exploring item
recommendations. ASONAM ’23, November 6-9, 2023, Kusadasi, Turkey
© 2023 Copyright is held by the owner/author(s). I. INTRODUCTION ACM ISBN 979-8-4007-0409-3/23/11. http://dx.doi.org/10.1145/3625007.3627486
This work is licensed under a Creative Commons Attribution International 4.0 License. 335 In this paper, we make the following contributions: In this paper, we make the following contributions: here f(γu, γi) is a function of Euclidean distance between user
u and user i: • We propose a holistic model of user preferences, review
topics, and visualization for visualizing and understand-
ing user preferences. Our proposed model, named SeeP,
embeds users, items, and review topics in a 2- or 3-
dimensional space for visualization and explanation. Both
user preferences and semantic aspects of ratings are
preserved as much as possible in the visualization space. We derive an algorithm to estimate model parameters
based on the variational inference approach. f(γu, γi) = R exp
−α2 ∥γu −γi∥2
(3) (3) where R is the max rating that a user could give to an item
(i.e., 5 if the 5-point Likert scale is used) and α2 is a positive
scaling factor that will be learned. Intuitively, users give higher
ratings to the items that are closer to them in the visualization. Given the above assumptions, the generative process of SeeP
is as follows: • We conduct extensive experiments on several large real-
world datasets. The experimental results show the effi-
ciency and effectiveness of our proposed model. 1) For each document wi corresponding to item i, i =
1, . . . , Ni: ,
,
a) Draw a document coordinate: γi ∼N
0, σ2
i I
II. SEMANTIC VISUAL LATENT FACTOR MODEL
A. Problem Definition L (η, σi, σr, U, Φ, α, β; wi)
= −
Ni
X
i=1
DKL [q(γi|wi, η)∥p(γi|σi)]
+
Ni
X
i=1
Eq(γi|wn,η) [log p (wi|γi, Φ, β)]
+
Nu
X
u=1
Ni
X
i=1
Eq(γi|wn,η)
log
N(ru,i|f(γu, γi), σ2
r)
(4) • In addition, our model learns Z latent topics, their visu-
alization coordinates Φ = {ϕz}Z
z=1, and their word dis-
tributions β = {βz}Z
z=1. The topic distributions of item-
documents are denoted as Θ = {θi}Ni
i=1. The distances
between an item and topics reflect the topic distribution
of reviews for that item. Since the distance from a user to
an item expresses the rating, we can rely on topics near
to that item to explain why that user gives such a rating
to the item. • In addition, our model learns Z latent topics, their visu-
alization coordinates Φ = {ϕz}Z
z=1, and their word dis-
tributions β = {βz}Z
z=1. The topic distributions of item-
documents are denoted as Θ = {θi}Ni
i=1. The distances
between an item and topics reflect the topic distribution
of reviews for that item. Since the distance from a user to
an item expresses the rating, we can rely on topics near
to that item to explain why that user gives such a rating
to the item. (4) here q(γi|wi, η) is a variational distribution to approximate
the intractable true posterior p(γi|wi). q(γi|wi, η) can take
a Gaussian form whose parameters are parameterized by an
inference neural network: II. SEMANTIC VISUAL LATENT FACTOR MODEL
A. Problem Definition b) For each word wi,m in document wi: i) Draw a topic: z ∼Multi
{p (z|γi, Φ)}Z
z=1
The considered problem can be stated as follows: Given Nu
users, and Ni items. Let R be the rating matrix where Ru,i
represents the rating of user u for item i. Let wu,i be the
review by user u for item i. To extract and visualize review
topics, since an item i can be described by all reviews given to
it, we integrate all reviews for an item i into a single document
wi. Let D be the set of these item-documents, D = {wi}Ni
i=1,
and V is the vocabulary of the text reviews. We want to find:
ii) Draw a word: wi,m ∼Multi (βz) a) Draw a rating: ru,i ∼N
f(γu, γi), σ2
r
here γi, γu are the latent vectors of item i and user u or
their visualization coordinates. We treat α, U = {γu}Nu
u=1,
topic visualization coordinates Φ = {ϕz}Z
z=1, and topic word
distributions β = {βz}Z
z=1, as model parameters that can
be learned by maximizing the following lower bound on the
marginal log likelihood: i
• U = {γu}Nu
u=1 ∈RH×Nu and I = {γi}Ni
i=1 ∈RH×Ni
which are the latent factor matrices for all users and
items respectively. Here H = 2 or 3 for visualization,
and column γu and column γi represent user u and item
i respectively. The distances between users and items
reflect the ratings. A user close to an item would give
a high rating to that item. • U = {γu}Nu
u=1 ∈RH×Nu and I = {γi}Ni
i=1 ∈RH×Ni
which are the latent factor matrices for all users and
items respectively. Here H = 2 or 3 for visualization,
and column γu and column γi represent user u and item
i respectively. The distances between users and items
reflect the ratings. A user close to an item would give
a high rating to that item. ru,i ∼N
f(γu, γi), σ2
r
(2) B. Generation and Inference We model the topic distribution of an item-document i as: We model the topic distribution of an item-document i as: q(γi|wi, η) = N(γi|fµ0(wi), f 2
σ2
0(wi))
(5) (5) θiz = p (z|γi, Φ) =
exp
−1
2 ∥γi −ϕz∥2
PZ
z′=1 exp
−1
2 ∥γi −ϕz′∥2
(1) here fµ0, fσ2
0 can be multilayer perceptrons. The expectations
in Eq. 4 can be approximated by using a Monte Carlo estimator
[12]. More specifically, we sample γ(l)
i
from q(γi|wi, η) by
sampling an auxiliary noise sample ϵ(l) from N(0, I) and
computing γ(l)
i
= fµ0(wi) + ϵ(l) · fσ2
0(wi). The expectations
can then be approximated as: (1) here γi is the visualization coordinate of item i. It is clear
from the equation that we want to keep the topics of item i
close to it in the visualization space. To visualize user preferences toward items, we model the
ratings using Euclidean distances between users and items. We assume the following Gaussian distribution over the rating
ru,i: Eq(γi|wn,η) [log p (wi|γi, Φ, β)]
≈1
L
L
X
l=1
log p
wi|γ(l)
i , Φ, β
(6) (6) ru,i ∼N
f(γu, γi), σ2
r
(2) (2) 336 Dataset
#Users
#Items
#Reviews
#Classes
Software
1818
797
11732
15
Arts Crafts and Sewing
55964
22919
431443
10
Music Instruments
27449
10615
216105
14
Yelp Tucson
113974
9250
379719
743
Office Products
101065
27947
727635
3
Industrial and Scientific
10984
5321
70612
25
TABLE I: Dataset statistics and LDA12 [22]. For PLSV, γi is learned by using only item-
documents. For LDA, we extract topic proportions of item-
documents and γi is then obtained by embedding these topic
proportions into the visualization space using t-SNE [23]. Finally, with γi fixed, we learn γu by NCF trained on ratings. For visualization, γu and γi are learned with two dimensions
for all methods in our experiments. The hyperparameters of
methods are set based on the original implementations. All
experimental results are averaged across 5 separate runs. Eq(γi|wn,η)
log
N(ru,i|f(γu, γi), σ2
r)
≈1
L
L
X
l=1
log
h
N(ru,i|f(γu, γ(l)
i ), σ2
r)
i
≈−1
2σ2r
Nu
X
u=1
Ni
X
i=1
(ru,i −αd(γu, γi))2
(7) B. Visualization and Rating Prediction In this section, we evaluate the performance of our method
based on both rating prediction and visualization quality. For
rating prediction, Mean Squared Error (MSE) is a widely used
metric. γu and γi in a good visualization should produce a
low MSE and thus we can visually explore the items that
a user may like. For visualization quality, we also rely on
labels of items for evaluation. The intuition is that a good
visualization should group items of the same label together. Therefore, we calculate k-nearest neighbors (k-NN) accuracy
in the visualization space. A higher k-NN accuracy means a
better visualization. Plug these approximated expectations into Eq. 4, we have a
differentiable objective function which can be optimized by
stochastic gradient descent1. The main steps of the inference
algorithm are described in Algorithm 1. Plug these approximated expectations into Eq. 4, we have a
differentiable objective function which can be optimized by
stochastic gradient descent1. The main steps of the inference
algorithm are described in Algorithm 1. A. Datasets and Comparative Methods We use several real-world datasets from different on-
line product and review platforms: Amazon2, Yelp3. We
adopt 5 product categories from Amazon with different
sizes: Software, Arts Crafts and Sewing, Music Instruments,
Office Products,
and
Industrial and Scientific. For
Yelp
dataset, we keep only reviews for all businesses in Tucson
city. The preprocessing step including removal of all duplicate
reviews, and following [13]–[15], we use the 5-core version
of these datasets in which every user and item has less than 5
reviews are removed. The statistics of all datasets are shown
in Table I. The last column shows the number of classes for
items in each dataset. We randomly use 90% of each dataset
for training and the rest for testing. We only use the text
reviews in the training set for training. For text preprocessing,
we remove stopwords, stem words, and keep the 5000 most
frequent words as the vocabulary. We compare SeeP with models that do not utilize textual
reviews including Probabilistic Matrix Factorization4 (PMF)
[16], Non-negative Matrix Factorization4 (NMF) [17], Neural
Collaborative Filtering5 (NCF) [18], DGCF6 [19]. For models
that leverage textual reviews, we compare SeeP with HFT7 [3],
TransNet-Ext8 [5], NARRE9 [20], RGCL10 [21], PLSV11 [10], 1We use L = 1 in our experiments
2https://nijianmo.github.io/amazon/
3https://www.yelp.com/dataset
4https://surprise.readthedocs.io/en/stable/index.html
5https://github.com/hexiangnan/neural collaborative filtering
6https://tinyurl.com/github-dgcf
7https://cseweb.ucsd.edu/∼jmcauley/
8https://github.com/winterant/TransNets
9https://github.com/chenchongthu/NARRE
10https://github.com/JarenceSJ/ReviewGraph
11https://github.com/dangpnh2/plsv vae 12https://github.com/akashgit/autoencoding vi for topic models III. EXPERIMENTS We plot k-NN vs. MSE in Figures 1 to demonstrate how
methods balance between k-NN and MSE for visualization and
rating prediction tasks. Figure 1 reports results with k = 5 and
Z = 10. As we can see in the figures, TransNet-Ext, NARRE,
and RGCL often have the lowest MSEs because they utilize
textual reviews for improving the recommendation. There is a
trade-off between k-NN accuracy and MSE. Recommendation
methods such as NCF, DGCF, TransNet-Ext, NARRE, RGCL,
HFT, PMF, and NMF have low MSEs (i.e., they perform well
in the rating prediction task). However, they do not produce
good visualizations, indicated by very low k-NN accuracies,
because these models are not for visualization. In contrast,
PLSV and LDA have high k-NN accuracies because they
visualize the items based on topic models trained on textual
reviews, which can group well the items of the same label. However, since they are not recommendation models, they
do not perform well in the rating prediction task, indicated
by their very high MSEs. For our proposed model, SeeP, it
balances well the k-NN accuracy and MSE where it has low
MSEs and high k-NN accuracies in most settings. By mod-
eling ratings, reviews, and visualization in a unified model,
SeeP can achieve a good performance in most of the datasets
in terms of visualization and rating prediction. This shows
that the visualization by SeeP preserves well user preferences,
which is useful for visually exploring and making sense of
user rating behavior. Visualization examples in Figures 3 will
demonstrate this point further. IV. RELATED WORK In recommender systems, latent factor models are widely
used for rating prediction [1], [16]–[18]. These models con-
sider only the numeric ratings and ignore all other sources of
information given either by users or items. Recent approaches
including deep learning exploit textual reviews to enhance
the performance of recommendation systems [5], [6], [20],
[21], [24], [25]. One of methods closely related our method
is HFT where it adds a corpus likelihood based on LDA [22]
topic models to the traditional latent factor model [3]. HFT
uses topics as regularizers for more accurately fitting user and
item latent factors. None of the above methods can generate
a visualization for effectively presenting user preferences and
exploring item recommendations. 1.2
1.4
Fig. 1: k-NN vs. MSE across all datasets with k = 5, Z = 10 Scientific
in reviews, SeeP also gains a competitive performance on topic
coherence. Since we use topics to explain the ratings in the
visualization, their coherence is very important. To evaluate
topic coherence, we use Normalized Pointwise Mutual Infor-
mation (NPMI) that can be estimated using an external large
corpus. For a topic, the NPMI score will be an average over
all possible pairs of words of that topic. Figure 2 shows NPMI
scores of all methods across different numbers of topics. As
shown in the figure, NPMI scores of SeeP are comparable
to topic model-based methods, PLSV and LDA. Since, for
visualization, HFT can only run with two topics, we do not
show it in Figure 2. For visualization in recommender systems, it has shown
several important applications in different tasks such as pre-
senting and manipulating user preferences [7], exploring social
recommendations [8], and visualizing explanations in recom-
mender systems [9]. In these methods, one possible way for
visualization is to embed user and item latent vectors into a 2-
or 3-dimensional space using a dimension reduction method
such as MDS [7] or t-SNE [23]. However, it is not trivial
to incorporate reviews into these methods when performing
the dimension reduction of user and item vectors. Some
recent document visualization methods can visualize topics in
reviews [10], [26]. However, they do not model users, items,
and ratings. Therefore, the output visualization may not reflect
well user rating patterns. There have been other works for
visualization recommender systems which is different from the
considered problem in this paper. IV. RELATED WORK Visualization recommender
systems aim to recommend visualisation methods to users
given specific contexts [27], [28]. 5 10
20
50
#Topics
a) Software
0.05
0.08
NPMI
5 10
20
50
#Topics
b) Arts Crafts and Sewing
0.04
0.06
5 10
20
50
#Topics
c) Music Instruments
0.04
0.06
NPMI
5 10
20
50
#Topics
g) Yelp Tucson
0.04
0.06
5 10
20
50
#Topics
e) Office Products
0.04
0.06
NPMI
5 10
20
50
#Topics
i) Industrial and Scientific
0.04
0.06
PLSV
LDA
SeeP
Fig. 2: Topic coherence 5 10
20
50
#Topics
a) Software
0.05
0.08
NPMI
5 10
20
50
#Topics
b) Arts Crafts and Sewing
0.04
0.06 #Topics
b) Arts Crafts and Sewing #Topics
a) Software 5 10
20
50
#Topics
c) Music Instruments
0.04
0.06
NPMI
5 10
20
50
#Topics
g) Yelp Tucson
0.04
0.06 #Topics
g) Yelp Tucson #Topics
c) Music Instruments D. Visualization Examples Due to limited space, we show some visualization examples
by SeeP, HFT, and PLSV on Arts Crafts and Sewing dataset
in Figure 3. In this visualization, each user is represented by a
black cross, the colored pluses are items whose colors express
the classes of items, and the large red points are topics. In
the visualization of HFT, the items are mixed up and groups
of items are not well-preserved. In contrast, SeeP and PLSV
generate different clusters of items that are consistent with the
class labels of items. However, in PLSV, all γu are grouped
close to the center of the visualization, which does not result
in a compelling visualization of user-item relationships. a) Software
b) Arts Crafts and Sewing
0.55
0.60
0.65
0.70
0.75
k-NN
c) Music Instruments
1.00
1.50
MSE
0.20
0.25
0.30
k-NN
d) Yelp Tucson
2.00
2.50 C. Topic Coherence In this section, we evaluate the quality of topics generated
by different methods. The goal is to show that besides achiev-
ing good performance in rating prediction by utilizing topics 337 0.4
0.5
0.6
0.7
k-NN
a) Software
1.50
2.00
2.50
MSE
0.5
0.6
0.7
k-NN
b) Arts Crafts and Sewing
0.80
1.00
0.55
0.60
0.65
0.70
0.75
k-NN
c) Music Instruments
1.00
1.50
MSE
0.20
0.25
0.30
k-NN
d) Yelp Tucson
2.00
2.50
0.88
0.90
0.92
0.94
k-NN
e) Office Products
1.00
1.25
1.50
MSE
0.4
0.5
k-NN
f) Industrial and Scientific
1.00
1.25
1.2
1.4
E
SeeP
PLSV
LDA
NCF
DGCF
TransNet-Ext
NARRE
RGCL
HFT
PMF
NMF
Fig. 1: k-NN vs. MSE across all datasets with k = 5, Z = 10 0.4
0.5
0.6
0.7
k-NN
a) Software
1.50
2.00
2.50
MSE
0.5
0.6
0.7
k-NN
b) Arts Crafts and Sewing
0.80
1.00 1REFERENCES [16] A. Mnih and R. R. Salakhutdinov, “Probabilistic matrix factorization,”
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Transactions on Information Systems (TOIS), vol. 37, no. 3, 2019. [21] J. Shuai, K. Zhang, L. Wu, P. Sun, R. Hong, M. Wang, and Y. 1REFERENCES Li, “A
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Management, 2020, pp. 1645–1654. This research is sponsored by NSF #1757207 and NSF
#1914635. (c) PLSV Beading & Jewelry Making
Fa V. CONCLUSION #Topics
c) Music Instruments
#Topics
g) Yelp Tucson
5 10
20
50
#Topics
e) Office Products
0.04
0.06
NPMI
5 10
20
50
#Topics
i) Industrial and Scientific
0.04
0.06
PLSV
LDA
SeeP
Fig. 2: Topic coherence )
g)
p
5 10
20
50
#Topics
0.04
0.06
NPMI
5 10
20
50
#Topics
0.04
0.06 We propose a probabilistic semantic latent factor model,
named SeeP, that embeds users, items, and topics of reviews
for visualization and explanation. The user-item interactions
are modeled by the distances between users and items in
the visualization space. We conduct experiments using several
datasets to show the effectiveness of our proposed method
in rating prediction, item classification, topic coherence, and
generating visualization. #Topics
i) Industrial and Scientific Fig. 2: Topic coherence Fig. 2: Topic coherence 338 (a) SeeP
(b) HFT
(c) PLSV
Sewing
Storage & Transport
Crafting
Printmaking
Needlework
Drawing & Art Supplies
Scrapbooking & Stamping
Beading & Jewelry Making
Topics
Knitting & Crochet
Fabric Decorating
Users (c) PLSV (b) HFT
(c) PLSV
Printmaking
Needlework
Drawing & Art Supplies
Scrapbooking & Stamping
Beading & Jewelry Making
Topics
Knitting & Crochet
Fabric Decorating
Users 1REFERENCES [28] X. Qian, R. A. Rossi, F. Du, S. Kim, E. Koh, S. Malik, T. Y. Lee,
and J. Chan, “Learning to recommend visualizations from data,” in
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https://link.springer.com/content/pdf/10.1007/s11864-021-00920-6.pdf
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English
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Allogeneic Chimeric Antigen Receptor Therapy in Lymphoma
|
Current treatment options in oncology
| 2,022
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cc-by
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This article is part of the Topical Collection on Lymphoma This article is part of the Topical Collection on Lymphoma Keywords CAR-T I Allogeneic CAR-T I Non-Hodgkin lymphoma Keywords CAR-T I Allogeneic CAR-T I Non-Hodgkin lymphoma Address
* Published online: 25 February 2022
* The Author(s) 2022 Published online: 25 February 2022
* The Author(s) 2022 DOI 10.1007/s11864-021-00920-6
Curr. Treat. Options in Oncol. (2022) 23:171–187 DOI 10.1007/s11864-021-00920-6
Curr. Treat. Options in Oncol. (2022) 23:171–187 Lymphoma (JL Muñoz, Section Editor) Allogeneic Chimeric Antigen
Receptor Therapy
in Lymphoma
Arushi Khurana, MBBS
Yi Lin, MD, PhD* Address
*Division of Hematology, Mayo Clinic, 200 First Street, SW, Rochester, MN, 55905,
USA
Email: Lin.Yi@mayo.edu Address
*Division of Hematology, Mayo Clinic, 200 First Street, SW, Rochester, MN, 55905,
USA
Email: Lin.Yi@mayo.edu Introduction receive tisagenlecleucel CAR-T therapy in the JULIET
trial, most of whom died due to progressive disease as
the median time from leukapheresis to infusion was 54
days [13]. Additionally, while waiting for the cells to be
manufactured, some patients require additional bridg-
ing therapy to maintain disease control which is associ-
ated with poor outcomes [6•, 9, 14]. Delay in CAR-T
therapy and increase in the lines of therapy before CAR-T
have both been shown to worsen outcomes along with
an increase in tumor burden, markers of inflammation,
and doubling time of CAR-T cells [15, 16, 17]. Autolo-
gous CAR-Ts are expensive and laborious to manufac-
ture due to patient specificity, vary in quality based on
patient’s disease factors and prior treatments, limit
redosing due to small-scale individual production, and
may not be effective if generated from dysfunctional T
cells due to an immunosuppressive tumor microenvi-
ronment seen in many lymphoma types [15, 18, 19]. To
overcome these issues related to cost, accessibility, effi-
cacy, and efficiency, “off the shelf” allogeneic (allo)
CAR-Ts are being explored. While they resolve some of
the challenges encountered with autologous CAR-T, they
also come with inherent hurdles such as the risk of graft
versus host disease (GvHD) and rejection of the CAR-Ts
by the host terminating their treatment effect [20, 21]. We herein review the different strategies of generating,
overcoming challenges associated with allo CAR-Ts,
their clinical experience in lymphoma, and implications
of their use. Chimeric antigen receptor T cell (CAR-T) therapy has
been transformative for the treatment of relapsed/
refractory (R/R) large B cell lymphoma (LBCL) patients
who have relapsed post 2 lines of therapy or autologous
stem cell transplant (ASCT). These patients were other-
wise subject to dismal outcomes [1]. Since 2017, three
different CAR-T products have been approved by the US
Food and Drug Administration (FDA) for the treatment
of R/R LBCL after two or more prior lines of therapy [2•,
3•, 4•]. All three boast a relatively similar durable re-
sponse rate of around 40% at 2 years in the registration
trials and real-world settings [5–8, 9]. Two additional
FDA approvals for lymphoma in the last year have been
for mantle cell lymphoma (brexucabtagene autoleucel)
and follicular lymphoma (axicabtagene ciloleucel) [10,
11•]. Opinion statement The therapeutic armamentarium has significantly expanded since the approval of various
CD19-targeting chimeric antigen receptor T cell (CAR-T) therapies in non-Hodgkin lym-
phoma (NHL). These CAR-Ts are patient-specific and require a complex, resource, and
time-consuming process. While this appears promising, autologous CAR-Ts are limited due
to the lack of accessibility, manufacturing delays, and variable product quality. To
overcome these, allogeneic (allo) CARs from healthy donors appear appealing. These can
be immediately available as “off the shelf” ready-to-use products of standardized and
superior quality exempt from the effects of an immunosuppressive tumor microenviron-
ment and prior treatments, and potentially with lower healthcare utilization using indus-
trialized scale production. Allogeneic CARs, however, are not devoid of complications and
require genomic editing, especially with αβ T cells to avoid graft versus host disease
(GvHD) and allo-rejection by the recipient’s immune system. Tools for genomic editing
such as TALEN and CRISPR provide promise to develop truly “off the shelf” universal CARs
and further advance the field of cellular immunotherapy. Several allogeneic CARs are
currently in early phase clinical trials, and preliminary data is encouraging. Longer follow-
up is required to truly assess the feasibility and safety of these techniques in the patients. This review focuses on the strategies for developing allogeneic CARs along with cell
sources and clinical experience thus far in lymphoma. Lymphoma (JL Muñoz, Section Editor) 172
172 Introduction All the commercially approved CAR-T therapies
are directed against CD19, a protein that is frequently
expressed in B cell lymphomas, and in a relatively higher
proportion than some other targets such as CD22 or
CD20 [12]. The commercial production of autologous
CAR-T cells requires the collection of T cells from the
patient (leukapheresis), followed by activation and
transduction with the CAR constructs using viral vectors,
and eventually expansion before reinfusion into the
patient after a short course of lymphodepleting chemo-
therapy [12]. This bespoke manufacturing process can
take up to 2–5 weeks, a delay that may be particularly
problematic for those with aggressive and rapidly pro-
liferative lymphomas. Up to 30% of patients could not Strategies to generate “off the shelf” or universal allogeneic
CAR T Strategies to generate “off the shelf” or universal allogeneic
CAR-T For a successful generation of “off the shelf” or “universal” allo CAR-Ts, two
important hurdles need solutions. First is the GvHD, which is mediated by the
alloreactivity of the T cell receptor (TCR) on the CAR-T cells, which recognize
the recipient’s human leukocyte antigens (HLA) as foreign and mount an
immune attack. Contrary to the first issue is the risk of rejection of allo CAR-
Ts by the recipient’s immune system, resulting in their destruction. An essential
requirement for the allo CAR-Ts to succeed is their persistence in the recipient,
and lack thereof would not allow for sustained responses. While this does not
apply to autologous CAR-Ts as seen in aggressive NHL with CD28 co-
stimulatory domain in axi-cel, allo CAR-Ts are subject to rapid elimination by
the host immune system, further shortening their persistence. Additionally,
with allo CAR-Ts, there is also a risk for alloimmunization, which may lead to 173 Allogeneic Chimeric Antigen Receptor Therapy in Lymphoma
Khurana and Lin issues with redosing similar products due to donor-specific antibodies (DSA). Similar problems due to DSA have been encountered in patients undergoing
solid organ transplantation and haploidentical stem cell transplantation [22,
23]. Comparison of features and implications between autologous and alloge-
neic CAR-T is outlined in Table 1. Several strategies to overcome these issues
outlined above are in development and discussed here (Fig. 1). Developing CAR-T from a stem cell transplant donor Differences between the autologous and allogenic CAR-Ts
Characteristic
Autologous
Allogeneic
Cell source
Self
Non-self healthy donor, iPSC derived
Production time
2–5 weeks
Ready to use/off the shelf
Cell type
Autologous T cells (may or may not be manipulated
for T cell composition)
Gene-edited αβ T cells, γδ T cells, NK cells, iNKT
cells
Manufacturing
process
• Individualized manufacturing for each patient
• Reduced accessibility due to manufacturing wait
and potential impact of patients’ native cells on
manufacturing success
• High variability in product composition due to
inherent patient heterogeneity in T cell composition
and immune profile
• Single manufacturing from one donor or cell
source potentially used to treat many patients
• Pre-made and ready to use to remove the
manufacturing wait time
• Possible standardization and control over T cell
composition in the product
Side Effects
• Cytokine release syndrome
• Immune effector cell-associated neurotoxicity
syndrome
• B cell aplasia
• CAR gene editing-related oncogenic potential
• Same as autologous PLUS
• More immune suppression and risk for infection
from more intense lymphodepletion for prevention
of allo rejection
• Graft versus host disease
• Allo rejection of the infusion product
Redosing
Response with the same CAR-T unfavorable in
aggressive lymphoma, possibly more promising in
FL
More studies needed to understand mechanisms of
relapse and resistance to redosing
Benefit to be investigated
Risk of alloimmunization—donor-specific
antibodies
Persistence
Months to years
Weeks to months
Cost
High
Unknown (projected to be lower) Characteristic
Autologous Developing CAR-T from a stem cell transplant donor αβ T cells are the main drivers of GvHD in allogeneic stem cell trans-
plant recipients [24–26, 27–29]. One strategy to decrease the risk of
GvHD for patients who have already received an allogeneic stem cell
transplantation and have subsequently relapsed is to derive CAR-Ts from
the same donor T cells. This was attempted in a clinical trial where 20
patients with B cell malignancies that had relapsed after allo stem cell
transplant received a single infusion of donor-derived CAR-Ts targeting
CD19 with no prior lymphodepletion chemotherapy. Eight of the 20
patients achieved a response with 6 complete remissions (CR). No
GvHD was reported, and CAR-T expansion was higher for those with a Table 1. Differences between the autologous and allogenic CAR-Ts
Characteristic
Autologous
Allogeneic
Cell source
Self
Non-self healthy donor, iPSC derived
Production time
2–5 weeks
Ready to use/off the shelf
Cell type
Autologous T cells (may or may not be manipulated
for T cell composition)
Gene-edited αβ T cells, γδ T cells, NK cells, iNKT
cells
Manufacturing
process
• Individualized manufacturing for each patient
• Reduced accessibility due to manufacturing wait
and potential impact of patients’ native cells on
manufacturing success
• High variability in product composition due to
inherent patient heterogeneity in T cell composition
and immune profile
• Single manufacturing from one donor or cell
source potentially used to treat many patients
• Pre-made and ready to use to remove the
manufacturing wait time
• Possible standardization and control over T cell
composition in the product
Side Effects
• Cytokine release syndrome
• Immune effector cell-associated neurotoxicity
syndrome
• B cell aplasia
• CAR gene editing-related oncogenic potential
• Same as autologous PLUS
• More immune suppression and risk for infection
from more intense lymphodepletion for prevention
of allo rejection
• Graft versus host disease
• Allo rejection of the infusion product
Redosing
Response with the same CAR-T unfavorable in
aggressive lymphoma, possibly more promising in
FL
More studies needed to understand mechanisms of
relapse and resistance to redosing
Benefit to be investigated
Risk of alloimmunization—donor-specific
antibodies
Persistence
Months to years
Weeks to months
Cost
High
Unknown (projected to be lower) Table 1. Differences between the autologous and allogenic CAR-Ts Table 1. Allogeneic Gene-edited αβ T cells, γδ T cells, NK cells, iNKT
cells • Single manufacturing from one donor or cell
source potentially used to treat many patients • Pre-made and ready to use to remove the
manufacturing wait time • Possible standardization and control over T cell
composition in the product • Same as autologous PLUS • More immune suppression and risk for infection
from more intense lymphodepletion for prevention
of allo rejection • B cell aplasia • Graft versus host disease • Allo rejection of the infusion product Benefit to be investigated Risk of alloimmunization—donor-specific
antibodies More studies needed to understand mechanisms of Weeks to months
Unknown (projected to be lower) Lymphoma (JL Muñoz, Section Editor) 174
174 Fig. 1. Various allogeneic CARs with genomic editing techniques (abbreviations: CAR, chimeric antigen receptor; TAA, tumor
associated antigen; B2M, β2 microglobulin; Ab, antibody; TCR, T cell receptor; FasL, Fas ligand; iPSC, induced pluripotent stem cells;
TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; IL, interleukin; NKG2D, natural killer group 2 member D). Fig. 1. Various allogeneic CARs with genomic editing techniques (abbreviations: CAR, chimeric antigen receptor; TAA, tumor
associated antigen; B2M, β2 microglobulin; Ab, antibody; TCR, T cell receptor; FasL, Fas ligand; iPSC, induced pluripotent stem cells;
TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; IL, interleukin; NKG2D, natural killer group 2 member D). response than non-responders, and no CAR-T cells persisted beyond 3
weeks [30–32]. This study proved the feasibility, safety, and initial
efficacy of such an approach albeit, with small patient numbers. Addi-
tionally, it highlights the use of allo CAR-Ts to generate a graft versus
leukemia/lymphoma effect without significantly increasing the risk for
GvHD. Another recent phase I clinical trial of 10 patients with relapsed
or refractory B-acute lymphoblastic leukemia or aggressive lymphoma
described the generation of CD19-targeting CAR-T from HLA-matched
sibling donors using the high-capacity piggy-Bac transposon method for
genetic modification. At a median follow-up of 18 months, 5 patients
were in CR [33]. Interestingly, 2 of the 10 patients developed malignant
lymphoma derived from CAR gene-modified T cells, and therefore, this
strategy requires further follow-up [34]. It remains patient-specific and
limited to those who have previously undergone allo stem cell trans-
plant with available T cells and not with a history of GvHD. response than non-responders, and no CAR-T cells persisted beyond 3
weeks [30–32]. Selecting non-alloreactive T cells as a source for CAR-T Another disease where this approach has been promising is
the Epstein-Barr virus (EBV)-associated malignancies, such as post-
transplant associated lymphoproliferative disorder (PTLD). This approach
combines the antigen specificity of CAR-Ts with the TCR specificity towards
EBV. Recently, Prockop and colleagues reported on a single-center cohort of
46 patients with rituximab refractory PTLD developing after allo transplant
(n = 33) or solid organ transplant (n = 13) who were treated with banked
third-party partially HLA-matched EBV-specific cytotoxic T cells. Each
treatment cycle consisted of 3 weekly infusions, followed by a 3-week
observation period. This product named tabelecleucel showed a 1-year
estimated overall survival (OS) for patients with PTLD following allo stem
cell transplant and solid organ transplant of 68 and 64%, respectively. The
median OS for post allo transplant PTLD cohort had not been reached after
a median follow-up of 23.3 months. The median OS for post solid organ
transplant PTLD cohort was 21.3 months. Treatment-related serious ad-
verse events were reported in 1 patient each in both the allo and solid organ
transplant cohorts, respectively [38, 39, 40]. Tabelecleucel is currently in
phase 2 (NCT04554914) and 3 trials for EBV+ PTLD following allo stem
cell transplant post rituximab failure (NCT03392142, ALLELE study) and
solid organ transplant after the failure of rituximab or rituximab and
chemotherapy (NCT03394365). Memory T cells These have a more restricted TCR repertoire as compared to naïve T cells
and are therefore associated with less GvHD [41, 42]. They have shown to
generate an effective anti-tumor response both in the preclinical and in
autologous setting in non-Hodgkin lymphoma [43, 44]. However, the
studies suggest a correlation between the presence of less-differentiated T
cells, such as naïve T cells (Tn), central memory T cells (Tcm), and stem cell-
like memory T cells (Tscm) with CAR-T efficacy. These less-differentiated T
cell subsets are essential for in vivo expansion, survival, and persistence [45,
46, 47]. Various T cell subtypes exist with varied phenotypes and functions
in the tumor microenvironment of lymphoma [18, 48, 49]. A T cell subset
expressing CD8, CD45RA, and chemokine receptor 7 (CCR7) with features
of Tn cells or Tscm cells was found to be associated with increased in vivo
expansion of CAR-T cells in lymphoma [50]. Fraietta et al. Selecting non-alloreactive T cells as a source for CAR-T Selecting non-alloreactive T cells as a source for CAR-T
Virus-specific T cells
These have been used for several years now in patients with allo stem cell
transplants for the treatment of viral infections without additional risk of
GvHD [35–37]. This is thought to be due to the restricted repertoire of these
memory T cells. Another disease where this approach has been promising is
the Epstein-Barr virus (EBV)-associated malignancies, such as post-
transplant associated lymphoproliferative disorder (PTLD). This approach
combines the antigen specificity of CAR-Ts with the TCR specificity towards
EBV. Recently, Prockop and colleagues reported on a single-center cohort of
46 patients with rituximab refractory PTLD developing after allo transplant
(n = 33) or solid organ transplant (n = 13) who were treated with banked
third-party partially HLA-matched EBV-specific cytotoxic T cells. Each
treatment cycle consisted of 3 weekly infusions, followed by a 3-week
observation period. This product named tabelecleucel showed a 1-year
estimated overall survival (OS) for patients with PTLD following allo stem
cell transplant and solid organ transplant of 68 and 64%, respectively. The
median OS for post allo transplant PTLD cohort had not been reached after
a median follow-up of 23.3 months. The median OS for post solid organ
transplant PTLD cohort was 21.3 months. Treatment-related serious ad-
verse events were reported in 1 patient each in both the allo and solid organ
transplant cohorts, respectively [38, 39, 40]. Tabelecleucel is currently in
phase 2 (NCT04554914) and 3 trials for EBV+ PTLD following allo stem
cell transplant post rituximab failure (NCT03392142, ALLELE study) and
solid organ transplant after the failure of rituximab or rituximab and
chemotherapy (NCT03394365). Memory T cells
Th
h
t i t d TCR
t i
d t
ï
T
ll Virus-specific T cells Virus-specific T cells These have been used for several years now in patients with allo stem cell
transplants for the treatment of viral infections without additional risk of
GvHD [35–37]. This is thought to be due to the restricted repertoire of these
memory T cells. Another disease where this approach has been promising is
the Epstein-Barr virus (EBV)-associated malignancies, such as post- These have been used for several years now in patients with allo stem cell
transplants for the treatment of viral infections without additional risk of
GvHD [35–37]. This is thought to be due to the restricted repertoire of these
memory T cells. Allogeneic This study proved the feasibility, safety, and initial
efficacy of such an approach albeit, with small patient numbers. Addi-
tionally, it highlights the use of allo CAR-Ts to generate a graft versus
leukemia/lymphoma effect without significantly increasing the risk for
GvHD. Another recent phase I clinical trial of 10 patients with relapsed
or refractory B-acute lymphoblastic leukemia or aggressive lymphoma
described the generation of CD19-targeting CAR-T from HLA-matched
sibling donors using the high-capacity piggy-Bac transposon method for
genetic modification. At a median follow-up of 18 months, 5 patients
were in CR [33]. Interestingly, 2 of the 10 patients developed malignant
lymphoma derived from CAR gene-modified T cells, and therefore, this
strategy requires further follow-up [34]. It remains patient-specific and
limited to those who have previously undergone allo stem cell trans-
plant with available T cells and not with a history of GvHD. Allogeneic Chimeric Antigen Receptor Therapy in Lymphoma
Khurana and Lin 175 Using genome editing of αβ T cells Novel genome editing tools have paved the way to using conventional αβ T
cells to generate allo CAR-T [20, 53, 54]. αβ TCR removal by disruption of the
gene encoding for alpha chain (TRAC) is sufficient and essential for preventing
TCR-dependent GvHD. Torikai et al. were the first to report the feasibility and
efficacy of αβ TCR(neg) CD19CAR(pos) T cells using the zinc finger nucleases
(ZFNs) for permanent deletion of α or β TCR chains in B cell malignancies
[55•]. Another genome editing tool used by Poirot et al. transcription activator-
like effector nuclease (TALEN) was able to perform multiplex gene editing to
manufacture T cells deficient in expression of both αβ TCR and CD52 [56]. These T cells were unable to cause GvHD in a mouse model and were resistant
to removal by alemtuzumab (anti-CD52 monoclonal antibody), which can be
used to eliminate host T cells and hence avoid rejection. Cluster regularly
interspaced short palindromic repeats (CRISPR)/Cas9 with its rapid and effi-
cient multiplex genome editing have been recently used to generate allogeneic
universal CAR-T with up to 4 disrupted genes [57, 58]. While these approaches
are very promising, they are not free from consequences. They may not result in
complete knock out of αβ TCR in all T cells resulting in GvHD [59]. Two, there
is a risk of “off-target” genome cleavage resulting in unwanted gene transloca-
tions or inactivation that could potentially confer oncogenic potential on the
CAR-Ts [56]. Three, these multiple genomic changes could result in increased
toxicity, poor transduction efficacy, or depending on the gene disruption, a
proliferation and survival advantage to the CAR-Ts [60]. Recently, CRISPR/Cas9 technology was used to integrate CAR construct into
the TRAC locus, to ensure that TCR is inactivated at the same time as CAR
introduction [61•, 62]. Other advantages of this approach are that it is safer
without insertional mutagenesis-related adverse effects. The CAR expression is
regulated by endogenous TCR promoter, preventing constant T cell activation,
differentiation, and exhaustion. These advantages render more potent anti-
tumor activity compared to the conventionally transduced CAR-Ts [61•, 63,
64]. UCART19 an allo CD19 CAR-T is currently under evaluation in adult and
pediatric ALL with promising result and up to 50% of patients able to proceed
to allogeneic transplant [65]. Selecting non-alloreactive T cells as a source for CAR-T performed
genomic, phenotypic, and functional testing to identify the determinants of
response in CD19-targeting autologous CAR-T in chronic lymphocytic
leukemia (CLL) and BCMA targeting autologous CAR-T in multiple mye-
loma. Sustained remission was associated with lymphocytes that possessed
memory-like characteristics and had CD27+CD45RO-CD8+ expressing T
cells before CAR-T generation [51, 52]. Therefore, further research is Lymphoma (JL Muñoz, Section Editor) 176
176 required to better understand the ideal composition of various T cell
subsets in the allo CAR-T product. Only selecting for Tem cells might lead to
ineffective therapy. No trials of allogeneic CAR-T with this strategy are
currently ongoing. Using non-αβ TCR T cells for CAR approach Several other immune cell types can generate CAR-based therapy if they possess
cytotoxic properties and are easily accessible from sources such as peripheral
blood mononuclear cells (PMBCs), or renewable stem cells. Natural killer (NK) cells are an attractive alternative as they are highly
cytolytic to cancer cells via granzyme B, perforin, and Fas ligand. They are an
integral part of tumor immunosurveillance and are often found to be dysfunc-
tional due to cancer’s immunosuppressive mechanisms for immune escape. Due to their innate anti-tumor activity, without causing GvHD, CAR-expressing
NK is appealing. This has been shown to be feasible and active in in vitro and
xenogeneic tumor models, as well as against patient-derived glioblastoma cell
lines [71, 72]. However, NK cells exist in low numbers in peripheral blood and
are less pliable to genetic manipulation which makes its use difficult [73, 74]. A
strategy uses the NK92 cell line, an activated human NK cell line derived from
an NHL patient [75, 76]. CAR-NK cells derived from the NK92 cell line targeting
CD20 have shown efficacy against lymphomas in vitro [77]. NK-92 cell line has
disadvantages as it is derived from a patient with NK lymphoma and has the
potential for tumor engraftment post infusion. In addition, these cells are EBV
positive, which carry various cytogenetic abnormalities. Due to these reasons,
the NK92 cell line needs to be irradiated before infusion into patients, which
can negatively impact its expansion and persistence, ultimately leading to low
efficacy [78]. Another strategy using umbilical cord blood (UCB) to obtain NK
cells has been used to create CD19 directed CAR-NK cells. These UCB-derived
NK cells were transduced with a retroviral vector incorporating the genes for
CAR-CD19, interleukin-15 (IL-15), and inducible caspase-9-based suicide gene
(iCasp9). Liu et al. reported efficient killing of CD19-expressing cell lines, with
marked prolongation of survival in a xenograft lymphoma murine model. IL-15
production by the transduced UCB-NK cells critically improved their function
over controls. The iCasp9 suicide gene upon pharmacologic activation resulted
in rapid elimination of the iCasp9/CAR.19/IL-15 UCB-NK cells, thereby incor-
porating a safety switch [79]. This approach was evaluated in a phase I/II clinical
trial where HLA-mismatched UCB-derived CAR-NK cells targeting CD19 were
infused in 11 patients with relapsed or refractory CD19-positive B cell NHL or
CLL. There were no cytokine release syndrome (CRS), neurotoxicity, or GvHD,
and no increase in inflammatory markers such as IL-6. Using genome editing of αβ T cells There is still the risk of allo-rejection with TCR-negative allogeneic CAR-Ts
due to the recognition of non-self HLA by the recipient’s T cells if there is a
mismatch. While strategies such as lymphodepletion and irradiation can help
delay the rejection, the CAR-T persistence which is necessary for efficacy will be
relatively short. For this, the removal of class I HLA molecules is required from
the T cells, which can be achieved by disrupting the beta-2 microglobulin
(β2M) gene locus [57, 66–68]. CD19 and CD70 (COBALT-LYM)-targeting
CAR-Ts with CRISPR/Cas9 disrupted TRAC and β2M genes (double knockout)
are currently under evaluation in B cell lymphomas and leukemias
(NCT03166878, NCT04502446). Further triple knockout of HLA classes I and
II and TRAC by CRISPR-based techniques has shown even better persistence of
CAR-Ts in vivo when compared to double knockout [67]. While the HLA class I 177 Allogeneic Chimeric Antigen Receptor Therapy in Lymphoma
Khurana and Lin knockout prevents allo-rejection from T cells, it does not contain the allo CAR-
Ts from potential rejection by NK cells. Avoiding recognition by NK cells can be
achieved by expression of non-classical HLA molecules such as HLA-E and G
[66, 69]. The use of HLA homozygous donors to generate a bank of universal
allo CARs can also help prevent rejection due to HLA mismatch [70]. Genome
editing techniques indeed open up a wide range of opportunities in the devel-
opment of allo CAR-Ts; clinical studies are needed to determine their efficacy
and safety for use. knockout prevents allo-rejection from T cells, it does not contain the allo CAR-
Ts from potential rejection by NK cells. Avoiding recognition by NK cells can be
achieved by expression of non-classical HLA molecules such as HLA-E and G
[66, 69]. The use of HLA homozygous donors to generate a bank of universal
allo CARs can also help prevent rejection due to HLA mismatch [70]. Genome
editing techniques indeed open up a wide range of opportunities in the devel-
opment of allo CAR-Ts; clinical studies are needed to determine their efficacy
and safety for use. Using non-αβ TCR T cells for CAR approach Of the 11 patients, 8
(73%) had a response, with seven patients achieving a CR; however, the full
clinical activities of the NK cells were difficult to interpret as many patients
received subsequent systemic therapy or stem cell transplant as consolidation/
maintenance. The CAR-NK cells were detectable in some subjects at low levels
for up to 12 months post infusion [80•]. To overcome the limited amount of Lymphoma (JL Muñoz, Section Editor) 178
178 NK cell present even in the UCB, a Good Manufacturing Practice (GMP)-
compliant procedure has been developed which reliably generates clinically
relevant doses of GMP-grade NK cells from a UCB unit [81]. These studies
support the use of NK cells as a source of allo CAR-Ts, and several products are
currently in early phase clinical trials (Table 2). NK cell present even in the UCB, a Good Manufacturing Practice (GMP)-
compliant procedure has been developed which reliably generates clinically
relevant doses of GMP-grade NK cells from a UCB unit [81]. These studies
support the use of NK cells as a source of allo CAR-Ts, and several products are
currently in early phase clinical trials (Table 2). Induced pluripotent stem cells (iPSCs) are another source for CARs. These
can be genetically modified at a clonal level and used to create a homogeneous
population of uniformly engineered T or NK cells. These have certain advan-
tages, such as their unlimited division potential, and are more amenable to
genetic modification [82]. Any somatic cell can be reprogrammed into iPSC;
single clones from these cells can be expanded to create an iPSC cell line. T cells
generated from iPSC still need genetic editing to prevent GvHD; however, once
modified, a single cell can then be used to generate a clonal population of CAR-
Ts with desired knockouts. Clinical trials are already underway using iPSC-
derived CAR-Ts and CAR-NK cells in B cell lymphomas (NCT04023071,
NCT04245722, NCT04629729; Table 2) [83]. NK T (NKT) cells are a subset of T cells that express NK cell markers. A subset
of NKT cells called the “invariant NKT” (iNKT) cells expressed a highly restricted
TCR, which can recognize lipid antigens presented by CD1d (HLA class I-like
molecule) on B cells, antigen-presenting cells, and some epithelial cells [84, 85]. Using non-αβ TCR T cells for CAR approach Allogeneic or donor iNKT cells have also shown to be protective against GvHD
due to the production of IL-4 and promotion of a Th2-based immune response
in preclinical models and clinical acute GvHD [86–88]. CD19-targeting CAR-
iNKT cells showed a significantly improved efficacy over conventional CD19
CAR-Ts in a murine model of CD1d+ CD19+ B cell malignancy [89]. Protective
effect against GvHD and improved efficacy over conventional CD19 CAR-Ts
make this an explorable approach which is currently in phase I trial in B cell
malignancies (NCT03774654). γδ T cells are another potential candidate under investigation as allogeneic
CAR-Ts. They represent a small percentage of circulating lymphocytes (1–5%),
however, in abundance in certain tissue sites such as gut mucosa, reproductive
organs, tongue, and skin [90–92]. γδ T cells form a key component of the innate
immune system and tumor immunosurveillance and can target tumor antigens
not recognized by αβ T cells [93]. Additionally, their tissue residency gives them
an advantage in efficacy over αβ CAR-Ts which have poor penetrance into non-
inflamed tumors. They are also less likely to induce GvHD as their TCR
activation is not MHC restricted [94]. CD19-targeting CAR-T developed from
γδ T cells has already shown efficacy in CD19+ B cell malignancies in vitro and
in vivo and also in an early phase clinical trial (NCT02656147) [95]. Clinical experience with allogeneic CAR-T in lymphoma A list of completed and ongoing clinical trials using allo CAR-Ts in lymphoma is
shown in Table 2. ALLO-501 is a genetically modified anti-CD19 CAR-T in
which the TCR α gene is disrupted to reduce the risk of GvHD, and the CD52
gene is disrupted to permit the use of ALLO-647 an anti-CD52 monoclonal
antibody (mAb), for selective and prolonged host lymphodepletion. The phase
I ALPHA study (NCT03939026) was first presented at the ASCO 2020 meeting. This included patients with R/R LBCL and follicular lymphoma (FL) after two or 179 Allogeneic Chimeric Antigen Receptor Therapy in Lymphoma
Khurana and Lin more lines of therapy. Prior exposure to CD19-directed therapy was allowed. The data were presented for 22 patients with age range 34–73 years, 64% with
LBCL, and 41% with prior autologous stem cell transplant, and 18% with
autologous CAR-T. The overall response rate (ORR) was 63%, with a 37%
complete remission rate (CR) in the 19 evaluable patients. Retreatment was
noted to be feasible. The most common grade ≥3 adverse events (AEs) were
cytopenias. CRS occurred in 32% (7 patients), and none with grade ≥3; no dose-
limiting toxicities or GvHD was observed [96•, 97]. This was further updated for
42 patients (infused 41; 11 LBCL, and 21 FL) at the ASCO 2021 meeting,
including 9 patients that had previously received autologous CAR-T. The median
time from enrollment to start of therapy was 5 days in 98% of the enrolled
patients. The efficacy analysis was available for 32 patients, and ORR and CR rates
were 75% (24) and 50% (16), respectively. Redosing led to clinical responses,
with an overall treatment failure-free survival for autologous CAR-T naïve patients
of 64% and 61% at 6 months for FL and LBCL. No dose-limiting toxicities or
GvHD was observed, and one grade 3 neurotoxicity was reported. CRS occurred
in 27% of patients, and none with grade ≥3. There were 5 treatment-emergent
deaths, one each from pneumonia, arrhythmia, stroke, and two instances of
COVID-19 infection. The ALPHA2 study (NCT04416984), an open-label,
single-arm study of ALLO-501A in non-HLA, matched patients with R/R LBCL
after two or more lines of treatment also presented at the ASCO 2021 meeting. Prior autologous CD19 CAR-T was allowed if the tumor remained CD19+. As of
the April 19th, 2021 data cutoff, 13 patients were enrolled, and 12 patients were
treated with ALLO-501A. Clinical experience with allogeneic CAR-T in lymphoma One patient that developed central nervous system
involvement before infusion was not treated. Nine patients evaluable for efficacy
were CAR-T naïve, except for one who received prior autologous CAR-T and
previously had a 16-week CR followed by relapse. Of the 9 patients eligible for
efficacy analysis (n = 5, in consolidation cohort), both the ORR and CR rates were
56%. No dose-limiting toxicities, GvHD, or neurotoxicity was seen, and CRS
occurred in 2 patients, both grade G 3 [98]. PBCAR0191, an off-the-shelf, allogeneic CAR-T product, is also being eval-
uated in the CD19+ R/R B cell NHL (NCT03666000). The preliminary safety
and efficacy data from the ASCO 2021 meeting on 13 patients have been
presented. The median time from eligibility confirmation to infusion was 6.5
days. No grade ≥3 CRS or neurotoxicity was observed, and no GvHD was noted
either. The ORR at day+28 was 77% with a 54% CR rate [99]. The safety and
efficacy of CTX110, an allo anti-CD19 CAR-T cell product modified by using
CRISPR/Cas9 editing to disrupt the endogenous TRAC locus and disrupt β2M,
which eliminates major histocompatibility complex (MHC) class I expression,
are under evaluation in R/R LBCL, double/triple hit lymphoma, transformed FL,
and FL grade 3b. The phase 1 CARBON trial (NCT04035434) is an open-label,
multicenter, global study enrolling patients with 2 or more lines of treatment. Prior allogeneic stem cell transplant and autologous CD19 CAR-T are excluded. This trial is open and enrolling at this time, and the data are awaited [100]. Implications of allogeneic CAR-T Allogeneic “off the shelf” CAR-T offers the promise of a readily available,
hopefully at a reduced cost and more accessible to the patients and resource- Lymphoma (JL Muñoz, Section Editor) 180
180 Table 2. Clinical experience with allogeneic CAR-T in lymphoma (Continued)
Trial ID/Phase
Company/product
name
CAR Cell type
CAR Target
Gene Editing technology
and modification
Location
by β2M disruption and CAR
inserted into TRAC locus
Canada
NCT04502446/Phase I
(COBALT-LYM)
CRISPR Therapeutics
AG/CTX130
T-cells
CD70
CRISPR-Cas9 Gene-Editing –
CAR inserted into TRAC
9locus, MHC-1 Knockout
by β2M disruption
US, Australia,
Canada
NCT03939026/Phase I
Allogene
Therapeutics/ALLO-501
T-cells
CD19
TRAC and CD52
Genes disruption by TALEN
US
NCT04416984/Phase
I-II**
Allogene Therapeutics
(ALPHA 2
study)/ALLO-501A
T-cells
CD19
TALEN mediated TCR and CD52
knockout
US
NCT03166878/Phase
I-II
Chinese PLA General
Hospital/UCART019
T-cells
CD19
CRISPR-Cas9 Gene-Editing
disruption of endogenous
TCR and β2M genes
China
NCT03229876/Safety
and efficacy study
Bioray Laboratories/
CD19-UCART
T-cells
CD19
CRISPR-Cas9 Gene-Editing
disruption of endogenous
TCR and β2M genes
China
NCT03666000/Phase
I-IIa (Cohort N)**
Precision BioSciences,
Inc./ PBCAR0191
T-cells
CD19
ARCUS mediated gene editing –
insertion of CAR at the TRAC
gene locus
US
NCT04030195/Phase
I-IIa
Precision BioSciences,
Inc./
PBCAR20A
T-cells
CD20
CAR inserted into
TCR locus
US
NCT04637763/Phase I
(ANTLER study)
Caribou Biosciences,
Inc./CB-010
T-cells
CD19
CRISPR-Cas9 Gene-Editing –
CD19 CAR inserted into T cell
genome, TRAC and PD 1 gene
disrupted
US
NCT04264078/early
phase I
Xinqiao Hospital of
Chongqing, Gracell
Biotechnology Shanghai
Co., Ltd. T-cells
CD7
CRISPR-Cas9 Gene-Editing –
TRAC gene locus, and CD7
knockout to prevent
fratricide
China
Other “off the shelf” CARs
NCT03774654/Phase I
(ANCHOR study)
Baylor College of
Medicine/
CD19.CAR-aNKT cells
KUR-502
NKT cells
CD19
Single gamma retroviral vector
– CD19 CAR, IL15 and short
hairpin RNA expression
(downregulates HLA I, II)
US
Allogeneic Chimeric Antigen Receptor Therapy in Lymphoma
Khurana and Lin
1 Allogeneic Chimeric Antigen Receptor Therapy in Lymphoma
Khurana and Lin 181 Table 2. Clinical experience with allogeneic CAR-T in lymphoma Ongoing clinical trials of allogeneic CARs in non-Hodgkin’s lymphoma
Trial ID/Phase
Company/product
name
CAR Cell type
CAR Target
Gene Editing technology
and modification
Location
CAR-Ts with no TCR modification
NCT04176913/Phase I
Nanjing Legend
Biotechnology Co.,
Ltd./LUCAR-20S
T-cells
CD20
Not specified
China
NCT04384393/Phase I
Fundamenta Therapeutics,
Ltd./ThisCART19
T-cells
CD19
Not specified
China
NCT04601181/Phase I
Fundamenta Therapeutics,
Ltd./ThisCART22
T-cells
CD22
Not specified
China
NCT04288726/Phase I
CD30.CAR-EBV Specific T
cells
EBV-CTLs
CD30
Not specified
US
NCT01430390/Phase I
Memorial Sloan Kettering
Cancer Center/ EBV-CTLs
EBV-CTLs
CD19
Not specified
US
NCT03768310/Phase I
Baylor College of Medicine
Multivirus-Specific
Cytotoxic T
Lymphocytes
CD19
Not specified
US
NCT02050347/Phase I
(CARPASCIO study)
Baylor College of Medicine
Donor derived
T-cells post Allo
transplant
CD19
Not specified
US
NCT04538599/open
label safety
He Huang, Zhejiang
University/ RD13-01
T-cells
CD7
Not specified
China
Genome edited CAR-Ts
NCT04264039/Phase I
Xinqiao Hospital of
Chongqing; Gracell
Biotechnology Shanghai
Co., Ltd./Universal
CD19CAR-T
T-cells
CD19
Not specified
China
NCT03398967/Phase
I-II
Chinese PLA General
Hospital/ Universal
CRISPR-Cas9
Gene-Edited Dual CAR-T
Cells
T-cells
CD19 and CD20/
CD22
CRISPR-Cas9 Gene-Editing,
disruption of endogenous
TCR and β2M genes
China
NCT04026100 /Phase I
Nanjing Bioheng Biotech
Co., Ltd./CTA101
T-cells
CD19/CD22
CRISPR-Cas9 Gene-Editing
TRAC region and CD52 gene
disruption
China
NCT04035434/Phase I
CRISPR Therapeutics
AG/CTX 110
T-cells
CD19
CRISPR-Cas9 Gene-Editing
MHC-1 Knockout
US,Australia,
Germany, Table 2. Clinical experience with allogeneic CAR-T in lymphoma T-cells
CD7
CRISPR-Cas9 Gene-Editing –
TRAC gene locus, and CD7
knockout to prevent
fratricide
China
Other “off the shelf” CARs
NCT03774654/Phase I
(ANCHOR study)
Baylor College of
Medicine/
CD19.CAR-aNKT cells
KUR-502
NKT cells
CD19
Single gamma retroviral vector
– CD19 CAR, IL15 and short
hairpin RNA expression
(downregulates HLA I, II)
US
Allogeneic Chimeric Antigen Receptor Therapy in Lymphoma
Khurana and Lin
1 Gene Editing technology
and modification
Location
by β2M disruption and CAR
inserted into TRAC locus
Canada
CRISPR-Cas9 Gene-Editing –
CAR inserted into TRAC
9locus, MHC-1 Knockout
by β2M disruption
US, Australia,
Canada
TRAC and CD52
Genes disruption by TALEN
US
TALEN mediated TCR and CD52
knockout
US
CRISPR-Cas9 Gene-Editing
disruption of endogenous
TCR and β2M genes
China
CRISPR-Cas9 Gene-Editing
disruption of endogenous
TCR and β2M genes
China
ARCUS mediated gene editing –
insertion of CAR at the TRAC
gene locus
US
CAR inserted into
TCR locus
US
CRISPR-Cas9 Gene-Editing –
CD19 CAR inserted into T cell
genome, TRAC and PD 1 gene
disrupted
US
CRISPR-Cas9 Gene-Editing –
TRAC gene locus, and CD7
knockout to prevent
fratricide
China
Single gamma retroviral vector
– CD19 CAR, IL15 and short
hairpin RNA expression
(downregulates HLA I, II)
US 2
Lymphoma (JL Muñoz, Section Editor)
2 182
182 Table 2. (Continued)
Trial ID/Phase
Company/product
name
CAR Cell type
CAR Target
Gene Editing technology
and modification
Location
NCT04245722/Phase I
Fate Therapeutics/FT596
iPSC derived NK
cells
CD19
US
NCT04023071/Phase
I-Ib
Fate Therapeutics/FT516
iPSC derived NK
cells
non-cleavable CD16
(hnCD16) Fc
receptor
US
NCT04673617/PhaseI-II
Artiva Biotherapeutics,
Inc. cord blood-derived
NK cells
selected for B-KIR
haplotype and the
homozygous
polymorphism of
CD16
US
NCT04629729/Phase I
Fate Therapeutics/FT819
iPSC derived T cells
CD19
CAR inserted into
TRAC locus
US
NCT02892695/Phase
I-II
PersonGen
BioTherapeutics
(Suzhou) Co.,
Ltd./PCAR-119
NK cells
CD19
Derived from NK-92 cells line
China
NCT04887012/Phase I
Second Affiliated Hospital,
School of Medicine,
Zhejiang University/CAR
NK019
HLA Haploidentical
NK cells
CD19
China
NCT04639739/Early
phase I
Chongqing Precision
Biotech Co., Ltd
NK cells
CD19
China
NCT02656147/Phase I
Beijing Doing Biomedical
Co., Ltd. γδ T cells
CD19
China Table 2. (Continued)
Trial ID/Phase
Company/product
name
CAR Cell type
CAR Target
Gene Editing technology
and modification
Location
NCT04245722/Phase I
Fate Therapeutics/FT596
iPSC derived NK
cells
CD19
US
NCT04023071/Phase
I-Ib
Fate Therapeutics/FT516
iPSC derived NK
cells
non-cleavable CD16
(hnCD16) Fc
receptor
US
NCT04673617/PhaseI-II
Artiva Biotherapeutics,
Inc. Clinical experience with allogeneic CAR-T in lymphoma (Continued)
Trial ID/Phase
Company/product
name
CAR Cell type
CAR Target
Gene Editing technology
and modification
Location
by β2M disruption and CAR
inserted into TRAC locus
Canada
NCT04502446/Phase I
(COBALT-LYM)
CRISPR Therapeutics
AG/CTX130
T-cells
CD70
CRISPR-Cas9 Gene-Editing –
CAR inserted into TRAC
9locus, MHC-1 Knockout
by β2M disruption
US, Australia,
Canada
NCT03939026/Phase I
Allogene
Therapeutics/ALLO-501
T-cells
CD19
TRAC and CD52
Genes disruption by TALEN
US
NCT04416984/Phase
I-II**
Allogene Therapeutics
(ALPHA 2
study)/ALLO-501A
T-cells
CD19
TALEN mediated TCR and CD52
knockout
US
NCT03166878/Phase
I-II
Chinese PLA General
Hospital/UCART019
T-cells
CD19
CRISPR-Cas9 Gene-Editing
disruption of endogenous
TCR and β2M genes
China
NCT03229876/Safety
and efficacy study
Bioray Laboratories/
CD19-UCART
T-cells
CD19
CRISPR-Cas9 Gene-Editing
disruption of endogenous
TCR and β2M genes
China
NCT03666000/Phase
I-IIa (Cohort N)**
Precision BioSciences,
Inc./ PBCAR0191
T-cells
CD19
ARCUS mediated gene editing –
insertion of CAR at the TRAC
gene locus
US
NCT04030195/Phase
I-IIa
Precision BioSciences,
Inc./
PBCAR20A
T-cells
CD20
CAR inserted into
TCR locus
US
NCT04637763/Phase I
(ANTLER study)
Caribou Biosciences,
Inc./CB-010
T-cells
CD19
CRISPR-Cas9 Gene-Editing –
CD19 CAR inserted into T cell
genome, TRAC and PD 1 gene
disrupted
US
NCT04264078/early
phase I
Xinqiao Hospital of
Chongqing, Gracell
Biotechnology Shanghai
Co., Ltd. Clinical experience with allogeneic CAR-T in lymphoma Lastly, determination of the optimal subset of T cells, significantly
enriched with less differentiated naïve, and stem cell-like memory cells would
be critical for optimal expansion, survival, and long-term persistence. As more
clinical data are generated by bringing these promising therapies to the bedside
for efficacy and side effects, further advances will be made to create an ideal “off
the shelf” CAR-T product. Conclusions CAR-T has revolutionized the treatment landscape of lymphomas and other
cancers. Developing low-cost, readily available, easily accessible, safe, and
effective products using genome editing tools and non-alloreactive cells will
help generate true “off the shelf products.” With the number of active investi-
gations, more transformative updates are anticipated soon to advance the
capacity of this novel therapy. CAR-T has revolutionized the treatment landscape of lymphomas and other
cancers. Developing low-cost, readily available, easily accessible, safe, and
effective products using genome editing tools and non-alloreactive cells will
help generate true “off the shelf products.” With the number of active investi-
gations, more transformative updates are anticipated soon to advance the
capacity of this novel therapy. Clinical experience with allogeneic CAR-T in lymphoma Lastly, determination of the optimal subset of T cells, significantly
enriched with less differentiated naïve, and stem cell-like memory cells would
be critical for optimal expansion, survival, and long-term persistence. As more
clinical data are generated by bringing these promising therapies to the bedside
for efficacy and side effects, further advances will be made to create an ideal “off
the shelf” CAR-T product. Conclusions
CAR-T has revolutionized the treatment landscape of lymphomas and other
cancers. Developing low-cost, readily available, easily accessible, safe, and
effective products using genome editing tools and non-alloreactive cells will
help generate true “off the shelf products.” With the number of active investi-
gations, more transformative updates are anticipated soon to advance the
capacity of this novel therapy. Declarations limited settings. In addition, the ability to generate CAR-Ts from healthy donor
cells with better quality without exposure to prior chemotherapies or the
immunosuppressive NHL microenvironment could be transformative. It would
also allow for redosing and standardized scale production and quality mainte-
nance. These could also permit targeting multiple antigens simultaneously or
sequentially. Genome editing tools enable a broader application of these “off
the shelf” products with multiplex gene knockout and targeted transductions
focused on making them safer (less GvHD) and persistent (less allo rejection)
for effective use. Still, many challenges remain in enhancing the efficacy of allo
CAR-Ts, such as the degree of in vivo expansion and persistence of CAR-Ts for
efficacy and effective lymphodepletion strategies, which is likely of greater
importance in this setting. Also, the impact of allo CAR-T persistence on efficacy
is yet to be determined as these are more likely to be eliminated by the
recipient’s immune system. Redosing with the same or another allo CAR
targeting different antigens may be the solution, carefully considering donor-
specific antibodies. The association of persistence of CAR-T cells in aggressive
lymphoma is still debatable, as autologous CAR-T with CD28 signaling domain
(axi-cel) with shorter persistence can still ensue durable responses in aggressive
lymphomas. On the other hand, redosing with the axi-cel in indolent lympho-
ma such as FL or marginal zone lymphoma (MZL) as done in the ZUMA-5 trial
in 13 patients (11 FL, 2 MZL) showed an ORR rate of 100% and a 77% CR rate. The median duration of response after redosing had not been reached, and 8
and 4 patients developed CRS and neurotoxicity, respectively but no grade ≥3
[101]. Declarations Conflict of Interest
Arushi Khurana declares that she has no conflict of interest. Yi Lin has received research funding (paid to Mayo
Clinic) from Kite/Gilead, Celgene/Bristol-Myers Squibb, bluebird bio, Janssen, Legend Biotech, Takeda, Merck, and
Boston Scientific; has served as a consultant for Kite/Gilead, Celgene/Bristol-Myers Squibb, Juno/Bristol-Myers
Squibb, Janssen, Legend Biotech, Gamida Cell Ltd, Novartis, Iovance Biotherapeutics, Takeda, and Fosun Kite Clinical experience with allogeneic CAR-T in lymphoma cord blood-derived
NK cells
selected for B-KIR
haplotype and the
homozygous
polymorphism of
CD16
US
NCT04629729/Phase I
Fate Therapeutics/FT819
iPSC derived T cells
CD19
CAR inserted into
TRAC locus
US
NCT02892695/Phase
I-II
PersonGen
BioTherapeutics
(Suzhou) Co.,
Ltd./PCAR-119
NK cells
CD19
Derived from NK-92 cells line
China
NCT04887012/Phase I
Second Affiliated Hospital,
School of Medicine,
Zhejiang University/CAR
NK019
HLA Haploidentical
NK cells
CD19
China
NCT04639739/Early
phase I
Chongqing Precision
Biotech Co., Ltd
NK cells
CD19
China
NCT02656147/Phase I
Beijing Doing Biomedical
Co., Ltd. γδ T cells
CD19
China China
China 183 Allogeneic Chimeric Antigen Receptor Therapy in Lymphoma
Khurana and Lin limited settings. In addition, the ability to generate CAR-Ts from healthy donor
cells with better quality without exposure to prior chemotherapies or the
immunosuppressive NHL microenvironment could be transformative. It would
also allow for redosing and standardized scale production and quality mainte-
nance. These could also permit targeting multiple antigens simultaneously or
sequentially. Genome editing tools enable a broader application of these “off
the shelf” products with multiplex gene knockout and targeted transductions
focused on making them safer (less GvHD) and persistent (less allo rejection)
for effective use. Still, many challenges remain in enhancing the efficacy of allo
CAR-Ts, such as the degree of in vivo expansion and persistence of CAR-Ts for
efficacy and effective lymphodepletion strategies, which is likely of greater
importance in this setting. Also, the impact of allo CAR-T persistence on efficacy
is yet to be determined as these are more likely to be eliminated by the
recipient’s immune system. Redosing with the same or another allo CAR
targeting different antigens may be the solution, carefully considering donor-
specific antibodies. The association of persistence of CAR-T cells in aggressive
lymphoma is still debatable, as autologous CAR-T with CD28 signaling domain
(axi-cel) with shorter persistence can still ensue durable responses in aggressive
lymphomas. On the other hand, redosing with the axi-cel in indolent lympho-
ma such as FL or marginal zone lymphoma (MZL) as done in the ZUMA-5 trial
in 13 patients (11 FL, 2 MZL) showed an ORR rate of 100% and a 77% CR rate. The median duration of response after redosing had not been reached, and 8
and 4 patients developed CRS and neurotoxicity, respectively but no grade ≥3
[101]. Conflict of Interest Lymphoma (JL Muñoz, Section Editor) 184
184 Biotechnology Co., Ltd; and has served on a Data Safety Monitoring Board (DSMB) for Sorrento Therapeutics. All
funds were paid directly to Mayo Clinic. Dr. Lin received no personal compensation. Biotechnology Co., Ltd; and has served on a Data Safety Monitoring Board (DSMB) for Sorrento Therapeutics. All
funds were paid directly to Mayo Clinic. Dr. Lin received no personal compensation. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use,
sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate
credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if
changes were made. The images or other third party material in this article are included in the article's Creative
Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the
article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds
the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this
licence, visit http://creativecommons.org/licenses/by/4.0/. Biotechnology Co., Ltd; and has served on a Data Safety Monitoring Board (DSMB) for Sorrento Therapeutics. All
funds were paid directly to Mayo Clinic. Dr. Lin received no personal compensation. References and Recommended Reading Papers of particular interest, published recently, have been
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Bee Venom in Wound Healing
|
Molecules/Molecules online/Molecules annual
| 2,020
|
cc-by
| 10,321
|
Anna Kurek-Górecka 1,*
, Katarzyna Komosinska-Vassev 2
, Anna Rzepecka-Stojko 3
and Paweł Olczyk 1 Anna Kurek-Górecka 1 1
Department of Community Pharmacy, Faculty of Pharmaceutical Sciences in Sosnowiec, Medical University
of Silesia in Katowice Kasztanowa 3 41-200 Sosnowiec Poland; polczyk@sum edu pl of Silesia in Katowice, Kasztanowa 3, 41 200 Sosnowiec, Poland; polczyk@sum.edu.pl
2
Department of Clinical Chemistry and Laboratory Diagnostics, Faculty of Pharmaceutical Sciences in
Sosnowiec, Medical University of Silesia in Katowice, Jedno´sci 8, 41-200 Sosnowiec, Poland;
kvassev@sum.edu.pl p
y
p
2
Department of Clinical Chemistry and Laboratory Diagnostics, Faculty of Pharmaceutical Sciences in
Sosnowiec, Medical University of Silesia in Katowice, Jedno´sci 8, 41-200 Sosnowiec, Poland;
kvassev@sum.edu.pl p
3
Department of Drug Technology, Faculty of Pharmaceutical Sciences in Sosnowiec, Medical University of
Silesia in Katowice, Jedno´sci 8, 41-200 Sosnowiec, Poland; annastojko@sum.edu.pl
*
Correspondence: akurekgorecka@sum.edu.pl p
3
Department of Drug Technology, Faculty of Pharmaceutical Sciences in Sosnowiec, Medical University of
Silesia in Katowice Jedno´sci 8 41-200 Sosnowiec Poland; annastojko@sum edu pl 3
Department of Drug Technology, Faculty of Pharmaceutical Sciences in Sosnowiec, Medical University of
Silesia in Katowice, Jedno´sci 8, 41-200 Sosnowiec, Poland; annastojko@sum.edu.pl
*
Correspondence: akurekgorecka@sum.edu.pl *
Correspondence: akurekgorecka@sum.edu.pl Abstract: Bee venom (BV), also known as api-toxin, is widely used in the treatment of different
inflammatory diseases such as rheumatoid arthritis or multiple sclerosis. It is also known that BV can
improve the wound healing process. BV plays a crucial role in the modulation of the different phases
of wound repair. It possesses anti-inflammatory, antioxidant, antifungal, antiviral, antimicrobial and
analgesic properties, all of which have a positive impact on the wound healing process. The mentioned
process consists of four phases, i.e., hemostasis, inflammation, proliferation and remodeling. The
impaired wound healing process constitutes a significant problem especially in diabetic patients, due
to hypoxia state. It had been found that BV accelerated the wound healing in diabetic patients as well
as in laboratory animals by impairing the caspase-3, caspase-8 and caspase-9 activity. Moreover, the
activity of BV in wound healing is associated with regulating the expression of transforming growth
factor (TGF-β1), vascular endothelial growth factor and increased collagen type I. BV stimulates the
proliferation and migration of human epidermal keratinocytes and fibroblasts. In combination with
polyvinyl alcohol and chitosan, BV significantly accelerates the wound healing process, increasing
the hydroxyproline and glutathione and lowering the IL-6 level in wound tissues.
Citation: Kurek-Górecka, A.;
Komosinska-Vassev, K.;
Rzepecka-Stojko, A.; Olczyk, P. Bee
Venom in Wound Healing. Molecules
2021, 26, 148. https://doi.org/
10.3390/molecules26010148 Keywords: bee venom; wound healing; biological properties; molecular mechanism Academic Editors: Raffaele Capasso
and Lorenzo Di Cesare Mannelli
Received: 30 November 2020
Accepted: 24 December 2020
Published: 31 December 2020 Academic Editors: Raffaele Capasso
and Lorenzo Di Cesare Mannelli
Received: 30 November 2020
Accepted: 24 December 2020
Published: 31 December 2020 molecules molecules molecules molecules Review Anna Kurek-Górecka 1,*
, Katarzyna Komosinska-Vassev 2
, Anna Rzepecka-Stojko 3
and Paweł Olczyk 1 The effect of BV
on the wounds has been proved by numerous studies, which revealed that BV in the wound healing
process brings about a curative effect and could be applied as a new potential treatment for wound
repair. However, therapy with bee venom may induce allergic reactions, so it is necessary to assess
the existence of the patient’s hypersensitivity to apitoxin before treatment. , Katarzyna Komosinska-Vassev 2
, Anna Rzepecka-Stojko 3
and Paweł Olczyk 1 Anna Kurek-Górecka 1,*
, Katarzyna Komosinska-Vassev 2 Bee Venom in Wound Healing Anna Kurek-Górecka 1,*
, Katarzyna Komosinska-Vassev 2
, Anna Rzepecka-Stojko 3
and Paweł Olczyk 1 1. Introduction The stage of proliferation is connected with the reconstruction of damaged tissue by the
proliferation of fibroblasts, epithelial cells and keratinocytes. Fibroblasts excrete: IGF-
1, bFGF, TGF-β, PDGF, EGF. Epithelial cells produce: VEGF, bFGF, PDGF. Keratinocytes
excrete TGF-α, TGF-β, KDAF (keratinocyte-derived autocrine growth factors). The secreted
mediators take part in the biosynthesis of the extracellular matrix, epithelialization and
angiogenesis. About four days after injury, the granulation tissue consists of collagen (type I
and type III), elastin, proteoglycans, glycosaminoglycans and noncollagenous proteins. Initially, the matrix contains a great amount of hyaluronic acid and fibronectin, however,
eventually hyaluronic acid is replaced by collagen. The matrix of granulation tissue is
enriched in heparan-sulfate proteoglycans and chondroitin–dermatan proteoglycans [2,9]. p
p
g y
p
g y
The granulation tissue replaces the dermis and transforms into a scar during the
next phase—remodeling. Epithelialization is based on the renewal of the epithelium after
damage. EGF, KGF and TGF-α influence the migration and proliferation of epithelial cells. The huge role in the epithelialization process is also played by the matrix metallopro-
teinases such as MMP-2 (gelatinase-A) and MMP-9 (gelatinase-B) and MMP-I (interstitial
collagenase), which are responsible for the degradation of collagen type IV, VII. Endothelial
progenitor cells derived from hematopoietic stem cell linage take part in neovascularization. Growth factors regulate the inflammatory process, play a chemotactic role for neutrophils,
monocytes, macrophages, fibroblasts, keratinocytes and initiate angiogenesis and the for-
mation of an extracellular matrix [10]. Angiogenesis is an important phase of the healing
process as it creates new blood vessels. Angiogenesis stimulates the tissue repair process. It is stimulated by: bFGF, TGF-β, TNF-α and VEGF [2]. y
The proliferation phase is connected with the activity of fibroblasts and the formation
of new blood vessels and extracellular matrix. After the proliferation phase, the process
of remodeling begins. In the last-fourth phase, the extracellular matrix is rebuilt and the
production of collagen type I increases. Myofibroblasts which occur in the wound close
the wound. The scar is the end result of fibroblast apoptosis and extracellular matrix
degradation [11]. Disorders in the functioning of stem cells and in the synthesis and degradation of
growth factors may lead to an impaired wound healing process as well as to hypertrophic
scar creation. Bee venom is a promising component of wound dressing due to its anti-inflammatory
effect and enhanced wound repairing capacity. 1. Introduction Wound healing is a complex process consisting of four overlapping phases: hemostasis
and the formation of a clot, inflammation, proliferation, which involves the biosynthesis
of the extracellular matrix (ECM), epithelialization and angiogenesis and the last stage—
tissue remodeling [1–3]. The first stage starts after an injury appears. The narrowing of
the vessels is caused by serotonin, thromboxane A2 and adrenaline. The platelets undergo
adhesion, aggregation and activation. After 24 h from the injury, a temporary matrix in
wound is created. The aggregated platelets release from alfa-granules as growth factors,
namely platelet derived growth factor (PDGF), transforming growth factor α (TGF-α),
transforming growth factor β (TGF-β), fibroblast growth factor (bFGF), insulin-like growth
factor 1 (IGF-1), vascular endothelial growth factor (VEGF) [1,4,5]. PDGF and TGF-β recruit
monocytes and neutrophils and initiate an inflammatory response and start the second
stage of the healing process—the inflammatory phase. TGF-α, bFGF and VEGF activate
endothelial cells to angiogenesis [1,4]. Publisher’s Note: MDPI stays neu-
tral with regard to jurisdictional clai-
ms in published maps and institutio-
nal affiliations. Copyright: © 2020 by the authors. Li-
censee MDPI, Basel,
Switzerland. This article is an open access article
distributed under the terms and con-
ditions of the Creative Commons At-
tribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/). The inflammatory phase begins after 24 h from injury and lasts for up to 48 h. This
stage is characterized by vessels widening and increasing their permeability. The process is https://www.mdpi.com/journal/molecules Molecules 2021, 26, 148. https://doi.org/10.3390/molecules26010148 Molecules 2021, 26, 148 2 of 13 supported by histamine, kinins, prostaglandins, leukotrienes, nitrogen oxide, hydrolases
and reactive oxygen species (ROS). Neutrophils and macrophages clean the wound bed
and release cytokines and growth factors, which are responsible for initiating the healing
process. Neutrophiles releasing IL-1 and TNF-α quicken the inflammatory process. After
two or three days, neutrophils undergo apoptosis and are replaced by monocytes. Due
to the influence of TGF-β and the products of the degradation of fibrin and fibronectin,
monocytes are transformed into macrophages. Macrophages take part in phagocytosis,
clean wounds from bacteria and dead tissue and release proinflammatory cytokines such
as IL-1, IL-6, Il-8, TNF-α and growth factors like PDGF, TGF-α, TGF-β, IGF-1,FGF [6–8]. Growth factors regulate the inflammatory process. Additionally, cytokines and growth
factors activate fibroblasts and epithelial cells to the third stage of healing, i.e., proliferation. 2. Chemical Composition of Bee Venom Bee venom is a liquid mixture which contains 88% of water and only 0.1 g of dry weight
of a complex mixture of peptides, enzymes and nonpeptide components in one drop [16]. The composition of dry and fresh bee venom differs in contents of volatile components but
the biological properties are similar. The composition of bee venom varies depending on
the species. The bee species mainly responsible for human envenoming are Apis mellifera
melifera and Apis mellifera ligustica in Europe or Apis mellifera scutellate in Africa. Africanized bee hybrids inhabit both Americas. They are a genetic mix of the honeybee
from Africa and they spread across the Americas. Data relating to toxic envenoming by
species other than A. mellifera are rare [17]. Moreover, the composition of apitoxin depends
on bee age, geographical localization, seasonal changes and social condition. Especially,
the level of mellitin, apamin, hyaluronidase or PLA2 is very susceptible to variability. In
addition, the methods collecting bee venom influence the content of volatile components. During bee venom collecting by electrical stimulation, histamine can disappear [17–21]. g
p
During bee venom collecting by electrical stimulation, histamine can disappear [17–21]. Bee venom contains several biologically active molecules such as peptides, bioactive
amines and enzymes, which have an advantageous potential in healing the wounds. Bee
venom contains different peptides including melittin, apamin, adolapin, secapin and its
isomers (i.e., secapin-1 and -2), procamine, tertiapin and mast cell degranulating peptide
(MCD-peptides) [22,23]. Peptides are the main components of bee venom. The concentration of small proteins
and peptides is about 48–50% in dry venom. Among these peptides, melittin especially
plays an important role in inducing reactions associated with bee stings. Melittin is a
26-amino acid peptide and constitutes the main biological active component in bee venom. Its percentage content in dry bee venom is about 50% [24]. It possesses an amphipathic
property, due to the carboxyl-terminal region of the peptide, which is hydrophilic and the
amino-terminal region which is hydrophobic. The amphipathic property of melittin allows
it to be inserted into membranes by disrupting the phospholipid bilayers. Melittin induces
membrane permeabilization and lyses of the cells. Depending on the dose, melittin induces
transient or stable pores. In the case of the transient pore, the membranes are permeable
for ions. In the case of the stable pore, the membranes are permeable for large molecules
like glucose [25]. 1. Introduction It was found to significantly accelerate
the healing of diabetic wounds [12,13]. The slowed wound repair process is connected
with hypoxia [12,14]. Insufficient recruitment of macrophages and neutrophiles into the
wound environment delay the start of the inflammatory phase and in this way impair the
wound healing process. On the other hand, hypoxia amplify the early inflammatory re-
sponse through the prolonged release of inflammatory cytokines. Moreover, hyperglycemia
increases oxidative stress and causes many dysfunctions such as defective T cell immu-
nity, defects in phagocytosis and dysfunctions of fibroblasts. The accumulation of reactive
oxygen species (ROS) increases the cellular damage and impairs neovascularization. In ad-
dition, faster apoptosis, reduced angiogenesis, and impaired lymphangiogenesis influence
the impaired healing process [12,15]. Molecules 2021, 26, 148 3 of 13 3 of 13 The impaired diabetic wound healing is characterized by a lower collagen production,
downregulated expression of TGF-β, VEGF, as well as the upregulated expression of ATF-3
(activating transcription factor 3), iNOS, and an increased activity of caspase-3, caspase-8
and caspase-9. Interestingly, during hypoxia, the mobilization of endothelial progenitor
cells (EPCs) is activated by VEGF secreted by macrophages, fibroblasts and epithelial cells,
while in diabetic wounds there is a VEGF deficiency. Thus, impaired endothelial progenitor
cells (EPCs) mobilization is responsible for endothelial progenitor cell deficiency [11]. 2. Chemical Composition of Bee Venom Melittin also induces the formation of pores responsible for its hemolytic,
antimicrobial, antifungal action. At low doses, it exhibits anti-inflammatory, antibacterial,
antifungal, antiviral effects and increases capillary permeability by increasing the blood cir-
culation. However, at high doses melittin initiates local pain, itching, inflammation [26,27]. Apamin is the second very important active peptide in bee venom. It accounts for 1% of dry
bee venom. It is a polypeptide which consists of 18 amino acids with two disulfide bridges. Apamin exhibits an anti-inflammatory, antinociceptive effect and increases the defense
capability. It crosses the blood-brain barrier and affects the nervous system. In the vascular
wall, apamin inhibits vascular smooth muscle cell proliferation and migration via the Akt
and Erk signaling pathways. It inhibits PDGF-BB-induced phosphorylation of Akt and
Erk1/2. PDGF, produced by activated macrophages, vascular smooth muscle cells (VSMCs)
and endothelial cells or released from platelets in thrombi, plays an essential role in the
abnormal proliferation and migration of VSMCs. Five different dimeric isoforms have been
described including PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC and PDGF-DD. Among the
isoforms of the PDGF family, the PDGF-BB-treated proliferation of VSMCs has been well
demonstrated. Apamin has been found to suppress the G0/G1 cell cycle by the PDGF-BB Molecules 2021, 26, 148 4 of 13 signaling pathway and thus plays a key role in the prevention of vascular proliferation and
migration constituting a promising candidate for the therapy of atherosclerosis. Apamin
plays a key role in the inhibition of vascular smooth muscle cell (VSMC) proliferation and
migration by the PDGF signaling pathway by preventing atherosclerosis [16,28]. signaling pathway and thus plays a key role in the prevention of vascular proliferation and
migration constituting a promising candidate for the therapy of atherosclerosis. Apamin
plays a key role in the inhibition of vascular smooth muscle cell (VSMC) proliferation and
migration by the PDGF signaling pathway by preventing atherosclerosis [16,28]. g
y
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g p
y
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[
]
Adolapin is an important component of bee venom. It is a polypeptide with 103 amino
acid residues. It occurs at a concentration of 1% in dry bee venom (BV). Adolapin comprises
2–5% of the peptides in bee venom. Adolapin exhibits anti-inflammatory, antinociceptive
and antipyretic effects by blocking prostaglandin synthesis and inhibiting the cyclooxyge-
nase activity. It inhibits lipoxygenase from human platelets and may cause an analgesic
effect [29–31]. 2. Chemical Composition of Bee Venom Secapin is another polypeptide composed of 25 amino acids containing
a large proportion of proline and one disulphide bridge. It is a non-toxic polypeptide
which comprises only 0.5% of dry bee venom. Lee et al. showed that secapin (AcSecapin-1)
from the bee venom of the Asiatic honeybee exhibits antifibrinolytic, anti-elastolytic, and
antimicrobial activities [32,33]. Procamine is the next polypeptide in bee venom, which
inhibits the activity of proteases like trypsin, chymotrypsin, plasmin and thrombin, thus
decreasing inflammation. Tertiapin is a 21-amino acid bee venom peptide. It contains two
disulphide bridges and a C-terminal residue in an amidated form. It belongs to neurotoxin
peptides like apamin. Tertiapin consists of a very minor component of bee venom, compris-
ing <0.1% of the BV dry weight. Tertiapin blocks potassium channels in the human body. It is used as a potassium channel modulator [34,35]. The mast cell degranulation peptide
(MCDP) is chemically similar to apamin and comprises 22 amino acids and contains two
disulfide bridges. MCDP is known as peptide 401. Its amount in bee venom is about 2–3%
of dry api-toxin [27]. It exhibits immunological and pharmacological activities. It shows
anti-inflammatory activity at higher concentrations, but in low concentration it plays a role
as a strong mediator of mast cell degranulation and histamine release. MCDP shows an
analgesic and nociceptive effect [16]. Bee venom also possesses biologically active amines
like histamine, epinephrine, dopamine, norepinephrine. Bee venom contains enzymes like
phospholipase A2, hyaluronidase, alfa-glucosidase, acid phosphatase, acid phosphomo-
noesterase, lysophospholipase. Phospholipase A2 is an enzyme hydrolyzing phospholipids
and at the same time is a very strong allergen. This exhibits inflammatory effects, destroys
phospholipids and disrupts the cell membrane. The concentration of phospholipase A2 in
a sting is 0.23 nM. In dry venom, the percentage content is about 12–15%. Phospholipase B
exhibits detoxifying activity. Hyaluronidase catalyzes the hydrolysis of hyaluronic acid
by attacking the tissue hyaluronic acid polymers. Hydrolyzed hyaluronan fragments ex-
hibit the proinflammatory, proangiogenic and immunostimulatory action. As bee venom
hyaluronidase increases the capillary permeability and catalyzes the protein hydrolysis, it
penetrates better into tissues [36]. Bee venom also contains dipeptidylpeptidase IV (Api m 5), Api m 6—a new bee
allergen exists as four isoforms of 7190, 7400, 7598, and 7808 d, respectively (Api m 6),
CUB serine protease (Api m 7), icarapin (Api m 10), major royal jelly proteins (MRJPs
8 and 9) [37–41]. 2. Chemical Composition of Bee Venom )
Bee venom also contains components other than peptides including carbohydrates
and free amino acids and minerals. Among carbohydrates, there are glucose and fructose
in bee venom. Among amino acids, the main components are γ-aminobutyric acid and
B-aminoisobutyric acid. Bee venom also contains minerals such as magnesium, calcium
and phosphor. Moreover, bee venom contains volatile compounds including pheromones,
which are represented by complex ethers. Isopentyl acetate and (Z)-11-eicosen-l-ol are
pheromones that warn bees of danger and stimulate the stinging reaction [16,21,26]. 3. Therapeutic Action of Bee Venom on Wounds Bee venom possesses antioxidant, antimicrobial, antifungal, antiviral, anti-inflammatory
and analgesic properties, so it might be a therapeutic agent in treatment of wounds. The
antioxidant activity of bee venom is caused by the inhibition of the production of superoxide
anion and hydrogen peroxide production in human neutrophils [16]. Moreover, bee venom Molecules 2021, 26, 148 5 of 13 was found to decrease the levels of reactive oxygen species (ROS) in animal blood and
wounds, thus accelerating wound healing as reactive oxygen species lead to widespread
cellular damage and to impaired neovascularization [11]. In addition to antioxidant activity,
bee venom possesses an antimicrobial effect due to the content of melittin and secapin. Melittin exhibits the antibacterial effect against Gram-positive and Gram-negative bacteria,
due to the formation of cell membrane channels [42]. Secapin directly binds to the bacteria
cell walls, resulting in antimicrobial action [32]. As it was reported by Park and Lee, bee
venom exhibits antifungal property via an apoptosis mechanism [43]. Additionally, the
major compound of bee venom—melittin presents antiviral activity. The mechanism of
melittin antiviral action is based upon the interferon type I stimulation and in this way,
the inhibition of viral replication in the host cell. The conducted studies confirmed that
melittin has antiviral activity against vesicular stomatitis virus, influenza A virus and
herpes virus [44]. Anti-inflammatory and antinociceptive properties of bee venom are connected with
decreased of expression of COX-2 and PLA2 and a decreased expression of TNF-α, IL-1,
IL-6, NO, as well as intracellular calcium. Melittin, adolapin and tertiapin are responsi-
ble for the anti-inflammatory and analgesic effect of bee venom. Melittin and adolapin
inhibit prostaglandin synthesis, hence bee venom inhibits cyclooxygenase and lipoxyge-
nase activities [16]. Tertiapin exhibits anti-inflammatory activity by blocking potassium
channels [45]. Moreover, MCDP possesses anti-inflammatory activity which was found
in animal models. Many researchers indicate that BV injections lead to initial nociceptive
action and prolonged antinociceptive effects [16]. Pharmacological activities of bee venom allow to use bee venom in wound healing. Multiple molecular mechanisms of bee venom explain the impact of bee venom on the
mentioned process. Various growth factors and cytokines are involved in each phase of
the wound healing process. These active biomolecules are secreted by inflammatory cells,
keratinocytes and fibroblasts. The study conducted by Han et al. confirmed that bee venom
had a significant wound-healing activity associated with decreasing levels of TGF-β1,
fibronectin and VEGF [46]. 4. Therapeutic Action of Bee Venom in Diabetic Wounds Impaired diabetic wound healing involves pathophysiological mechanisms and con-
stitutes a major problem in diabetic patients. Many studies indicate that bee venom can
be used in diabetic patients because it significantly restores the levels of inflammatory
cytokines, growth factors, and reduces the level of free radicals and accelerates wound
closure [11,49,50]. Moreover, Badr et al. confirmed that bee venom therapy affects all phases
of the wound healing process [11]. The impaired wound healing in diabetic patients is con-
nected with the reduced expression of TGF-β and VEGF, the significant elevation caspase
activity and the upregulated expression of ATF-3 (activating transcription factor-3) and
iNOS (inducible nitric oxide synthase). Bee venom injected subcutaneously to diabetic mice
for 15 days in a dose of 50 µL upregulated the expression of TGF-β and VEGF. Bee venom
restores the levels of TGF-β and VEGF, which regulates different phases of wound healing. TGF-β is released by platelets in the early stage of the healing process and stimulates the
chemotaxis of inflammatory and immune cells to the wounds, enhances extracellular ma-
trix deposition and influences the granulation tissue formation. Additionally, TGF-β plays
a crucial role in the remodeling process. At this stage, TGF-β supports the replacement
of collagen type III with collagen type I and stimulates reepithelization. The significant
restoration of the expression of VEGF influences the hemostasis, proliferation and remodel-
ing phases. VEGF is responsible for stimulating platelet aggregation and makes the blood
clot protect the environment of wounds, stimulates the proliferation of endothelial cells,
activates angiogenesis and regulates the collagen fibers production and transforms collagen
fibers into scar [10]. Summing up, bee venom restores the levels of TGF-β, and VEGF
regulates different phases of healing. It has also been reported that bee venom acceler-
ates the healing process in experimental animals by impairing the caspase-3, caspase-8,
caspase-9 activity. The activity of caspase-3, -8, -9 was studied using a fluorometric protease
assay. The conducted study indicated that the caspase cascade was decreased by therapy
with bee venom. Caspase cascades play a crucial role in the induction of apoptosis and
constitute an important factor in the impaired wound healing process. Due to inhibiting
caspase-3, -8, -9 activity, bee venom stimulates endothelial progenitors cells in tissues. This study also revealed that bee venom treatment decreased the expression of ATF-3 and
iNOS. Thus, bee venom reduces oxidative stress and decreases the level of free radicals. 3. Therapeutic Action of Bee Venom on Wounds TGF-β1, fibronectin and VEGF play a key role in angiogenesis,
endothelial cell proliferation, smooth muscle cell growth and wound healing. The results
of the conducted study indicated that bee venom effectively inhibits the accumulation of
ECM and angiogenesis. According to the results of Han S. et al., bee venom decreased
TGF-β1, fibronectin, and VEGFmRNA levels and increased the collagen type I mRNA level. Collagen type I is responsible for the tensile strength of wounds. Histological analyses
indicate that collagen type I enhances the wound healing process [46]. The repair capacity
of bee venom evaluated in an animal model was found to be satisfactory since the wound
size was reduced. Topical application of bee venom has an impact on reepithelization. The mentioned
process involves the restoration of an intact epidermal barrier. Necessary for wound ep-
ithelialization is the keratinocytes migration. Hang et al. showed the effects of bee venom
on keratinocyte migration in vitro. The distance of human epidermal keratinocytes migra-
tion after 48 h incubation was increased in a group treated with bee venom. This result
indicates that bee venom stimulates the proliferation and migration of human epidermal
keratinocytes. Moreover, the mentioned study indicates that bee venom decreases the
expression of IL-8 and TNF-α in human epidermal keratinocytes from 24 to 74 h after BV
treatment. These cytokines are secreted to suppress the proliferation of keratinocytes [47]. Toxic substances stimulate the expression of TNF-α and IL-8. These cytokines are secreted
especially during the infection of wounds. This study shows that BV in a concentration be-
low 100 µg/mL is not cytotoxic and its topical application accelerates cell regeneration and
wound treatment [24]. Additionally, bee venom inhibits MMP-1 and MMP-3 production. MMP-1 is known as interstitial collagenase and supports the migration of keratinocytes
and breaks down collagen type I, II, III and VI. MMP-3 known as matrix metalloproteinase-
3 or stromelysin-1 is responsible for the hydrolysis of components ECM. Therefore, the
inhibition of matrix metalloproteinase production prevents collagen damage [48]. Molecules 2021, 26, 148 6 of 13 4. Therapeutic Action of Bee Venom in Diabetic Wounds CD31
is a marker of neovascularization and angiogenesis in injured tissues. The expression of
antigen CD31 occurs on the blood vessel endothelium. It is a protein that takes part in
the wound healing process. Treatment with bee venom enhances expression of CD31 and
in this way activates macrophage recruitment in bed wounds. Macrophages play a huge
role in the healing process, as they take part in phagocytosis and influence the release of
mediators: TGF-β, PDGF, TGF-α, FGF, Il-1, Il-6, TNF-α, which control the inflammatory
process and modulate epithelialization and angiogenesis. An impaired healing process in
diabetic wounds is connected with the reduction activity of enzymes such as: glutathione
peroxidase (GSH-Px), manganese superoxide dismutase (MnSOD) and catalase (CAT). Many researchers indicate that oxidative stress and a large production of ROS delay the
repair process. The therapy with the subcutaneous injection of bee venom protects against
oxidative stress by activating antioxidant enzymes [49]. Deveci et al. indicate that oxida-
tive stress is responsible for decreased keratinocyte proliferation and their migration and
increased apoptosis [50]. p p
Another study indicates that bee venom is a promising therapeutic agent in wound
dressing. Amin and Abdel-Raheem (2014) revealed that bee venom-loaded wound dressing
composed of polyvinyl alcohol (PVA), chitosan and bee venom enhances the healing of
wounds in diabetic rats. In the case of wound dressing with a concentration of bee venom
at 3 and 4% the researchers obtained a closure of wounds at 84 and 91%, respectively,
after 14 days of treatment [12]. Moreover, the scar was clean, smooth and was not con-
taminated. In the described experimental study the content of hydroxyproline in wound
tissues was found to be increased. The hydroxyproline is a marker of collagen content. Hydroxyproline is the end product of collagen breakdown, hence it is used in the quantity
assessment of collagen in tissues. Increasing production of collagen is necessary to create
an extracellular matrix in the stage of proliferation. Badr et al. also confirmed that bee
venom treatment significantly increased collagen content in the extracellular matrix during
the proliferative phase [11]. Amin and Abdel-Raheem (2014) [12] indicated that a bee
venom-loaded dressing increases the glutathione level in wound tissues of diabetic rats. Bee venom at a concentration of 4% as a component of wound dressing showed antioxidant
activity and exhibited the beneficial action in the wound repair process. 4. Therapeutic Action of Bee Venom in Diabetic Wounds The increased activation of ATF-3 and iNOS leads to enhance oxidative stress and it is
responsible for the prolonged wound healing process in diabetic mice. Therefore, it limits
prolonged inflammation and accelerates wound healing in BV-treated diabetic mice. The
regulated expression of ATF-3 and iNOS in diabetic mice by their impact on oxidative stress
improves cellular differentiation and wound remodeling. Furthermore, the subcutaneous
injection of bee venom restores the levels of the pro-inflammatory cytokines such as IL-1β,
IL-6, TNF-α, released early during the healing process, and they play a key role as effectors
for keratinocytes and fibroblasts stimulating epithelialization [11]. Further research on the
influence of the subcutaneous injection of bee venom on the wound healing process in
an animal model has been studied by Hozzein et al. [49]. In this study, bee venom was
subcutaneously injected at the dose of 50 µL, for 15 days into the wounded area in diabetic
mice. These results indicated that bee venom increased the content of collagen type I and
β-defensin-2 (BD2) in diabetic wounds. Collagen is necessary for the formation of granula-
tion tissue, thus BD2 is a very important factor to regulate the wound closure. Moreover,
β-defensin-2 is a factor of inflammation, re-epithelization and angiogenesis. BD2 increases
the production of proinflammatory cytokines and stimulates keratinocyte migration and
proliferation. In this way, bee venom stimulates the wound healing process in diabetic
wounds. Moreover, the therapy with bee venom decreased apoptotic macrophages which
resulted in increasing the phagocytic index. In addition, bee venom activates angiogenesis
via recovering Ang-1/Tie-2 signaling and activates the expression of Nrf2, beta-defensin-2. Tie-2 is an endothelial specific receptor tyrosine kinase which plays a key role as a regu-
lator of wound healing. Angiopoietin-1 and angiopoietin-2 bind the Tie-2 receptor and
induce angiogenesis. The connection of Ang-1 to Tie-2 in endothelial cells leads to the Molecules 2021, 26, 148 7 of 13 production of PDGF and the recruiting of smooth muscle cells to new capillaries. Impaired
wound healing is caused by disrupting Ang-1/Tie-2 signaling. Additionally, the nuclear
E2-related factor is an agonist ligand to Tie-2. After the subcutaneous injection of BV, the
levels of Ang-1 Nrf2 were restored in wounds. Thus, Ang-1 and Nrf2 have an impact on
neovascularization and accelerate the wound healing process. The molecular mechanism
of bee venom action on neovascularization is connected with the expression of CD31. 4. Therapeutic Action of Bee Venom in Diabetic Wounds Component
Effect on Healing Wounds
References
Adolapin
Antinociceptive action;
antibacterial action;
anti-inflammatory action; inhibition of cyclooxygenase activity and
PLA2
[46]
Apamin
Antinociceptive action;
anti-inflammatory action;
antibacterial action
[46]
Hyaluronidase
Selectively degrade tissue hyaluronic acid polymers;
increases the capillary permeability
[51]
Melittin
Induces membrane permeabilization by reorganizing phospholipid
bilayer; increases blood circulation; anti-inflammatory, antibacterial,
antifungal and antiviral activity; amphipathic properties
[26,27,46]
Peptide 401
The mast cell
degranulation peptide
Anti-inflammatory activity;
analgesic effect;
nociceptive effect
[16]
Secapin
Antibacterial properties;
antifungal properties;
antifibrinolytic;
anti-elastolytic
[32]
Tertiapin
Anti-inflammatory activity by blocking potassium channels
[45] Table 1. Effect of compounds of bee venom on healing wounds. Table 2. Molecular mechanisms of bee venom on skin wound healing process. Phase of Healing
Molecular Mechanism of Bee Venom
References
Hemostasis
Upregulates the expression of TGF-β and VEGF in diabetic mice
[11]
Inflammation
Limits prolonged inflammation
Reduces levels of proinflammatory cytokines IL-1, IL-6, TNF-α
Decreases the expression of ATF-3 and iNOS
[11]
Biosynthesis of the
extracellular matrix
Increases collagen formation
[11,12]
Reepithelization
Stimulates human epidermal keratinocytes proliferation and migration;
Decreases level of IL-8, TNF-α
[24]
Neovascularization and
angiogenesis
Decreases activity of caspase-3, -8 and -9;
Upregulates the expression of TGF-β and VEGF in diabetic mice
[11]
Remodeling
Decreases levels of TGF-β1, fibronectin and VEGF;
Increases collagen-I mRNA level;
Decreases expression of ATF-3 and iNOS
[11,46] 4. Therapeutic Action of Bee Venom in Diabetic Wounds Phase of Healing
Molecular Mechanism of Bee Venom
References
Hemostasis
Upregulates the expression of TGF-β and VEGF in diabetic mice
[11]
Inflammation
Limits prolonged inflammation
Reduces levels of proinflammatory cytokines IL-1, IL-6, TNF-α
Decreases the expression of ATF-3 and iNOS
[11]
Biosynthesis of the
extracellular matrix
Increases collagen formation
[11,12]
Reepithelization
Stimulates human epidermal keratinocytes proliferation and migration;
Decreases level of IL-8, TNF-α
[24]
Neovascularization and
angiogenesis
Decreases activity of caspase-3, -8 and -9;
Upregulates the expression of TGF-β and VEGF in diabetic mice
[11]
Remodeling
Decreases levels of TGF-β1, fibronectin and VEGF;
Increases collagen-I mRNA level;
Decreases expression of ATF-3 and iNOS
[11,46] Table 1. Effect of compounds of bee venom on healing wounds. Component
Effect on Healing Wounds
References
Adolapin
Antinociceptive action;
antibacterial action;
anti-inflammatory action; inhibition of cyclooxygenase activity and
PLA2
[46]
Apamin
Antinociceptive action;
anti-inflammatory action;
antibacterial action
[46]
Hyaluronidase
Selectively degrade tissue hyaluronic acid polymers;
increases the capillary permeability
[51]
Melittin
Induces membrane permeabilization by reorganizing phospholipid
bilayer; increases blood circulation; anti-inflammatory, antibacterial,
antifungal and antiviral activity; amphipathic properties
[26,27,46]
Peptide 401
The mast cell
degranulation peptide
Anti-inflammatory activity;
analgesic effect;
nociceptive effect
[16]
Secapin
Antibacterial properties;
antifungal properties;
antifibrinolytic;
anti-elastolytic
[32]
Tertiapin
Anti-inflammatory activity by blocking potassium channels
[45]
Table 2. Molecular mechanisms of bee venom on skin wound healing process. Phase of Healing
Molecular Mechanism of Bee Venom
References
Hemostasis
Upregulates the expression of TGF-β and VEGF in diabetic mice
[11]
Inflammation
Limits prolonged inflammation
Reduces levels of proinflammatory cytokines IL-1, IL-6, TNF-α
Decreases the expression of ATF-3 and iNOS
[11]
Biosynthesis of the
extracellular matrix
Increases collagen formation
[11,12]
Reepithelization
Stimulates human epidermal keratinocytes proliferation and migration;
Decreases level of IL-8, TNF-α
[24]
Neovascularization and
angiogenesis
Decreases activity of caspase-3, -8 and -9;
Upregulates the expression of TGF-β and VEGF in diabetic mice
[11]
Remodeling
Decreases levels of TGF-β1, fibronectin and VEGF;
Increases collagen-I mRNA level;
Decreases expression of ATF-3 and iNOS
[11,46]
5. Allergic Reaction to Bee Venom Table 1. Effect of compounds of bee venom on healing wounds. 4. Therapeutic Action of Bee Venom in Diabetic Wounds Furthermore, the
therapy with bee venom-loaded hydrogel may bring significant advantages in the case of
skin wounds under hyperglycemia. Glutathione plays a key role as a scavenger of free
radicals and protects against oxidant damage in tissues. The antioxidant role of glutathione
is based on its reactivity with free organic radicals and allows for the regeneration of
damaged molecules. BV, PVA and chitosan in combination possess the anti-inflammatory
effect through the inhibition of the production of IL-6. Bee venom at a concentration of 4%
reduced carrageenan-induced rat paw edema at similar degree as 1% diclofenac [12]. g
p
g
[
]
Other studies showed that bee venom at a concentration of 6% with chitosan film
demonstrates a similar anti-inflammatory effect like indomethacin. BV in one topical
formulation reduced PGE2 in the serum of rats [13]. Thus, bee venom-loaded wound
dressing with PBA and chitosan accelerated wound healing. The implementation of bee
venom into dressing as a therapeutic strategy demonstrates that the topical application of
BV can promote cell regeneration and wound treatment. Tables 1 and 2 summarize the effect
of bee venom’s compounds on healing wounds process and the molecular mechanisms of
bee venom on the skin wound healing process. Molecules 2021, 26, 148 8 of 13 Table 1. Effect of compounds of bee venom on healing wounds. Component
Effect on Healing Wounds
References
Adolapin
Antinociceptive action;
antibacterial action;
anti-inflammatory action; inhibition of cyclooxygenase activity and
PLA2
[46]
Apamin
Antinociceptive action;
anti-inflammatory action;
antibacterial action
[46]
Hyaluronidase
Selectively degrade tissue hyaluronic acid polymers;
increases the capillary permeability
[51]
Melittin
Induces membrane permeabilization by reorganizing phospholipid
bilayer; increases blood circulation; anti-inflammatory, antibacterial,
antifungal and antiviral activity; amphipathic properties
[26,27,46]
Peptide 401
The mast cell
degranulation peptide
Anti-inflammatory activity;
analgesic effect;
nociceptive effect
[16]
Secapin
Antibacterial properties;
antifungal properties;
antifibrinolytic;
anti-elastolytic
[32]
Tertiapin
Anti-inflammatory activity by blocking potassium channels
[45]
Table 2. Molecular mechanisms of bee venom on skin wound healing process. 5. Allergic Reaction to Bee Venom In a large local reaction of the bee sting, pain can persist for several days
and the area of redness and swelling may cover a diameter greater than 10 cm. This reaction
is caused by the late phase of an allergic reaction. In these cases, topical corticosteroids and
H-1 and H-2 blockers are applied orally. However, in patients with allergic reactions to bee
venom, there not only large local reactions but also systemic symptoms including anaphy-
lactic shock may occur. Patients with asthma and allergic rhinitis and allergy to bee stings
in the family may experience a systemic reaction [62]. Patients may suffer from symptoms
of the respiratory, digestive, circulatory and nervous systems. Among allergic patients,
some could occur systemic urticaria, pruritus, angioedema, vomiting or diarrhea [63,64]. In practice, the classification of the severity of the systemic reaction was developed by Ring
and Messmer [65]. This classification divides the severity of the systemic reaction into four
degrees. The first degree includes generalized cutaneous manifestations and angioedema. The second degree includes symptoms of the respiratory, circulatory and digestive systems. The third degree includes an anaphylactic shock and loss of consciousness. The last degree
manifests itself through cardiac arrest and apnea. In anaphylaxis, the first choice of action
should be administered intramuscular adrenaline. Then, the stinger should be removed. Some authors indicated that the first line of action to scrap off and take out the sting in a
way that prevents the venom from entering the body. However, it is safer to administer
adrenaline first [66]. In addition, short-acting β2 agonists are inhaled. Then, H1- and H2-
blockers and corticosteroids are given [66–68]. g
Venom immunotherapy treatment (VIT) is the preventive treatment of hymenoptera
venom anaphylaxis. VIT provides long-lasting immunoprotection against severe reactions
to subsequent stings. Immunotherapy consists of increasing the doses of an allergen (in
this case bee venom) over a period of time in order to obtain tolerance to this allergen. VIT can be conducted with different venom products (purified and non-purified, aqueous
or depot). Bee venom products can be administered by the subcutaneous or sublingual
routes. The effective immunotolerance of allergens results from the different mechanisms. 5. Allergic Reaction to Bee Venom Bee venom may induce allergic reactions following the sting. Allergic reactions may oc-
cur in the skin, the respiratory track, the cardiovascular system, and the gastrointestinal sys-
tem. Moreover, an anaphylactic reaction can lead to cerebral or myocardial ischemia [52,53]. Bee venom contains 12 main allergens, which are multiple protein allergens possessing an
enzymatic activity and being responsible for an allergic response. Among the main aller-
gens there are: phospholipase A2 (PLA2) called Api m1, hyaluronidase called Api m2, acid
phosphatase called Api m3, melittin called Api m4, dipeptidyl-peptidase IV called Api m5,
Api m6 which was w new bee venom allergen, protease called Api m7, carboxyl-esterase
called Api m8, carboxyl-peptidase called Api m9, incarapin called Api m10, major royal
jelly protein called Api m11 and vitellogenin called Api m12 [38,54–56]. Among the most Molecules 2021, 26, 148 9 of 13 important allergens mentioned are Api m1, Api m2, Api m3 and Api m4. Phospholipase
A2 is the most allergenic and immunogenic protein. Hyaluronidase is known as spreading
factor because it takes part in the distribution of apitoxin in the body. Acid phosphatase is
capable of releasing histamine from sensitized human basophils. Mellitin is responsible
for inducing minor allergic reactions [57,58]. Among people who are hypersensitive to bee
venom, 97% possess specific antibodies directed against phospholipase A2, while 50% of
allergic patients have sIgE for hialuronidase. In people with confirmed bee venom allergy,
a specific IgE for acid phosphatase among 60% patients and 25–50% of them possess a
specific IgE for melittin [54]. These allergens are responsible for allergic reactions, but bee
venom also contains allergens which are involved in IgE-independent reactions, such as
the bradykinin (BK) mediator, and in this way give various anaphylactic symptoms [59–61]. A non-immune mediator can be produced after induction by melittin. Melittin constitutes
of an activator of PLA2 that can imitate BK’s effects on tracheal tone [27]. Moreover, MCD
peptides can exchange disulfate between IgE on the mast cell surface and in this way MCD
may inhibit the release of histamine. Due to this action, MCD can play a pivotal role as an
anti-allergic agent [16]. The reaction to the bee sting may occur as a normal reaction. The
normal reaction after bee sting manifests by appearing pain, redness and edema. The pain
persists for 24 h. 5. Allergic Reaction to Bee Venom The desensitization of mast cells and basophils, inhibition of innate lymphoid cells, the
activation of regulatory T cells (which caused the increasing level of IL-10 and TGF-β),
immunoglobulin cell-switch to IgG4 and IgA induced by regulatory T cells cytokines, are
responsible for restoring the immunotolerance to bee venom. VIT should be performed
under medical care due to the possibility occurring due to an allergic reaction [56,58,69]. A study found that in some cases, during the administration of maintenance immunother-
apy, a patient had an allergic reaction. Anaphylactic shock in the 31-year-old woman in her
premenstrual period appeared despite the lack of complications in the course of initiating
immunotherapy and the first six maintenance doses. Anaphylactic shock was most likely
influenced by the premenstrual period and withdrawal of oral contraceptives [70]. Molecules 2021, 26, 148 10 of 13 It is worth drawing attention to bee venom therapy in patients treated with angiotensin
converting enzyme inhibitors and β-blockers. It was confirmed that angiotensin converting
enzyme inhibitors and β-blockers used in therapy increase the risk of large allergic reac-
tion after stinging. Therefore, before the therapy of bee venom, the problem of potential
interactions between bee venom with medications taken should be considered [71]. Due to the allergic reaction to bee venom in patients, before proper bee venom therapy
a basic diagnostic could be applied such as: skin test as a prick test and assays specific IgG4
levels. However, in individual situations, in case the negative results of prick test or specific
IgG4 levels additional tests as component-resolved diagnosis, cell-based assays can be used. Moreover, in risk assessment systemic allergy reactions (SAR) tryptase assays should be
conducted. Currently, in the diagnosis of allergy to bee venom there are conducted assays
of concentration of specific IgG4 for phospholipase A2 and lymphocytes B and T [54]. 6. Conclusions The presented studies reveal that bee venom treatment influences all the phases of the
healing process. Bee venom can be applied subcutaneously or topically in the area of wounds. Based on research conducted on animal models, the bee venom seems to be promising
the potential repair agent of wounds in diabetic patients. Bee venom stimulates insulin
release and lowers glycemia in the animal model. Moreover, bee venom inhibits prolonged
inflammation, reduces levels of proinflammatory cytokines and reduces oxidative stress. This
plays an important role in restoring delayed wound healing as it decreases caspase-3, caspase-
8 and caspase-9 activity. Additionally, treatment with BV regulates the level of TGF-β, which
is responsible for keratinocytes migration. This also restores the VEGF level which is a crucial
factor for tissue repair. p
Bee venom restores the expression of ATF-3 and iNOS, which play a crucial role in
damaged tissue remodeling. The presented molecular mechanisms confirm that bee venom
accelerates the wound healing process. In the light of the information presented, it will be
justified to conduct research on the possibility of using a regenerative system containing a
polymer with bee venom, similarly to the case of a polymer dressing with propolis [72,73]. Further studies are needed to evaluate the advantages and disadvantages of treating wounds
with bee venom. Despite that the apitoxin may cause an allergic effect, therapy with bee
venom brings a satisfactory effect, and therefore a venom dressing should be developed in
order to minimize the systemic side effects. The therapeutic value of bee venom is huge
due to antioxidant, antimicrobial, antifungal, antiviral, anti-inflammatory and analgesic
properties, so it might be an attractive therapeutic agent in the treatment of wounds. Author Contributions: The authors of the publication have read and agreed to this version of the
paper and have contributed to the conceptualization and writing of this review. Funding: This review received no external funding. Acknowledgments: The authors would like to thank the Medical University of Silesia in Katowice
for their financial support. Conflicts of Interest: The authors declare no conflict of interest. 8.
Koh, T.J.; DiPietro, L.A. Inflammation and wound healing: The role of the macrophage. Expert Rev. Molecul 1.
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English
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A NEW SPECIES OF THE GENUS MALACONOTHRUS (ACARI, ORIBATIDA, MALACONOTHRIDAE) FROM ECUADOR
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Ecologica Montenegrina
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Introduction This work is a part of my continuing study of the oribatid mite fauna of Ecuador (e.g. Ermilov & Kalúz
2012; Ermilov et al. 2014; 2016). The present study includes data on Malaconothridae (Acari, Oribatida). This family comprises two genera and more than 170 species, which has a cosmopolitan distribution
collectively (Colloff & Cameron 2013; Subías 2004, online version 2016). During taxonomic study of the Ecuadorian malaconothrids, I found one new species belonging to the
genus Malaconothrus Berlese, 1904. The main goal of the paper is to describe and illustrate this species
under the name M. paraweigmanni Ermilov sp. nov. ISSN 2336-9744 (online) | ISSN 2337-0173 (print)
The journal is available on line at www.biotaxa.org/em
https://zoobank.org/urn:lsid:zoobank.org:pub:493BA1D0-298F-4933-9F77-754D6DC41408
Research Artic ISSN 2336-9744 (online) | ISSN 2337-0173 (print)
The journal is available on line at www.biotaxa.org/em
https://zoobank.org/urn:lsid:zoobank.org:pub:493BA1D0-298F-4933-9F77-754D6DC41408
Research Artic Research Article Research Article https://zoobank.org/urn:lsid:zoobank.org:pub:493BA1D0-298F-4933-9F77-754D6DC41408 Abstract Abstract
A new species of oribatid mites of the genus Malaconothrus (Oribatida, Malaconothridae) is described from forest litter
in Ecuador. Malaconothrus paraweigmanni Ermilov sp. nov. differs from the most similar species, M. weigmanni
Colloff, 2013 by the smaller body size, barbed notogastral setae and comparatively long anal setae. Key words: oribatid mite, Malaconothrus, new species, systematics, morphology, Neotropical region. SERGEY G. ERMILOV Tyumen State University, Semakova str., 10, 625003 Tyumen, Russia. E-mail: ermilovacari@yandex.ru Tyumen State University, Semakova str., 10, 625003 Tyumen, Russia. E-mail: ermilovacari@yandex.ru Received: 10 October 2016 │ Accepted by V. Pešić: 8 November 2016 │ Published online: 10 Novemb A new species of the genus Malaconothrus (Acari, Oribatida,
Malaconothridae) from Ecuador SERGEY G. ERMILOV Material and Methods Material. The collection locality and habitat are given in the "Material examined" section. Methods. Specimens were mounted in lactic acid on temporary cavity slides for measurement and
illustration. Body length was measured in lateral view, from the tip of the rostrum to the posterior edge of the
ventral plate. Notogastral width refers to the maximum width in dorsal aspect. All body measurements are
presented in micrometers. Formulas for leg setation are given in parentheses according to the sequence
trochanter–femur–genu–tibia–tarsus (famulus included). Formulas for leg solenidia are given in square
brackets according to the sequence genu–tibia–tarsus. Morphological terminology used in this paper follows that of F. Grandjean: see Travé & Vachon
(1975) for references, Norton (1977) for leg setal nomenclature, and Norton & Behan-Pelletier (2009), for
overview. Drawings were made with a camera lucida using a Carl Zeiss transmission light microscope
―Axioskop-2 Plus‖. Images were obtained with an AxioCam ICc3 camera using a Carl Zeiss transmission
light microscope ―Axio Lab.A1‖. Ecol. Mont., 9, 2016, 31-37 Malaconothrus paraweigmanni Ermilov sp. nov.
(Figs 1–18) Diagnosis. Body size: 381–398 × 190–207. Body surface porose, covered by granular cerotegument,
opisthosoma covered by reticulate cerotegument. Prodorsal setae setiform, long, barbed, interlamellar setae
considerably longer than exobothridial setae ex1. Lateral parts of prodorsum and notogaster with scale-like
ledges. Notogastral ridges absent. Notogastral setae long, setiform, barbed; c1, d1, e1 longer than other setae. Epimeral, genital and anal setae thin, smooth. Four pairs of genital setae present, anal setae well-developed. Adanal setae setiform, barbed, ad1 longer than ad2; ad3 shortest. Legs tridactylous. Figures 1–4. Malaconothrus paraweigmanni sp. nov.: 1 — dorsal view; 2 — subcapitulum, ventral view; 3 — palp,
right, antiaxial view; 4 — chelicera, left, paraxial view. Scale bar 50 μm (1), 20 μm (2, 4), 10 μm (3). Figures 1–4. Malaconothrus paraweigmanni sp. nov.: 1 — dorsal view; 2 — subcapitulum, ventral view; 3 — palp,
right, antiaxial view; 4 — chelicera, left, paraxial view. Scale bar 50 μm (1), 20 μm (2, 4), 10 μm (3). 32 ERMILOV ERMILOV E
l M
t 9 2016 31 37
33
Figures 5–8. Malaconothrus paraweigmanni sp. nov.: 5 — ventral view (gnathosoma and legs except trochanters not
shown); 6 — lateral view (gnathosoma and legs except basal parts not shown); 7 — posterior view; 8 — famulus,
solenidia and setae ft on leg tarsus I. Scale bar 50 μm (5–7), 10 μm (8). Figures 5–8. Malaconothrus paraweigmanni sp. nov.: 5 — ventral view (gnathosoma and legs except trochanters not
shown); 6 — lateral view (gnathosoma and legs except basal parts not shown); 7 — posterior view; 8 — famulus,
solenidia and setae ft on leg tarsus I. Scale bar 50 μm (5–7), 10 μm (8). Ecol. Mont., 9, 2016, 31-37 33 A NEW SPECIES OF MALACONOTHRUS FROM ECUADOR igures 9–12. Malaconothrus paraweigmanni sp. nov.: 9 — tibia and tarsus of leg I, right, antiaxial view; 10 — tibia
nd tarsus of leg II, right, antiaxial view; 11 — genu, tibia and tarsus of leg III, left, antiaxial view; 12 — genu, tibia
nd tarsus of leg IV, left, antiaxial view. Scale bar 20 μm. Figures 9–12. Malaconothrus paraweigmanni sp. nov.: 9 — tibia and tarsus of leg I, right, antiaxial view; 10 — tibia
and tarsus of leg II, right, antiaxial view; 11 — genu, tibia and tarsus of leg III, left, antiaxial view; 12 — genu, tibia
and tarsus of leg IV, left, antiaxial view. Malaconothrus paraweigmanni Ermilov sp. nov.
(Figs 1–18) Scale bar 20 μm. Figures 9–12. Malaconothrus paraweigmanni sp. nov.: 9 — tibia and tarsus of leg I, right, antiaxial view; 10 — tibia
and tarsus of leg II, right, antiaxial view; 11 — genu, tibia and tarsus of leg III, left, antiaxial view; 12 — genu, tibia
and tarsus of leg IV, left, antiaxial view. Scale bar 20 μm. 34 ERMILOV Description Four pairs of genital setae (g1–g4, 20) setiform, smooth, g1–g3 directed
backwards, g4 – forwards. One pair of anal setae (an, 24) well developed, thin, smooth. Three pairs of adanal
setae (ad1, 61–65, ad2, 49–53, ad3, 36–41) setiform, barbed. Lyrifissures ian and iad distinct. (
1,
,
2,
,
3,
)
,
y
Legs (Figs 6, 8, 9–12, 18). Tridactylous. Median claw thicker than laterals, all barbed dorsally. Formulae of leg setation and solenidia: I (1–5–3–4–11) [1–1–3], II (1–5–3–4–10) [1–1–1], III (2–3–2–2–10)
[0–1–0], IV (0–2–2–2–10) [0–0–0]; homology of setae and solenidia indicated in Table 1. Setae p, u, a, s, pv
and ft” (on tarsi I, II) spiniform, ft’ (on tarsi I, II), ft (on tarsi III), ft” (on tarsi IV) thickened, tc thin, very
long, famuli tubercle-like, inserted near ω3, other setae setiform. All solenidia simple, blunt-ended. Table 1. Leg setation and solenidia of Malaconothrus paraweigmanni Ermilov sp. nov. Table 1. Leg setation and solenidia of Malaconothrus paraweigmanni Ermilov sp. nov. Roman letters refer to normal setae (ɛ – famulus), Greek letters refer to solenidia, dφ and dσ – seta and solenidion
coupled. One apostrophe (') marks setae on anterior and double apostrophe (") setae on posterior side of the given leg
segment. Parentheses refer to a pair of setae. Leg
Trochanter
Femur
Genu
Tibia
Tarsus
I
v'
d, (l), bv'', v''
dσ, (l)
dφ, (l), v'
(ft), (tc), (p), (u), (a), ɛ, ω1, ω2, ω3
II
v'
d, (l), bv'', v''
dσ, (l)
dφ, (l), v'
(ft), (tc), (p), (u), (a), ω
III
l', v'
d, l', ev'
d, v’
dφ, v'
(ft), (tc), (p), (u), (a)
IV
-
d, ev'
d, v’
d, v'
ft'', (tc), (p), (u), (a), s Roman letters refer to normal setae (ɛ – famulus), Greek letters refer to solenidia, dφ and dσ – seta and solenidion
coupled. One apostrophe (') marks setae on anterior and double apostrophe (") setae on posterior side of the given leg
segment. Parentheses refer to a pair of setae. Material examined. Holotype (female) and one paratype (female): Ecuador, 0°25'8.04''S,
79°0'14.04''W, Reserva de Bosque Integral Otonga, near San Francisco de las Pampas, 2000–2200 m a.s.l.,
sifted litter from forest, 7.XI.1996 (G. Onore). Type deposition. The holotype is deposited in the collection of the Senckenberg Institute, Görlitz,
Germany; one paratype is deposited in the collection of the Tyumen State University Museum of Zoology,
Tyumen, Russia. Type deposition. ERMILOV ERMILOV
Ecol. Mont., 9, 2016, 31-37 35
Figures 13–18. Malaconothrus paraweigmanni sp. nov., microscope images: 13 — granulate cerotegument in median
part of prodorsum; 14 — reticulate cerotegument in median part of notogaster; 15 — notogastral lateral ledge; 16 —
basal part of notogastral seta d2; 17 — palp and part of subcapitulum; 18 — claws of tarsi III and IV. Scale bar 10 μm
(13, 16), 20 μm (14, 15, 17, 18). Figures 13–18. Malaconothrus paraweigmanni sp. nov., microscope images: 13 — granulate cerotegument in median
part of prodorsum; 14 — reticulate cerotegument in median part of notogaster; 15 — notogastral lateral ledge; 16 —
basal part of notogastral seta d2; 17 — palp and part of subcapitulum; 18 — claws of tarsi III and IV. Scale bar 10 μm
(13, 16), 20 μm (14, 15, 17, 18). Ecol. Mont., 9, 2016, 31-37 35 Description Measurements. Body length: 398 (holotype, female), 381 (one paratype, female); notogaster width:
190 (holotype), 207 (one paratype). Integument (Figs 1, 5–7, 13, 14). Body color light grey. Body surface finely porose (clearly visible
under high magnification) and covered by granular cerotegument (granules rounded or slightly elongated,
their diameter or length up to 4). Opisthosoma with reticulate cerotegument. Prodorsum (Figs 1, 6). Rostrum rounded. Lateral carinae well developed, reach to insertions of
rostral setae, translamellar ridge absent between these setae. Each carina with medially-directed transverse
extension (located laterally to lamellar setae). Rostral (ro, 36–41), lamellar (le, 49–53), interlamellar (in, 90–
94) and exobothridial (ex1, 41–45) setae setiform, barbed, ro directed forwards. Exobothridial setae ex2
represented by alveoli. Lateral parts of prodorsum (located laterally to ex2) with scale-like ledges (Ld). p
y
p
p
(
y
)
g
(
)
Notogaster (Figs 1, 5–7, 15, 16). Anterior margin slightly convex medially. Lateral parts of
notogaster with scale-like ledges. Posterior part with two slightly visible concavities. Notogastral ridges
absent. Notogastral setae setiform, barbed; c1, d1, e1 (90–94) longer than c3, d2, cp (73–77), c2, e2, f2, h1, h2, h3
(61–65) and p1, p2, p3 (49–57). Lyrifissures well visible, ia located posteriorly to c2, im – posteriorly to d2, ip
– laterally to h1, ips and ih – on lateral parts of notogaster. y
p
p
g
Gnathosoma (Figs 2–4, 17). Subcapitulum slightly longer than wide: 90–98 × 82–90. Subcapitular
setae (h, 8; m, 16; a, 20) setiform, thin, smooth. Adoral setae (or1, or2, or3, 4) minute, smooth. Palps (41–45)
with setation 0–0–1–3–9(+ω). Setae cm setiform, longest on tarsi, other setae spiniform, from these, acm
shortest and thinnest. Postpalpal setae (ep, 8–10) thin, erect, barbed. Solenidia (6) thickened, blunt-ended. Chelicerae (86–90) with two setae, cha (4–6) spiniform, smooth, chb (8) fusiform, barbed mediodistally. Trägårdh’s organs not visible. Epimeral and lateral podosomal regions (Figs 5, 6). Epimeral plates I separated medially, plates II
and IV separated partially anteromedially, while plates III separated partially posteromedially. Epimeral setal
formula: 3–1–3–3. Epimeral setae setiform, thin, smooth, 3a, 3b, 4c (20–24) longer than other setae (4–6). Anogenital region (Figs 5–7). Four pairs of genital setae (g1–g4, 20) setiform, smooth, g1–g3 directed
backwards, g4 – forwards. One pair of anal setae (an, 24) well developed, thin, smooth. Three pairs of adanal
setae (ad1, 61–65, ad2, 49–53, ad3, 36–41) setiform, barbed. Lyrifissures ian and iad distinct. Anogenital region (Figs 5–7). y
Etymology. The specific name paraweigmanni refers to the similarity between the new species and
Malaconothrus weigmanni Colloff, 2013. ERMILOV Remarks. The new species is most similar to Malaconothrus weigmanni Colloff, 2013 from Papua
New Guinea in the main morphological traits (notogaster with reticulate cerotegument; notogastral ridges
absent; majority of notogastral setae long; interlamellar setae considerably longer than exobothridial setae
ex1; adanal setae ad1 longer than ad2, ad3 shortest; tridactylous legs). However, the present new species
differs from M. weigmanni by the smaller body size (381–398 × 190–207 vs. 444–450 × 246–253),
notogastral setae barbed (vs. smooth) and comparatively long anal setae (vs. minute). Acknowledgements I cordially thank Prof. Dr. Badamdorj Bayartogtokh (National University of Mongolia, Ulaanbaatar,
Mongolia) for the valuable comments; Dr. Giovanni Onore (Pontifical Catholic University, Ecuador) for his
help with collecting Ecuadorian oribatid mites; and Dr. Stanislav Kalúz (Institute of Zoology, Slovak
Academy of Sciences, Bratislava, Slovakia) for sending me the oribatid mite material. Description The holotype is deposited in the collection of the Senckenberg Institute, Görlitz,
Germany; one paratype is deposited in the collection of the Tyumen State University Museum of Zoology,
Tyumen, Russia. y
,
Etymology. The specific name paraweigmanni refers to the similarity between the new species and
Malaconothrus weigmanni Colloff, 2013. Etymology. The specific name paraweigmanni refers to the similarity between the new species and
Malaconothrus weigmanni Colloff, 2013. 36 y
pp
p
p
Vachon, M. (1975) François Grandjean. 1882–1975 (Notice biographique et bibliographique
rologia, 17 (1), 1–19. References Berlese, A. (1904) Acari nuovi. Manipulus III. Redia, 2, 10–32. p
Colloff, M.J. (2013) Species-groups and biogeography of the oribatid mite family Malaconothridae
(Oribatida: Malaconothroidea), with new species from the south-western Pacific region. Zootaxa,
3722 (4), 401–438. Colloff, M.J. (2013) Species-groups and biogeography of the oribatid mite family Malaconothridae
(Oribatida: Malaconothroidea), with new species from the south-western Pacific region. Zootaxa,
3722 (4), 401–438. Colloff, M.J. & Cameron, S.L. (2013) A phylogenetic analysis and taxonomic revision of the oribatid mite
family Malaconothridae (Acari: Oribatida), with new species of Tyrphonothrus and Malaconothrus
from Australia. Zootaxa, 3681 (4), 301–346. Ermilov, S.G., Friedrich, S. & Kalúz, S. (2016) A new species of Neoribates (Neoribates) (Acari: Oribatida:
Parakalummidae) with key to the Neotropical species of the subgenus. Biologia, 71 (6), 673–677. Ermilov, S.G., Friedrich, S. & Kalúz, S. (2016) A new species of Neoribates (Neoribates) (Acari: Oribatida:
Parakalummidae) with key to the Neotropical species of the subgenus. Biologia, 71 (6), 673–677. Ermilov, S.G. & Kalúz, S. (2012) Two new species of oribatid mites (Acari: Oribatida) from Ecuador. Systematic & Applied Acarology, 17 (3), 269–280. Ermilov, S.G. & Kalúz, S. (2012) Two new species of oribatid mites (Acari: Oribatida) from Ecuador. Systematic & Applied Acarology, 17 (3), 269–280. Ermilov, S.G., Sandmann, D., Marian, F. & Maraun, M. (2014) New species of oribatid mites of the genera
Hermannobates and Rhynchoribates (Acari: Oribatida: Hermanniellidae, Rhynchoribatidae) from
Ecuador. Systematic & Applied Acarology, 19 (3), 313–321. Norton, R.A. (1977) A review of F. Grandjean's system of leg chaetotaxy in the Oribatei (Acari) and its
application to the family Damaeidae. In: Dindal, D.L. (Editor), Biology of oribatid mites. SUNY
College of Environmental Science and Forestry, Syracuse, pp. 33–61. g
y
y
pp
Norton, R.A. & Behan-Pelletier, V.M. (2009) Oribatida. Chapter 15. In: Krantz, G.W. & Walter, D.E. (Eds.), A Manual of Acarology (TX). Lubbock, Texas Tech University Press, pp. 430–564. Subías, L.S. (2004) Listado sistemático, sinonímico y biogeográfico de los ácaros oribátidos (Acariformes:
Oribatida) del mundo (excepto fósiles). Graellsia, 60 (número extraordinario), 3–305. Online version
accessed in February 2016, 593 pp.; http://escalera.bio.ucm.es/usuarios/bba/cont/docs/RO_1.pdf 37 Ecol. Mont., 9, 2016, 31-37 37
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English
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Cox Regression of Factors Associated with the Onset of Chronic Kidney Disease in HIV/AIDS Patients in Albert Luthuli Municipality of South Africa
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International journal of nephrology and kidney failure
| 2,021
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cc-by
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Cox regression of factors associated with the onset
of chronic kidney disease in HIV/AIDS patients in
Albert Luthuli Municipality of South Africa Pepukai Bengura ( 31659322@mylife.unisa.ac.za )
University of South Africa
Principal Ndlovu
University of South Africa
Mulalo Annah Managa
University of South Africa Pepukai Bengura ( 31659322@mylife.unisa.ac.za ) Pepukai Bengura ( 3165932
University of South Africa
Principal Ndlovu
University of South Africa
Mulalo Annah Managa
University of South Africa Cox regression of factors associated with the onset of chronic kidney disease
in HIV/AIDS patients in Albert Luthuli Municipality of South Africa
Pepukai Bengura1*, Principal Ndlovu2 and Mulalo Annah Managa2 Pepukai Bengura1*, Principal Ndlovu2 and Mulalo Annah Managa2 Research Article Keywords: HIV/AIDS, Renal disease, Glomerular ¦ltration rate, ART, Prevalence, risk factors, Cox
regression, Kaplan-Meier estimator, hazard ratio
Posted Date: August 25th, 2020
DOI: https://doi.org/10.21203/rs.3.rs-61124/v2
License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Posted Date: August 25th, 2020 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Abstract Background: Current research indicates that chronic kidney disease is a global problem
which poses a major health threat to people of poor countries with HIV/AIDS and on
antiretroviral treatment. In this study, the prevalence of chronic kidney disease and the
factors associated with it were investigated among the HIV/AIDS patients in a rural
community of South Africa. Methods: A cohort of HIV/AIDS patients was retrospectively followed from 2010 to 2017
until chronic kidney disease was diagnosed or until the end of the observation period at two
hospitals (Carolina and Embhuleni). Patient information was obtained from the routine
hospitals’ records, and the data were analysed using Cox regression and survival analysis
(Kaplan-Meier hazard functions and ratios, and log-rank tests) methods. Results: Out of a random sample of 320 HIV/AIDS patients, 51 patients (15.9%) had
chronic kidney disease. The factors associated with chronic kidney disease were: gender (p-
value=0.0356), age (p-value=0.00077), baseline creatinine (p-value=0.00253), follow-up
alanine transaminase (p-value=0.0152), ART treatments (p-value<0.00193) and hospital (p-
value=0.00258). Discussion: Whilst antiretroviral treatment is associated with some improvement in
virology and immunology in HIV-infected patients, research is still needed for the
assessment of the impact of ART and other risk factors on renal function in marginalised
communities in Africa. Conclusion: The research findings on HIV/AIDS patients in Albert Luthuli Municipality
concurred with several previous research findings on risk factors to CKD. The expected
action to alleviate the health threat due to CKD in South Africa is to educate the nation on
prevention, early detection and on the management of the disease. Keywords: HIV/AIDS, Renal disease, Glomerular filtration rate, ART, Prevalence, Risk
factors, Cox regression, Kaplan-Meier estimator, Hazard ratio. 1 Background HIV/AIDS has been a major health problem worldwide for more than three decades now. According to the WHO global statistics: since the beginning of the epidemic, 75 million people
have been infected with the HIV virus, about 32 million people have died of HIV and globally,
37.9 million people were living with HIV at the end of 2018. According to United Nations
Agency for International Development (UNAIDS) AIDSinfo (2018), South Africa has the
biggest and most high-profile HIV epidemic in the world, with estimated 7.7 million living
with HIV in 2018. South Africa’s Mpumalanga province has the second highest HIV
prevalence rate after KwaZulu-Natal province. Gert Sibande district which is in Mpumalanga
province is leading all districts in the country with 46.1% HIV prevalence rate (Motsoaledi,
2013). Gert Sibande district has Albert Luthuli as one of its municipalities whose HIV
prevalence stood at 43.2% (Nkosi, 2017). Human Immunodeficiency virus (HIV) affects every organ system in the body by direct
damage or by rendering the host susceptible to opportunistic infections (Mekuria et al., 2016). The commonest sites of infection include kidneys. A kidney contains millions of nephrons
which are made up of blood vessels called glomeruli. The function of kidneys is to filter blood
of waste products such as creatinine and urea and extra water which is removed in form of
urine. Chronic kidney disease (CKD) is a slow and prolonged failure of the kidneys to filter
impurities from the blood. The progression of the kidney damage is marked by the rise in
creatinine and urea whose evaluation in serum helps to assess Glomerular Filtration Rate
(GFR). CKD poses a major health threat to people living in poor countries, especially when it
is combined with HIV, antiretroviral treatment (ART) or communicable and non-
communicable diseases (Glaser et al., 2016). According to November et al. (2017), CKD is a global problem with about 10% of the world’s
population suffering from the disease. Ekrikpo et al. (2018) in agreement with Eneyew et al. (2016) and Crum-Cianflone et al. (2010) pointed out that the prevalence of renal dysfunction
is projected to increase as the life expectancy of the HIV-infected individuals increases due to
drug therapy. This study which focuses on rural population was motivated by among other
factors by the fact that 93% of the studies on CKD in sub-Saharan Africa were done in urban
settings (Stanifer et al., 2016). Study design and setting A cohort of HIV+ terminal ill patients was retrospectively followed from 2010 to 2017 until
CKD was diagnosed or until the end of the observation period at the two hospitals (Carolina
and Embhuleni) in Albert Luthuli Local Municipality. This is a South African municipality
situated in the Gert Sibande District of Mpumalanga Province. Carolina and Embhuleni district
hospitals offer comprehensive health care services which include HIV/AIDS and tuberculosis
(TB) treatments to the surrounding communities in Albert Luthuli municipality. These
hospitals are accredited antiretroviral (ARV) treatment initiation and on-going treatment sites. Both Hospitals serve mostly the rural population in Albert Luthuli Municipality. The target
population which was used as a sampling frame included all HIV/AIDS patients (≥16 years)
admitted and started ART treatment in the two hospitals within the three-month period from
the 1st of January to the 31st of March 2010. The variables which form part of the routine
hospital records in Albert Luthuli municipality were used in this study and are described as
follows. CKD status is the dependent variable for the study. It was recorded in terms of CKD
status (yes, no) and time until its onset. The categorical independent variables were gender,
hospital (Carolina, Embhuleni), WHO stage (1, 2, 3, 4), marital status ( single, married, staying
together,
widowed/separated/divorced),
treatments
(regimen
1)(NVP+D4T+3TC,
EFV+D4T+3TC, EFV+AZT+3TC and EFV+3TC+TDF), ART adherence (poor, fair, good),
transferred from hospital (yes, no) and lost to follow-up (yes, no). The continuous independent
variables for the study which except age were classified into baseline and follow-up variables
were mass, CD4 cell count, haemoglobin, lymphocyte, white blood cell count, viral load,
creatinine, total protein, sodium and alanine transaminase. Diabetes and hypertension were
excluded because their records were found in very few patient files. HIV/AIDS patients with
missing essential records such as baseline creatinine, gender and date of birth or age were
excluded from the study. A cohort of HIV+ terminal ill patients was retrospectively followed from 2010 to 2017 until
CKD was diagnosed or until the end of the observation period at the two hospitals (Carolina
and Embhuleni) in Albert Luthuli Local Municipality. This is a South African municipality
situated in the Gert Sibande District of Mpumalanga Province. Carolina and Embhuleni district
hospitals offer comprehensive health care services which include HIV/AIDS and tuberculosis
(TB) treatments to the surrounding communities in Albert Luthuli municipality. These
hospitals are accredited antiretroviral (ARV) treatment initiation and on-going treatment sites. Background 2 2 The objective of this study is to use Cox regression method to determine the prevalence of
CKD and to identify the risk factors associated with CKD among HIV/AIDS patients on ART
by using data from Albert Luthuli municipality hospitals in South Africa. 3 3 Clinical determination of CKD status The patient’s CKD status was determined by referring to the South African National Health
Laboratory Service report on Chemical pathology. CKD status was determined from the
estimates of the Glomeruli Filtration Rate (GFR) using Modification of Diet in Renal Disease
(MDRD) formula (Iseki, 2008; Omuse et al., 2017). The renal impairment in this study was
classified according to the National Kidney Foundation clinical practice guideline with
estimated GFR values ≥ 90ml/min/1.73𝑚2, 60–89 ml/min/1.73𝑚2, 30–59 ml/min/1.73𝑚2,15-
29ml/min/1.73𝑚2 and < 15 ml/min/1.73𝑚2 interpreted as normal, mild, moderate, severe and
kidney failure. Iseki (2008) categorized a patient as having CKD if their GFR was less than
60ml/min/1.73𝑚2 for more than 3 months. The estimates of patient Glomerular Filtration Rate
(GFR) were determined using the equation by Omuse et al. (2017). Ethical considerations The ethical approval for this study was granted by UNISA Ethics Review Committee with the
approval number being 2017/SSR ERC/005. The permission to conduct the study at Carolina
and Embhuleni hospitals was obtained from Mpumalanga Department of Health with the
permission number being MP_201708_013. All data related to the patients were handled with
utmost confidentiality in all the stages of the research. In addition, no reference to an individual
respondent was made as all results were handled in aggregate format. The electronic documents
carrying confidential information on patients are all protected by some encryption and will be
destroyed as per research policy. Sample size and sampling procedure Sample size was determined by using sample size calculation formula for survival analysis by
considering the following statistical assumptions on HIV-infected patients: an average of 13%
prevalence rate of renal insufficiency among ART naïve patients (Kaze et al., 2018), 5%
precision or margin error, 95% level of confidence interval and 0.45 loss (Damtew et al., 2015). The sample size was calculated using the formula (Eneyew et al., 2016), 𝑁=
𝑍2𝑝(1−𝑝)
𝛼2
, where The sample size was calculated using the formula (Eneyew et al., 2016), 𝑁=
𝑍2𝑝(1−𝑝)
𝛼2
, where 4 N = sample size, Z = 1.96 (critical value at 95% level of confidence), p = proportion of renal
insufficiency and 𝛼 = type-1 error (0.05) was 320. The estimated total sample size was
proportionally and randomly allocated to the two study sites (Embhuleni and Carolina hospitals
with proportions of 78% and 22% respectively) and according to the age and gender
proportions (39.4% male) as per population proportions. ℎ𝑖(𝑡) = ℎ0(𝑡)𝑒𝑥𝑝{𝑿𝑖
𝑇𝜷= ∑
𝛽𝑗𝑋𝑖𝑗
𝑝
𝑗=1
}, (1) Statistical methods Let 𝑿𝑖
𝑇= (𝑋𝑖1, 𝑋𝑖2, … , 𝑋𝑖𝑝) be a p-dimensional vector of the values of the covariates associated
with the ith patient. Then, the Cox proportional hazards regression model is (Klein &
Moeschberger, 2003; Etikan & Babatope, 2018) ℎ𝑖(𝑡) = ℎ0(𝑡)𝑒𝑥𝑝{𝑿𝑖
𝑇𝜷= ∑
𝛽𝑗𝑋𝑖𝑗
𝑝
𝑗=1
}, (1) (1) 5 5 where 𝜷𝑇=(𝛽1, 𝛽2, … , 𝛽𝑝) is a p-dimensional vector of regression coefficients to be estimated
from the data, and ℎ0(𝑡) is the unspecified baseline hazard function that does not have to be
estimated. The hazard model in model (1) makes no assumptions about the shape of the hazard
function over time. A hazard function could be constant, increasing, decreasing, or it could be
a combination of two or three of these graph trends. Model (1) can be written in terms of the survivor function (Kleinbaum & Klein, 2012)
𝑆𝑖(𝑡) = 𝑆0(𝑡)𝑒𝑥𝑝{𝑿𝑖
𝑇𝜷}. (2) Model (1) can be written in terms of the survivor function (Kleinbaum & Klein, 2012) (2) 𝑆𝑖(𝑡) = 𝑆0(𝑡)𝑒𝑥𝑝{𝑿𝑖
𝑇𝜷}. The assumptions of model (1) which may be violated by the data are: (i) the covariates 𝑿𝑖
𝑇 do
not vary with time and hence the hazard rate ratios of pairs of patients do not vary with time;
(ii) censoring and survival are independent; and (iii) the log hazard rate is indeed a linear
function of the covariates. Model (1) was fitted to the data using coxph function in R Version
3.5.1 to determine the risk factors associated with CKD. Firstly, the variables in Table 1 were
tested one by one and variables with p-values < 0.25 were considered in the final Cox
regression analysis. The final multivariate multiple Cox regression analysis retained in the
model only variables with p-values ≤ 0.05. Regarding model selection, Hosmer et al. (2008)
suggested the consideration of issues such as clinical importance and adjustment for
confounding, as well as statistical significance. Let 0 < 𝑡1 < 𝑡2 < 𝑡3 < … < 𝑡𝑘< ∞ be 𝑘 observed times to the onset of CKD in HIV/AIDS
patients during the observation period; 𝑑𝑖𝑗 (𝑖= 1,2, … , 𝑟; 𝑗= 1,2, … , 𝑘) be the respective
number of HIV/AIDS patients in the ith group with CKD at each of these times; and 𝑛𝑖𝑗 be the
corresponding number of remaining patients in the cohort at the respective times. Prevalence of CKD From the sample of 320 HIV/AIDS patients: 51 patients (15.9%); 27 males (50.9%); and 46
patients (88.7%) from Embhuleni hospital had CKD. A follow-up of the patients with CKD
gave rise to the following vital outcomes: 23.5% were lost to follow-up; 41.2% were transferred
from the original hospitals and 35,3% remained attached at the hospitals. Out of 157 (49.1%)
HIV/AIDS who transferred from the hospitals, 13.4% had CKD while from 84 (26.3%) who
were lost to follow-up, 14.3% had CKD. Statistical methods Then Kaplan-
Meier (KM) estimate of the survivor function 𝑆𝑖(𝑡) (𝑖= 1,2, … , 𝑟) when times to the onset of
CKD in patients in the ith group are tied is (Etikan et al., 2017) 𝑆̂𝑖(𝑡) = ∏
1 −(
𝑑𝑖𝑗
𝑛𝑖𝑗)
𝑗|𝑡𝑗≤𝑡
, 𝑖= 1,2, … , 𝑟 1 −(
𝑑𝑖𝑗
𝑛𝑖𝑗)
𝑡𝑗≤𝑡
, 𝑖= 1,2, … , 𝑟 (3) (3) In this study, the 𝑟 groups were the levels of the significant categorical covariates or categorized
continuous covariates retained in the Cox regression analysis of CKD. The log-rank test was
used to test the null hypothesis (Etikan et al., 2017) 0: 𝑆1(𝑡) = 𝑆2(𝑡) = ⋯= 𝑆𝑟(𝑡) ≡ ℎ1(𝑡) = ℎ2(𝑡) = ⋯= ℎ𝑟(𝑡) (4) for 𝑡≥0, where ℎ𝑖(𝑡) is the hazard function of the ith group patients. Data for the study were
recorded on research tools and then captured on Microsoft excel database and checked against
original records by two competent individuals. Data was analysed using R Version 3.5.1. Cox model fit for risk factors associated with the onset of CKD The function ‘coxph()’ in R fits a Cox proportional hazards regression model. Table 1 shows
the results that were obtained from the fitted Cox regression model. A test for proportional
hazards for each covariate in Table 1 along with a global test for the model was done in R with
‘cox.zph( )’ function. The likelihood-ratio (p-value = 0.0000), Wald (p-value = 0.0000), and
Score (log rank)(p-value = 0.0000) statistics which test the omnibus null hypothesis that all of
the 𝛽𝑠 are zero are all asymptotically equivalent and are in close agreement in soundly rejecting
the omnibus null hypothesis. The model gives a high concordance of 81.6% which implies a
high degree of discrimination between patients with and patients without CKD. There is no
evidence of non-proportional hazards for all the covariates since p-value > 0.05 for each
covariate in the model. The global chi-square test (p-value = 0.5486) is non-significant, this
implies that the assumption of proportionality is not violated. The good model fit statistics
indicate that the model (Table 1) is ideal for all the variables. Table 1 which contains the results from Cox regression analysis of the data shows that the
significant factors which are positively associated with CKD hazard at 0.05 level and have
significant effects to CKD are: age (p-value = 0.0008), hospital (Embhuleni relative to
Carolina) (p-value = 0.0026) and baseline creatinine (p-value = 0.0025). The description that
follows for each covariate assumes that other covariates in the model are held constant. The
estimated 95% confidence interval of the hazard ratio for Age is (1.0232, 1.0908), which 7 implies that for every one-year increase in the age of an HIV/AIDS patient results CKD hazard
increase by a factor of 1.0565 or by about 5.7% and which is as much as 9.1% low to 2.3%
lower. The estimated 95% confidence interval of the hazard ratio for baseline creatinine is
(1.0060, 1.0286), which implies that for every one unit increase in the baseline creatinine level
of an HIV/AIDS patient, the CKD hazard rate is as much as 2.9% low to 0.6% lower. Furthermore, Embhuleni hospital has hazard to CKD which is about 4.4 times the hazard of
Carolina hospital to CKD and this hazard may be as high as 11.5 times and as low as 1.7 times. Cox model fit for risk factors associated with the onset of CKD On the other hand, the significant factors which are negatively associated with CKD hazard at
0.05 level are: gender (female relative to male) (p-value = 0.0356), follow-up alanine
transaminase (p-value = 0.0152), Treatment (EFV+D4T+3TC relative to NVP+D4T+3TC) (p-
value = 0.0003), Treatment (EFV+AZT+3TC relative to NVP+D4T+3TC) (p-value = 0.0019),
Treatment (EFV+3TC+TDF relative to NVP+D4T+3TC) (p-value = 0.0001) and Treatment
(NVP+3TC+TDF relative to NVP+D4T+3TC) (p-value = 0.0010). Explanations of the results
which are negatively associated with CKD follow. The estimated 95% confidence interval of
the hazard ratio for gender is (0.2747, 0.9561), which implies female relative to male has a
hazard reduction effect to CKD which is about 0.5 times and which is as much as 9.6% low to
2.7% lower. The estimated 95% confidence interval of the hazard ratio for follow-up alanine
transaminase is (0.9605, 0.9957), which implies that an increase in follow-up alanine
transaminase by one unit results in a hazard reduction effect to CKD by a factor of 0.9780 or
by about 2.2% and which is as much as 4.1% low to 0.4% lower. Patients taking Treatments
(EFV+D4T+3TC, EFV+AZT+3TC, EFV+3TC+TDF or NVP+3TC+TDF each relative to
EFV+3TC+TDF) are likely to experience CKD hazard reduction by about 0.15 times, 0.07
times, 0.13 times or 0.14 times respectively. 8 Table 1: The estimates of the final Cox PH model coef
exp(coef)
se(coef)
p
Lower
0.95CL
Upper
0.95CL
rho chisq
p -value
(PH test)
Follow-up CD4
-0.0009
0.9992
0.0011
0.4534
0.9970
1.0014
0.9187
Age
0.0549
1.0565
0.0163
0.0008
1.0232
1.0908
0.8031
EFV+D4T+3TC
relative to
NVP+D4T+3TC
-1.9022
0.1493
0.5232
0.0003
0.0535
0.4161
0.5239
EFV+AZT+3TC
relative to
NVP+D4T+3TC
-2.6245
0.0725
0.8464
0.0019
0.0138
0.3808
0.6105
EFV+3TC+TDF
relative to
NVP+D4T+3TC
-2.0227
0.1323
0.5074
0.0001
0.0489
0.3577
0.2283
NVP+3TC+TDF
relative to
NVP+D4T+3TC
-1.9836
0.1376
0.6024
0.0010
0.0422
0.4481
0.6034
Hospital(Embhuleni
relative to Carolina)
1.4778
4.3835
0.49039
0.00258
1.6765
11.4614
0.2136
Follow-up
alanine
transaminase
-0.0223
0.9779
0.0092
0.0152
0.9605
0.9957
0.0754
lnFollow-up
viralload
0.0640
1.0661
0.0587
0.27541
0.9503
1.1961
0.8447
Baseline creatinine
0.0171
1.0172
0.0057
0.0025
1.0060
1.0286
0.3802
Follow-up
lymphocyte
0.0320
1.0325
0.0301
0.2886
0.9733
1.0952
0.7279
Gender
(female
relative to male)
-0.6684
0.5125
0.3181
0.0356
0.2747
0.9561
0.4277 Nonparametric inferences about the survivor functions The cohort of HIV+ terminal patients in this study is not homogeneous with respect to their
characteristics that may affect their survival from CKD. Hence, it will be necessary to test the
equality of survivor functions among groups (strata) of patients. The function ‘survdiff()’ was
used to test for the differences in survival between two groups using a log-rank test. Log-rank
tests and the Kaplan-Meier functions presented in this section are for gender, age and
creatinine. These variables constitute Glomerular Filtration Rate (GFR) equation given by 9 9 9 Omuse et al. (2017). Treatment (regimen one) is also included in this section because of its
clinical significance among the HIV/AIDS patients. Kaplan-Meier survival functions and hazard ratios for baseline creatinine Baseline creatinine was categorised as done in the standard Laboratory report on creatinine in
South African hospitals. Figure 1 shows that survivor functions of the baseline creatinine strata
are statistically different at 0.05 significance level (p-value= 0.004, from the log-rank test)
and this confirms that baseline creatinine level is associated with CKD hazard. This is further
confirmed by the pairwise comparisons of the strata using the hazard ratios in Table 2 which
show that patients with baseline creatinine level which is above normal level are about 2.5
times likely to experience CKD relative to patients with baseline creatinine which is at normal
level. This risk can be as low as about 1.3 times and as high as about 4.7 times (Table 2). The
difference in CKD experience between patients with baseline creatinine level below normal
relative to patients with baseline creatinine at normal level is statistically not significant (Table
2). 2). Figure 1: Kaplan-Meier survival function
estimates for Baseline creatinine strata
Table 2: Hazard Ratios for baseline
creatinine strata
Description
Point
Estimate
95% Wald
Confidence
Limits
Below normal
level relative to
normal level
0.5947 0.2095 1.688
Above normal
relative
to
normal level
2.4897 1.3233 4.684 Figure 1: Kaplan-Meier survival function
estimates for Baseline creatinine strata
Table 2: Hazard Ratios for baseline
creatinine strata
Description
Point
Estimate
95% Wald
Confidence
Limits
Below normal
level relative to
normal level
0.5947 0.2095 1.688
Above normal
relative
to
normal level
2.4897 1.3233 4.684 Kaplan-Meier survival functions and hazard ratios for age groups Figure 3 shows that survivor functions of the age strata are not statistically different at 0.05
significance level (p-value= 0.08, from the log-rank test) and this is confirmed by the
confidence limits for strata in Table 4 which include ‘1’ which is a value of no effect. Figure
3:
Kaplan-Meier
survival
function estimates for Age groups
Table 4: Hazard Ratios for Age group
strata
Description
Point
Estimate
95% Wald
Confidence
Limits
31-50
years
relative to 16-30
years
1.468 0.7748 2.782
Above 50 years
relative to 16-30
years
2.660 1.1070 6.393 Figure
3:
Kaplan-Meier
survival
function estimates for Age groups
Table 4: Hazard Ratios for Age group
strata
Description
Point
Estimate
95% Wald
Confidence
Limits
31-50
years
relative to 16-30
years
1.468 0.7748 2.782
Above 50 years
relative to 16-30
years
2.660 1.1070 6.393 Figure
3:
Kaplan-Meier
survival
function estimates for Age groups Figure
3:
Kaplan-Meier
survival
function estimates for Age groups Figure
3:
Kaplan-Meier
survival
function estimates for Age groups Patients who are above 50 years relative to patients aged 16-30 years are about 2.7 times likely
to experience CKD (Table 4). Patients aged 31-50 years relative to patients aged 16-30 years
are about 1.5 times likely to experience CKD. However, this difference is not significant since
the 95% confidence limits include 1 (a result of no effect) and also the graphs of 31-50 and 16-
30 age groups cross each other as shown in Figure 3. Kaplan-Meier survival functions and hazard ratios for gender Kaplan-Meier survival functions and hazard ratios for gender Table 3 shows that female patients relative to male patients have a reduced risk to CKD of
about 0.6 times. However, the effect of gender is statistically not significant in the experience
of CKD hazard. This is confirmed by the survivor functions of gender strata which are not
statistically different at 0.05 significance level (p-value =0.05, from the log-rank test)(Figure
2) and also by the confidence limits for gender strata in Table 3 which include ‘1’ which is a
value of no effect. 10 Figure 2: Kaplan-Meier survival
function estimates for Gender
Table 3: Hazard Ratios for gender
Description
Point
Estimate
95% Wald
Confidence
Limits
Female relative
to Male
0.6183
0.3561 1.073 Figure 2: Kaplan-Meier survival
function estimates for Gender
Table 3: Hazard Ratios for gender
Description
Point
Estimate
95% Wald
Confidence
Limits
Female relative
to Male
0.6183
0.3561 1.073 Figure 2: Kaplan-Meier survival
function estimates for Gender Discussion This research has several strengths. Firstly, research follow-up time of 7.5 years is relatively
long to yield consistent records and hence reliable findings. Secondly, the research has diverse
covariates with most of them having paired baseline and follow-up covariates for exhaustive
comparisons of associations. Finally, this was not a single-center study, it involved two district
hospitals namely Embhuleni and Carolina for comparison and for completeness in coverage. The researched prevalence of CKD among HIV/AIDS patients in Albert Luthuli municipality
of South Africa was 15.9%. This prevalence is within the range 6% to 45% which is the CKD
prevalence in HIV patients in Africa as cited by Moosa et al. (2015). Similar studies which
were carried out in South Africa for example by Zachor et al. (2016) in Cape Town and by
Moosa et al. (2015) in South Africa showed the prevalence of 2%-6%. The difference between
the prevalence findings could be explained by study design, population variation, variables
considered and the criteria used for the diagnosis of CKD. The prevalence of CKD among the
lost and transferred patients in this research were 14.3% and 13.4% respectively. The patients
lost to follow-up and the patients who transferred from the hospital were presumed to be part
of the research and to be alive until the end of the research (Etikan et al 2017, 2018; Goel et al,
2010). The researched risk factors found to be associated with CKD by using Cox regression were
gender, age, baseline creatinine, hospital, treatment (regimen 1) and baseline alanine
transaminase. The finding in this research on risk factors to CKD concurs with the study
findings by Kalayjian et al. (2012), Menezes et al. (2011), Nishijima et al. (2017), Hilton
(2013), Boswell and Rossouw (2017) and Crum-Cianflone et al. (2010) who had HIV, race,
sex, age, low CD4, high viral load, diabetes, ART, weight, high sodium and hypertension as
the risk factors to kidney disease. However, there is no consensus among researchers on the
effect of TDF-containing ART regimens on CKD. Although ART may improve kidney
function, TDF-containing regimens have been found to have the potential to cause
nephrotoxicity and hence to significantly contribute to CKD among HIV-infected patients. This
finding agrees with Boswell and Rossouw (2017), Wyatt (2015) and Kalayjian et al. (2012). Wyatt (2015) pointed out that TDF is associated with kidney toxicity and decrease in creatinine
clearance or decrease in GFR. Kaplan-Meier survival functions and hazard ratios for treatment (regimen one) The five treatment groups are statistically different (log rank p-value=0.003)(Figure 4). Treatment EFV+D4T+3TC relative to treatment NVP+D4T+3TC has a reduced risk to CKD of
about 0.34 times, Treatment EFV+AZT+3TC relative to treatment NVP+D4T+3TC has a reduced
risk to CKD of about 0.1 times while treatment EFV+3TC+TDF relative to NVP+D4T+3TC has a
reduced risk to CKD of about 0.4 times. The effect of treatment NVP+3TC+TDF relative to
NVP+D4T+3TC is not statistically significant in experiencing the risk of CKD since the graphs cross
(Figure 4) and the 95% confidence internal includes a value of no effect ‘1’ (Table 5). 11 Table 5: Hazard Ratios for ART regimen strata
Description
Point
Estimate
95% Wald
Confidence
Limits
EFV+D4T+3TC
relative to
NVP+D4T+3TC
0.3443
0.1354
0.8759
EFV+AZT+3TC
relative to
NVP+D4T+3TC
0.1045
0.0221 0.4957
EFV+3TC+TDF
relative to
NVP+D4T+3TC
0.4019
0.1779
0.9079
NVP+3TC+TDF
relative to
NVP+D4T+3TC
0.3973
0.1550
1.0186 Table 5: Hazard Ratios for ART regimen strata
Description
Point
Estimate
95% Wald
Confidence
Limits
EFV+D4T+3TC
relative to
NVP+D4T+3TC
0.3443
0.1354
0.8759
EFV+AZT+3TC
relative to
NVP+D4T+3TC
0.1045
0.0221 0.4957
EFV+3TC+TDF
relative to
NVP+D4T+3TC
0.4019
0.1779
0.9079
NVP+3TC+TDF
relative to
NVP+D4T+3TC
0.3973
0.1550
1.0186
Figure 4: Kaplan-Meier survival function
estimates for ART regimen strata Table 5: Hazard Ratios for ART regimen strata Figure 4: Kaplan-Meier survival function
estimates for ART regimen strata 12 12 Discussion In this study, treatment (regimen 1) is negatively associated with
CKD hazard, in both univariate and multiple Cox regression forms. Unexpectedly, in this study,
TDF-containing regimens which happen to have a greater number of CKD cases (60.8%) in
comparison to other regimens have a negative association with CKD hazard relative to 13 NVP+D4T+3TC (reference regimen). Thus, despite most CKD cases being found among
patients taking TDF-containing drugs, TDF-containing drugs are still associated with a CKD
hazard reduction effect. Some further studies on the association between TDF-containing drugs
and CKD hazard in necessary. The baseline findings from Figure 1 and Table 2 show that patients with baseline creatinine
which is above normal level are about 2.5 times likely to experience CKD relative to patients
with baseline creatinine which is at normal level. In addition, patients with baseline creatinine
which is below normal level are about 0.6 times likely to experience CKD relative to patients
with baseline creatinine which is at normal level. These findings reinforce the need for accuracy
in creatinine testing and for the inclusion of creatinine in GFR equation. Gender is significant in modelling (Table 1) but not significant in the testing of the hypothesis
of no difference of homogeneity using the Log-rank test and Kaplan-Meier functions (Figure
2, Table 3). This difference is because Cox modelling accounts for the effects of other factors
whereas Kaplan-Meier is a univariate analysis which does not consider the effects of other
variables. Research study in Albert Luthuli found out that being a male increases the hazard of
having CKD by about 1.6 times. The significance of gender as a risk factor to CKD is
reinforced by its inclusion in the MDRD formula. Another significant researched risk factor to CKD and which happens to be included in the
GFR formula is age. The fitting of age in the Cox regression in this study reinforces its inclusion
in GFR formula. The effect of age on CKD is due to glomerular filtration rate which declines
at 6.3 ml/min/1.73m2 per decade as cited by Denic et al. (2016). Findings on age in Table 4
indicate that patients aged above 50 years relative to patients aged 16-30 years are about 2.7
times likely to experience CKD. The finding that CKD is associated with baseline creatinine, age and gender is not novel as these
are the components of the GFR formula (Iseki, 2008; Omuse et al., 2017). Discussion Therefore, the
concurrence of the regression results with the existing formula confirms the appropriateness
and accuracy of the research methods used. 14 14 On the management of CKD; Pedro et.al (2016) supported the use of TDF but hinted on its
nephrotoxic potential and pointed out the need for the adjustment of the dose when baseline
creatinine clearance is below 50ml/min. Boswell and Rossouw (2017) pointed out that current
local guidelines recommend that TDF be substituted by an alternative Nucleoside reverse
Transcriptase Inhibitors (NRTI) when a patient’s GFR is less than 50ml/min and when using
MDRD method. Moosa et al. (2015) gave some guidelines on dose adjustments for ART in
CKD cases for Lamivudine (3TC), Stavudine (d4T) and Tenofovir (TDF). Some other points
to note on the management of CKD as cited by several studies are: • All individuals should be assessed for kidney disease at the time of diagnosis of HIV/AIDS
and annually thereafter. • All individuals should be assessed for kidney disease at the time of diagnosis of HIV/AIDS
and annually thereafter. • Prevention of CKD at the population level through interventions that lead to a reduction in
BP, obesity, type 2 diabetes mellitus, smoking and salt ingestion. • Careful screening and monitoring of high-risk patients. • Increased awareness of CKD and knowledge on how to manage kidney disease should be
emphasized for general medical practioners. • Increased awareness of CKD and knowledge on how to manage kidney disease should be
emphasized for general medical practioners. November et al. (2017) cited main challenges in handling of CKD; firstly, the extent of HIV
epidemic and its associated burden on CKD, secondly there are no measures to prevent renal
disease in people living with HIV or to detect CKD early enough to treat it and lastly the
challenge of a rise in diet-related non-communicable diseases such as obesity and hypertension. Some other challenges include costly dialysis; in 2017 South Africa had only three public
hospitals which offered kidney transplants. The researcher acknowledges several limitations in this study. First, despite hypertension and
diabetes being regarded by many studies as important risk factors to CKD, they could not be
included because of missing data. However, the contribution of hypertension and diabetes to
the development CKD was not significant in similar studies by Mekuria et al. (2016). Conclusion This study, through the application of Cox regression, found the factors associated with CKD
as: gender, age, baseline creatinine, hospital, treatment (regimen 1) and Baseline alanine
transaminase. Given the HIV/AIDS background and the diversity of the worsening risk factors
to CKD in Africa and in South Africa, there is need for some concerted effort to alleviate health
and economic burden caused by CKD. This research has presented risk factors associated with
CKD in Albert Luthuli Municipality and the expected action is to educate the nation on
prevention, early detection and on informed management of CKD. The study established
diverse baseline statistics against which future research may be based. Discussion Second,
this research had predominantly black participants and hence it failed to evaluate the effect of
race on CKD as was done by Winston et al. (2008) and by Hilton (2013), who showed that
black race is the most important determinant of kidney disease prevalence in HIV-infected
persons. Lastly, Bukabau et al. (2019) in agreement with Ekrikpo et al. (2018) propose that the
MDRD which was used in this study as a creatinine-based formula is inadequate for African
population. 15 Authors’ contributions PB was involved in obtaining ethics approval, data collection/analysis and drafting of the
manuscript. PD and MAM contributed to data interpretation, project design and editing the
manuscript. All authors read and approved the final manuscript. Acknowledgement We are grateful to HIV/AIDS clinics staff for Carolina and Embhuleni hospitals. The Data
capturers assisted with data collection while the administrative staff swiftly performed data
collection permissions protocols. Author notes Pepukai Bengura, Prof Principal Ndlovu and Mulalo Annah Managa contributed equally to
this work hence the corresponding author is transferable if the need arises. Correspondence to Pepukai Bengura. Correspondence to Pepukai Bengura. Corresponding author Correspondence to Pepukai Bengura. Funding Not applicable. 16 Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Availability of data and materials The data that support the findings of this study are available from the Department of health,
but restrictions apply to the availability of these data, which were used under license for the
current study, and so are not publicly available. Data are however available from the authors
upon reasonable request and with permission of the Department of health. Ethics approval and consent to participate This study was conducted in accordance with the South African local and national research
guidelines. Ethics approval for this project was obtained from UNISA Ethics Review
Committee (ERC) with the approval number being (2017/SSR ERC/005). The permission to
conduct the study at Carolina and Embhuleni hospitals was obtained from Mpumalanga
Department of Health with the permission number being (MP_201708_013). The consent to
participate does not apply to a patient since no reference to an individual respondent was made,
all results were handled in aggregate format. Author details 1Department of Statistics, Unisa, Pretoria, South Africa 1Department of Statistics, Unisa, Pretoria, South Africa
2Department of Statistics, Unisa, Pretoria, South Africa 2Department of Statistics, Unisa, Pretoria, South Africa 17 17 REFERENCES 1. World Health Organization. Global Health Observatory (GHO) data. 2018. https://www.who.int/gho/hiv/en/. Accessed 27 Dec 2019. 2. UNAIDS 'AIDSinfo', 2019. http://aidsinfo.unaids.org/. Accessed 11 August 2019. 3. Motsoaledi, A. Mpumalanga's Gert Sibande district has highest HIV rate. Johannesburg:
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November 2018. 28 34. Bukabau JB, Yayo E, Gnionsahe A, Monnet D, Pottel H, Cavalier E, Nkodila A, Makulo
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filtration rate in sub-Saharan African populations. Kidney International, 2019; 95, 1181–
1189; https://doi.org/10.1016/j.kint.2018.11.045. Accessed 17 July 2019. REFERENCES 21 Figures Figure 1: Kaplan-Meier survival function estimates for Baseline
creatinine strata
Log-rank, p-value = 0.005 Log-rank, p-value = 0.005 Figure 1: Kaplan-Meier survival function estimates for Baseline
creatinine strata Figure 2: Kaplan-Meier survival function estimates for
Gender
Log-rank, p-value = 0.05 Log-rank, p-value = 0.05 Figure 2: Kaplan-Meier survival function estimates for
Gender 22 Log-rank, p-value = 0.08 Figure 3: Kaplan-Meier survival function estimates for
Age groups
Log-rank, p-value = 0.08 Figure 3: Kaplan-Meier survival function estimates for
Age groups Log-rank, p-value = 0.003
Figure 4: Kaplan-Meier survival function estimates for ART
regimen strata Log-rank, p-value = 0.003 Figure 4: Kaplan-Meier survival function estimates for ART
regimen strata 23 Figures Figure 1 Figure 1 Figure 1 Kaplan-Meier survival function estimates for Baseline creatinine strata Kaplan-Meier survival function estimates for Baseline creatinine strata Figure 2
Kaplan-Meier survival function estimates for Gender Figure 2 Figure 3 Kaplan-Meier survival function estimates for Age groups Kaplan-Meier survival function estimates for Age groups Kaplan-Meier survival function estimates for Age groups Kaplan-Meier survival function estimates for Age groups Figure 2 Kaplan-Meier survival function estimates for Gender Kaplan-Meier survival function estimates for Gender Kaplan-Meier survival function estimates for Gender Figure 3
Kaplan-Meier survival function estimates for Age groups
Figure 4
Kaplan-Meier survival function estimates for ART regimen strat Figure 3 Figure 3
Kaplan-Meier survival function estimates for Age groups
Figure 4
Kaplan-Meier survival function estimates for ART regimen strata Figure 3 Figure 4 Kaplan-Meier survival function estimates for ART regimen strata Kaplan-Meier survival function estimates for ART regimen strata
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Laser Generation of Colloidal Nanoparticles in Liquids: Key Processes of Laser Dispersion and Main Characteristics of Nanoparticles
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INTRODUCTION tion of nanoparticles in liquids with experimental
investigations aimed at filling the gaps in the literature
concerning the properties of generated dispersions. The first part of the work has been dictated by the need
to systematize numerous data reported in the litera-
ture, including those on the productivity of nanopar-
ticle generation as depending on the parameters of
laser action. The second part allows one, using, on the
one hand, the analysis of the physical processes
accompanying laser generation of particles and, on the
other hand, the data on the physicochemical parame-
ters of the resulting nanodispersions, to select optimal
methods for obtaining nanoparticles/dispersions for
solving these or those problems. Nanoparticles are the main building blocks of
modern nanotechnologies. Due to quantum and sur-
face effects, they have various unique properties and,
therefore, are used as nanomaterials in many fields of
science and technology [1, 2]. In particular, they have
found wide application as functional components of
subwavelength optical devices [3–5], sensor substrates
for surface-enhanced spectroscopy [6–11], biological
marking and probing systems [12–14], theranostics
[15–19], and antibacterial agents [20–25]. Along with other methods used for generating solu-
tions of colloidal nanoparticles, such as chemical
reduction [26–29], precipitation [30–32], hydrolysis
[33, 34], sol–gel synthesis [35–40], and hydrothermal
synthesis [41–43], laser ablation in liquids is one of the
most promising scalable methods for obtaining chem-
ically pure nanoparticles and is believed to be an envi-
ronmentally friendly process [44–51]. The relevance
and promise of this method are due to its simplicity,
the feasibility to use different targets and liquids, and
the purity of the ablation products. Moreover,
liquids are convenient media for collecting ablation
products [10]. Laser Generation of Colloidal Nanoparticles in Liquids:
Key Processes of Laser Dispersion and Main
Characteristics of Nanoparticles A. A. Nastulyavichusa, *, S. I. Kudryashova, A. M. Emelyanenkob, and L. B. Boinovichb
a Lebedev Physical Institute, Russian Academy of Sciences, Moscow, 119991 Russia
b Frumkin Institute of Physical Chemistry and Electrochemistry, Russian Academy of Sciences, Moscow, 119071 Russia
*e-mail: nastulyavichusaa@lebedev.ru
Received January 17, 2023; revised February 1, 2023; accepted February 2, 2023 A. A. Nastulyavichusa, *, S. I. Kudryashova, A. M. Emelyanenkob, and L. B. Boinovichb
a Lebedev Physical Institute, Russian Academy of Sciences, Moscow, 119991 Russia
b Frumkin Institute of Physical Chemistry and Electrochemistry, Russian Academy of Sciences, Moscow, 119071 Russia
*e-mail: nastulyavichusaa@lebedev.ru
Received January 17, 2023; revised February 1, 2023; accepted February 2, 2023 Abstract—The processes of laser dispersion of materials for high-performance generation of colloidal
nanoparticles in liquids have been considered. Various laser and material parameters affecting this process
have been studied. Efficiencies and ergonomics of the generation of colloidal nanoparticles with the help of
laser systems having nano-, pico-, and femtosecond pulse durations have been compared using optical and
mass criteria by the example of laser ablation of a chemically inert model material (gold) in distilled water
without the use of chemical stabilizers. The main characteristics of gold and silver nanoparticles obtained by
ablation in water using pulsed laser radiation of different durations have been comprehensively compared. The types of colloidal interactions between nanoparticles in aqueous media have been discussed, and the con-
tributions of structural and ion-electrostatic interactions to the long-term stability of gold and silver nanopar-
ticle dispersions have been analyzed. ISSN 1061-933X, Colloid Journal, 2023, Vol. 85, No. 2, pp. 233–250© The Author(s), 2023. This article is an open access publication.
Russian Text © The Author(s), 2023, published in Kolloidnyi Zhurnal, 2023, Vol. 85, No. 2, pp. 200–218. ISSN 1061-933X, Colloid Journal, 2023, Vol. 85, No. 2, pp. 233–250© The Author(s), 2023. This article is an open access publication. Russian Text © The Author(s), 2023, published in Kolloidnyi Zhurnal, 2023, Vol. 85, No. 2, pp. 200–218. ISSN 1061-933X, Colloid Journal, 2023, Vol. 85, No. 2, pp. 233–250© The Author(s), 2023. This article is an open access publication.
Russian Text © The Author(s), 2023, published in Kolloidnyi Zhurnal, 2023, Vol. 85, No. 2, pp. 200–218. Approaches to Estimating Colloidal Nanoparticle
Concentration in Liquids Approaches to Estimating Colloidal Nanoparticle
Concentration in Liquids Due to the large number of colloidal nanoparticles
n ∼ 103–106 with characteristic size R ∼ 100 nm (the
number of smaller particles is 1/R3 times more) gener-
ated even during one laser pulse with a relatively low
energy (1–103 μJ), it is, as a rule, actually impossible
to count them directly; so, macroscopic methods are
used for estimating this number. (4) Evolution of a (sub)millimeter vapor bubble
over the surface of the ablation region from bubble
growth to its collapse (of (sub)millisecond duration)
[68–71]. (4) Evolution of a (sub)millimeter vapor bubble
over the surface of the ablation region from bubble
growth to its collapse (of (sub)millisecond duration)
[68–71]. They include a rather convenient express method
for measuring optical transmission coefficient Tcol of a
dispersion of nanoparticles, with this method making
it possible to characterize colloidal nanoparticles
either by optical density OD = log(T0/Tcol) or by the
extinction coefficient, which is determined by the
joint contribution of absorption and scattering,
κ =ln(T0/Tcol)/d, where T0 is the transmittance of a
cell with a pure dispersion medium and d is its width
along the optical axis. In the general case, the optical
density and extinction coefficient are relative charac-
teristics of the concentration of colloidal nanoparti-
cles; however, if nanoparticles of only one chemical
and structural-phase type are present, it is possible to
determine total mass Mopt of nanoparticles in a solu-
tion by measuring the extinction coefficient in the
region of interband transition (IBT) of a material. Indeed, in contrast to the metallic (intraband) absorp-
tion of free electrons [72], in the spectral region of
interband transitions of strongly localized d electrons At present, the effect of laser pulse repetition fre-
quency f ∼ 1–107 Hz has been studied in greatest
detail. This frequency, in combination with the speed
of laser beam scanning over a target surface, ∼ 1–
109 μm/s, determines the shift of the beam along the
surface from pulse to pulse, Δ ≈ /f. If shift Δ turns out
to be much smaller than the size of a focal spot on the
surface, a screening effect may take place due to the
secondary absorption of nanoparticles formed by pre-
vious pulses [52]. Main Stages of Laser Ablation in Liquids Main Stages of Laser Ablation in Liquids At present, there are data in the literature on
achieving a high productivity of laser-ablative genera-
tion of nanoparticles in liquid media on the order of
several grams per hour [52–54]. In this case, various
targets are considered from bulk materials to thin
films. In addition, particles of different types and
materials (metals, dielectrics, etc.) have been
obtained, thus making it possible to carry out extensive This work is a combination of the analysis of com-
munications available in the literature on the studying
the effects of different laser parameters on the genera- 233 NASTULYAVICHUS et al. 234 material-dependent (wavelength) or trivial (the greater
the average radiation power, the higher ablation effi-
ciency [72, 73]). At the same time, a very important
laser parameter is the duration of the radiation pulses,
which, for most contemporary and available laser sys-
tems, varies by many orders of magnitude from a few
tens of femtoseconds to several hundreds of nanosec-
onds, thus determining, in particular, the features of
laser radiation energy transfer to a target surface (stage
1, see above). Published communications, devoted to
the comparison between laser systems with different
durations from a few femtoseconds to several nanosec-
onds during ablation of targets of the same materials,
e.g., gold, present greatly different data. In some works
[74], it is reported that the productivity of colloidal
nanoparticle generation of picosecond pulses is better. Others works [73] report a better productivity of nano-
second laser pulses. Here, it should also be taken into
account that the laser systems under consideration dif-
fer in not only pulse durations, but also wavelengths,
energies, repetition frequencies, focusing, and some-
times the volumes of produced nanoparticle disper-
sions [55]. Accordingly, in terms of optimizing and
scaling the generation of colloidal nanoparticles, a
problem arises concerning the correct comparing
experimental results obtained under different laser-
and material-related research conditions. laboratory studies on the use of nanomaterials in vari-
ous important technological areas mentioned above. Nevertheless, in most of the works presented in the lit-
erature, no directed optimization of the process for
producing particles was carried out, and the obtained
nanoparticles were characterized using a rather lim-
ited spectrum of methods. Main Stages of Laser Ablation in Liquids At the same time, the vari-
ety of laser processing parameters (wavelength,
energy, duration and repetition frequency of laser
pulses, immersion depth of an ablation source, and its
movement relative to a liquid) and types of liquid
media makes it possible to perform such optimization
based on the detailed characterization of resulting par-
ticles. It is necessary to single out the main physical stages
that successively determine the course of laser ablation
in liquids with the generation of nanoparticles and the
formation of dispersions. (1) Propagation of laser radiation to the surface of
a solid target. This stage can be complicated by the for-
mation of an ablation plasma and plasma screening
when using (sub)nanosecond laser pulses [55–58] or
by nonlinear focusing and filamentation of femto- and
picosecond laser pulses [55, 59–61]. (1) Propagation of laser radiation to the surface of
a solid target. This stage can be complicated by the for-
mation of an ablation plasma and plasma screening
when using (sub)nanosecond laser pulses [55–58] or
by nonlinear focusing and filamentation of femto- and
picosecond laser pulses [55, 59–61]. (2) Absorption of radiation by an electronic subsys-
tem, energy transfer to a lattice, and phase transition to
high-temperature states of a melt [62, 63]. (3) Nano- or subnanosecond emission of an abla-
tion torch in the form of a vapor–droplet mixture [64]. Note that the vapor–droplet mixture in the case of
nanosecond (ns) laser pulses undergoes optical break-
down, with the formation of screening subcritical
plasma [64]. As has been shown in the literature, such
plasma self-consistently controls the fraction of radia-
tion energy transmitted to the target and determines
the flux of low-molecular-weight ablation products
from the target surface [64] without taking into
account the microdroplet fraction [65–67]. (3) Nano- or subnanosecond emission of an abla-
tion torch in the form of a vapor–droplet mixture [64]. Note that the vapor–droplet mixture in the case of
nanosecond (ns) laser pulses undergoes optical break-
down, with the formation of screening subcritical
plasma [64]. As has been shown in the literature, such
plasma self-consistently controls the fraction of radia-
tion energy transmitted to the target and determines
the flux of low-molecular-weight ablation products
from the target surface [64] without taking into
account the microdroplet fraction [65–67]. Approaches to Estimating Colloidal Nanoparticle
Concentration in Liquids If, in this situation, the shift appears
to be smaller than the sizes of the vapor bubble, the
refraction/scattering of radiation by the bubble dis-
torts the distribution of the radiation energy over the
surface and reduces ablation efficiency [72]. v
v Other effects of laser radiation during laser ablation
in liquids are relatively poorly studied and are either COLLOID JOURNAL
Vol. 85
No. 2
2023 LASER GENERATION OF COLLOIDAL NANOPARTICLES 235 work, gold and silver in an aqueous medium are used
as model chemically inert materials, which give
nanoparticles of only one chemical type (metallic gold
or silver, respectively) during laser ablation, to show
the potentials of different typical femto-, pico-, and
nanosecond laser systems employed in the laser-abla-
tive production of nanoparticle dispersions in water. The application of such a system minimizes the num-
ber of chemical reactions and the formation of side
compounds characteristic of ablation in organic sol-
vents. to the conduction band, the particle size does not
affect the extinction coefficient up to nanometer sizes. In this case, the strong interband absorption domi-
nates over the plasmonic effects of absorption and
scattering (in the case of metal nanoparticles), and the
measured extinction coefficient may be compared
directly with the absorption coefficient in the IBT
region, and their ratio will characterize specific den-
sity ρcol of the material in a solution in comparison
with material density ρ0. For known volume V of a dis-
persion, total mass Mopt = ρcolV of nanoparticles is
obtained, while the determination of its distribution
over nanoparticles with different sizes requires the use
of additional methods, such as dynamic light scatter-
ing by nanoparticles [55, 75, 76]. Comparison of Femto-, Pico-, and Nanosecond Laser
Dispersion of Gold in Water Gold is a convenient model material for studying
the processes and parameters, as well as the productiv-
ity, of laser dispersion in liquids; therefore, it has been
repeatedly applied for these purposes using water as a
dispersion medium [60, 73, 88, 89]. At the same time,
the results of experiments on femto-, pico-, and nano-
second laser dispersion have turned out to be
extremely contradictory. Moreover, only the rough
mass criterion of dispersion efficiency has been used in
the cited works. Approaches to Estimating Colloidal Nanoparticle
Concentration in Liquids Just recently, such studies were carried
out under the maximum possible comparable condi-
tions, namely, at close laser radiation wavelengths
(1064 nm (ns) and 1030 nm (fs, ps)), focusing condi-
tions, and pulse repetition frequencies [55, 83], i.e.,
using actually the same experimental setup (Fig. 1). It
is also very important to use complementary optical
and mass criteria to analyze the productivity and ergo-
nomics of dispersion processes [83]. Meanwhile, in the case of laser generation of
nanoparticles of only one chemical type, i.e., in the
absence of chemical reactions of addition in a solu-
tion, a mass productivity criterion is also applied, i.e.,
the mass loss of an ablated material sample [55, 73] or
the mass of the obtained dried colloid [52, 74]. Although being convenient, the first approach is indi-
rect (part of the material ablated in the form of large,
micron-sized, particles can quickly undergo sedimen-
tation [77, 78]). The second approach directly charac-
terizes the total mass of nanoparticles in a solution;
however, it is less convenient. In general, the mass cri-
terion is additional to the optical one, and their com-
bined use makes it possible to minimize artifacts in the
measurement of the concentration of produced colloi-
dal nanoparticles. Finally, for laser ablation, in addition to the expo-
sure (the number of laser pulses spent for particle gen-
eration), a very important role is played by the energy
of laser pulses [52, 79–81], which, for different laser
systems, may vary within a fairly wide range, from a
few microjoules (femtosecond/picosecond lasers)
[82] to several joules (nanosecond lasers); hence, it is
one of the important factors for scaling the production
of colloidal nanoparticles in liquids by the laser abla-
tion method [52]. Therefore, the productivity of
obtaining nanoparticles, as determined according to
the optical or mass criteria, is usually recalculated per
one pulse [55] and normalized to the laser pulse energy
(ergonomics criterion [83]). Ergonomics characterizes
the energy consumption for the generation of a fixed
amount or mass of nanoparticles [83], thus reflecting
the optimality of the selected regimes. Moreover,
ergonomics characterizes the scalability of the process
with an increase in the energy of laser pulses by
increasing the local energy density at fixed focusing on
the material surface or by enlarging laser focusing area
at a fixed local energy density. Approaches to Estimating Colloidal Nanoparticle
Concentration in Liquids Only one of these pos-
sibilities turns out to be optimal (e.g., for nanosecond
laser pulses). In some cases, e.g., for ultrashort laser
pulses, the possibility of increasing the energy is lim-
ited by nonlinear self-focusing [84] or nonlinear ion-
ization (breakdown) of a medium [85–87]. In this In these studies, laser-ablative generation of
nanoparticles was carried out using two laser systems
(an ultrashort-pulse laser with laser radiation duration
varied from 300 fs to 10 ps and a nanosecond laser with
radiation duration of 100 ns). The laser beam was
focused using an f-theta lens with a focal distance of
≈100 mm onto a bulk gold target (purity of 99.99%)
placed into a cell containing distilled water (3 mL) (the
height of the water layer over the target was ≈1.5 mm). The experiment is schematically illustrated in Fig. 1. The influence of laser radiation time on the efficiency
of nanoparticle generation was studied in a range of
subpico-, pico-, and nanoseconds. Therewith, the
pulse frequency and scanning speed were constant and
amounted to 20 kHz and 100 mm/s, respectively. The
pulse energies were 2.5–6.5 μJ for subpico- and pico-
second pulses and 0.3–0.6 mJ for nanosecond ones. Minimum focus spot size (diameter 1/e) was ≈20 μm
for subpico- and picosecond laser pulses and ≈40 μm
for nanosecond laser pulses; the size of the scanning
area was 10 × 20 mm2. After the laser generation, a dispersion of gold
nanoparticles was sampled for subsequent determina-
tion of nanoparticle sizes by dynamic light scattering
and electron microscopy, extinction coefficient by
spectrophotometry, and mass loss by weighing a dry COLLOID JOURNAL
Vol. 85
No. 2
2023 NASTULYAVICHUS et al. 236 Fig. 1. Scheme of nanoparticle generation. 100 nm
0
Radius, nm
Intensity,
arb. units
0.02
0.04
0.06
0.08
10
100
0
Wavelength, nm
Extinction
coefficient, cm–1
0.2
0.6
0.4
0.8
1.0
1000
800
600
400
200
Dispersion of gold
nanoparticles
Galvanoscaner
Laser
1030 nm
0.3–10 ps
Laser
1064 nm
100 ps
X
Y
Z Dispersion of gold
nanoparticles Laser
1030 nm
0.3–10 ps Wavelength, nm Laser
1064 nm
100 ps Galvanoscaner Z Fig. 1. Scheme of nanoparticle generation. target before and after irradiation with an accuracy of
1 μg. Dispersion process productivities were compared
using the key characteristics of the experiments recal-
culated per one laser pulse. Approaches to Estimating Colloidal Nanoparticle
Concentration in Liquids The characteristics com-
prised total mass loss of a target in one experiment and
the “optical efficiency” (OE, the extinction coeffi-
cient of the dispersions in the region of interband tran-
sitions of bulk gold at about 380 nm, KIBT, multiplied
by fixed volume V of a dispersion and divided by num-
ber N of performed pulses). Figure 2 shows the depen-
dences of OE on the duration of laser radiation. supethreshold character of ablation in the “phase
explosion” regime,1 i.e., expansion of a supercritical
fluid for fentosecond/picosecond laser pulses [90, 91],
and homogeneous boiling up of a superheated liquid
in the near-critical region for nanosecond laser pulses
[92]. However, the dependence of the OE on the dura-
tion of laser pulses is more interesting. As the duration
of laser radiation increases within a range of 0.1–10 ps,
the OE initially grows; then, a local maximum is
observed in the region of several picoseconds, after
which, the OE decreases. The decrease in the effi-
ciency in the region of subpicosecond durations is
associated with the appearance of nonlinear effects,
i.e., self-focusing and filamentation [93–96]. The
decrease in efficiency in the region of several picosec-
onds is associated with the acoustic unloading of the
heated layer during its heating by a laser pulse in the
phase explosion regime [90, 97, 98]. Further, when
passing to nanosecond laser ablation, the OE values
increase several times (Fig. 2); however, the pulse
energy is almost two orders of magnitude higher; i.e.,
the energy efficiency (per unit energy, ergonomics) of
nanoparticle generation under the action of nanosec-
ond laser pulses is lower by more than an order of mag-
nitude. This is due to the screening effect of subcritical
ablation plasma,2 which almost inevitably arises under
the action of nanosecond laser pulses in an ablation
plume in the phase explosion regime [64] and self- The OE values are comparable for all used laser
pulse durations and increase monotonically with laser
pulse energy (Fig. 2), thereby indicating a stable and Fig. 2. Dependences of OE on the duration of laser radia-
tion for a gold target at different energies of laser pulses. 1 Phase explosion is the hydrodynamic expansion of a target material
at high, supercritical, internal pressures and temperatures.
2 Subcritical ablation plasma is the ablation plasma with a density
lower than the critical one for a given wavelength of laser radiation. Approaches to Estimating Colloidal Nanoparticle
Concentration in Liquids //
10–6
10–5
ns
1
10
100
0.1
, ps
2.5 J
3.5 J
6.5 J
0.3 mJ
0.4 mJ
0.5 mJ
0.6 mJ
КIBTV/N, сm2/puls //
10–6
10–5
ns
1
10
100
0.1
, ps
2.5 J
3.5 J
6.5 J
0.3 mJ
0.4 mJ
0.5 mJ
0.6 mJ
КIBTV/N, сm2/puls Fig. 2. Dependences of OE on the duration of laser radia-
tion for a gold target at different energies of laser pulses. COLLOID JOURNAL
Vol. 85
No. 2
2023 COLLOID JOURNAL
Vol. 85
No. 2
2023 LASER GENERATION OF COLLOIDAL NANOPARTICLES LLOIDAL NANOPARTICLES
237
Fig. 4. Dependence of OE recalculated per unit laser pulse
energy on the duration of laser radiation for a gold target. //
10–2
10–3
10–1
ns
1
10
100
0.1
, ps
2.5 J
3.5 J
6.5 J
0.3 mJ
0.4 mJ
0.5 mJ
0.6 mJ
КIBTV/E N,
сm2/J pulse 237 LASER GENERATION OF
Fig. 3. Dependences of gold target mass loss recalculated
per pulse, M/N, on the duration of laser radiation at differ-
ent pulse energies. The dashed curves represent the data
calculated from the extinction coefficients for mass frac-
tion Mopt of the solid in dispersion. //
102
101
100
103
ns
1
10
100
0.1
, ps
0.3 mJ
0.4 mJ
0.5 mJ
0.6 mJ
2.5 J
3.5 J
6.5 J
М/N, pg/pulse //
10–2
10–3
10–1
ns
1
10
100
0.1
, ps
2.5 J
3.5 J
6.5 J
0.3 mJ
0.4 mJ
0.5 mJ
0.6 mJ
КIBTV/E N,
сm2/J pulse //
102
101
100
103
ns
1
10
100
0.1
, ps
0.3 mJ
0.4 mJ
0.5 mJ
0.6 mJ
2.5 J
3.5 J
6.5 J
М/N, pg/pulse Fig. 3. Dependences of gold target mass loss recalculated
per pulse, M/N, on the duration of laser radiation at differ-
ent pulse energies. The dashed curves represent the data
calculated from the extinction coefficients for mass frac-
tion Mopt of the solid in dispersion. Fig. 4. Dependence of OE recalculated per unit laser pulse
energy on the duration of laser radiation for a gold target. the influence of nonlinear effects and the screening
effect of ablation plasma, provide a significantly
higher efficiency recalculated per unit energy of radia-
tion pulse. For example, it is one or two orders of mag-
nitude higher than that for nanosecond laser pulses. This tendency is also confirmed for the target mass
loss (mass efficiency, Fig. Approaches to Estimating Colloidal Nanoparticle
Concentration in Liquids 5). consistently determines the absorption of the target
and the ablation rate [58, 59, 99, 100]. It is noteworthy that a similar tendency is also
observed for the target mass loss (mass efficiency, ME)
recalculated per one laser pulse (Fig. 3). This indicates
that an almost constant fraction of the ablated sub-
stance passes into the dispersion of nanoparticles. Moreover, there is a good agreement (within 20–40%)
between mass loss per pulse M/N and mass Mopt of the
colloidal substance in the dispersion per radiation
pulse calculated from the value of the extinction coef-
ficient in the region of interband transitions of the
material (indicated in Fig. 3 by dashed lines). The
mass of gold in the dispersion of nanoparticles was cal-
culated by the following equation: It is clearly seen that the above tendencies and the
values of M/N (Mopt) for femto-, pico-, and nanosec-
ond laser dispersion of gold in water are in qualitative
agreement with the available fragmentary data of pre-
vious works [73, 88], as well as with similar systematic
results obtained for gold films of different thicknesses
[83]. Thus, it may be concluded that these results
reflect the general tendencies in the productivities of
different laser systems with respect to gold and, in
some cases, silver colloidal nanoparticles [102]. (1)
{
}
= ρ
IBT
I
opt
Au
BT,0
/
,
K
M
V
N
K (1) MAIN COLLOIDAL CHARACTERISTICS
OF NANOPARTICLE DISPERSIONS
PRODUCED BY LASER ABLATION
IN LIQUID MEDIA According to
the literature data, this leads to the fact that, e.g., gold
particles adsorb five times more oligonucleides than
do analogous particles obtained by chemical methods
[108]. It should be emphasized that the field of appli-
cation of nanoparticles essentially depends on the
temporal stability of nanodispersions containing these
particles, while the maintenance of the long-term sta-
bility of dispersions is always a difficult problem con-
cerning the controlled preparation and storage of nan-
odispersions [103, 104, 109]. Therefore, in this sec-
tion, we shall briefly consider the mechanisms of
stabilization of aqueous nanoparticle dispersions and
discuss the stability of dispersions obtained by the
methods described in this review. The character of the interparticle interaction
caused by steric forces depends on the interparticle
distance in a dispersion, the thickness of the layer of a
substance adsorbed on the particle surface, the energy
of its adsorption on the particles, and the type of inter-
action of the adsorbed molecules with a dispersion
medium. As a rule, dispersions of nanoparticles are
stabilized using long-chain surfactants or polymers
that are irreversibly adsorbed on dispersed phase par-
ticles and form brushlike structures. The formed
adsorption layer not only reduces the solubility of par-
ticles and their chemical activity upon the contact with
a dispersion medium, but also creates a steric barrier
that prevents the particles from coalescence in the dis-
persion. However, the application of such adsorption
layers can significantly change the chemical, catalytic,
and optical properties of the particles themselves and
affect their biocompatibility, bioavailability, and ther-
apeutic action [109]. Finally, the structural interaction
forces between nanoparticles can both cause an addi-
tional stabilizing effect, mainly for nanoparticles that
are highly wettable with a dispersion medium, and
induce rapid coalescence of hydrophobic particles in a
hydrophilic dispersion medium under the action of
hydrophobic attraction [112, 114]. Forces That Determine the Character
of Internanoparticle Interactions in Aqueous Media
and the Stability of Dispersions A large area of interfaces between particles and dis-
persion media makes particle dispersions thermody-
namically unstable. However, the switching-on of
interactions between individual particles can signifi-
cantly change the kinetic stability of a system. It
should be kept in mind that the state of a freshly pre-
pared highly dispersed system may vary in the follow-
ing directions. MAIN COLLOIDAL CHARACTERISTICS
OF NANOPARTICLE DISPERSIONS
PRODUCED BY LASER ABLATION
IN LIQUID MEDIA 238 238
NASTUL
//
10–1
100
101
ns
1
10
100
0.1
, ps
0.3 mJ
0.4 mJ
0.5 mJ
0.6 mJ
2.5 J
3.5 J
6.5 J
М/EN, pg/J pulse medium, both the transfer of a substance from smaller
particles to larger ones (Ostwald ripening) and the dis-
solution of particles at their low concentration in the
dispersion may occur. As a rule, for nanoparticles of
rather inert metals in deionized water, the last two
mechanisms of variations in the state of dispersions
play no significant role; however, they can be very
important for chemically active metals upon changes
in the pH of a dispersion medium [110, 111]. Prevention or inhibition of particle aggregation is
one of the main ways to increasing the kinetic stability
of nanodispersions, with this way being extensively
discussed in the literature. The stability of a dispersion
of nanoparticles may be governed by various types of
surface forces, including van der Waals, ion-electro-
static, steric, and structural forces [112]. The van der
Waals forces always contribute to the aggregation of
identical particles, thus destabilizing the dispersion. In
this case, for metal particles with a very high dielectric
permittivity, the contribution of the van der Waals
attractive forces in aqueous media can significantly
exceed interactions in systems with nonmetal parti-
cles. On the contrary, being a dispersion-stabilizing
mechanism, the ion-electrostatic interactions in
aqueous media with low ionic strengths cause the
repulsion between identical particles, and the higher
the charge of their surface in a liquid medium, the
stronger the repulsion [112]. An increase in the con-
centration of ions in a dispersion medium within a
range of their low concentration leads to screening the
ion-electrostatic repulsion, while, at a high concentra-
tion of ions, it can cause a correlation attraction
between the particles [113]. Fig. 5. Dependences of gold target mass loss per pulse on
the duration of laser radiation recalculated per unit pulse
energy. Dashed curves represent data calculated from
extinction coefficient for the mass fraction of the solid in
solution. vided that the substances used to modify the particles
form true or micellar solutions in the liquid media. It
should also be noted here that, due to the nonequilib-
rium state of the particles resulting from ablation, their
surface energy and adsorption activity for freshly
formed nanoparticles turn out to be significantly
higher than those in aged dispersions. MAIN COLLOIDAL CHARACTERISTICS
OF NANOPARTICLE DISPERSIONS
PRODUCED BY LASER ABLATION
IN LIQUID MEDIA where ρAu is the density of bulk gold;
is the solution
volume;
is the extinction coefficient in the region
of interband transitions for colloidal solutions of gold
nanoparticles;
is the absorption coefficient of
bulk gold in the region of 380 nm, which is equal to
≈6 × 105 cm−1 [101]; and N is the number of pulses. The observed agreement between the values of M/N
and Mopt indicates a high (almost 100%) efficiency of
the transfer of ablation products into the dispersion. V
IBT
K
IBT
K As has been shown above and discussed many
times in the literature (see, e.g., [45, 48, 103, 104]),
laser ablation in liquid media using lasers with differ-
ent pulse durations is a reliable and versatile approach
to the synthesis of rather stable dispersions of various
metal nanoparticles free of adsorbed substances that
modify their surface properties. In contrast to chemi-
cally synthesized nanoparticles, the surface of which is
usually covered with ligand molecules (long-chain
polymers or surfactants) [105–107], nanoparticle dis-
persions obtained by laser ablation demonstrate good
temporal stability in deionized water [104]. Moreover,
laser-based methods make it possible to realize simul-
taneous one-pot creation and dispersion of particles in
a liquid medium. Finally, this method implies no lim-
itations relevant to the choice and deposition of long-
chain ligands on the surface of resulting particles, pro- To estimate the energy efficiency of the nanoparti-
cle generation process, we considered the experimen-
tally determined values of the optical efficiency
(KIBTV)/N and mass loss M per unit energy E of laser
pulses spent for the generation of a dispersion
(KIBTV)/(EN) and M/(EN) (Figs. 4, 5), respectively. It
is quite indicative that, in terms of the energy effi-
ciency of the process of generating gold nanoparticles
in water, picosecond laser pulses, which are free from COLLOID JOURNAL
Vol. 85
No. 2
2023 238
NASTULY
Fig. 5. Dependences of gold target mass loss per pulse on
the duration of laser radiation recalculated per unit pulse
energy. Dashed curves represent data calculated from
extinction coefficient for the mass fraction of the solid in
solution. //
10–1
100
101
ns
1
10
100
0.1
, ps
0.3 mJ
0.4 mJ
0.5 mJ
0.6 mJ
2.5 J
3.5 J
6.5 J
М/EN, pg/J pulse NASTULYAVICHUS et al. MAIN COLLOIDAL CHARACTERISTICS
OF NANOPARTICLE DISPERSIONS
PRODUCED BY LASER ABLATION
IN LIQUID MEDIA It may be the sedimentation of large
particles under the action of gravity and the aggrega-
tion of small particles, including nanoparticles, which
is also followed by sedimentation. At a significant sol-
ubility of dispersed phase particles in a dispersion COLLOID JOURNAL
Vol. 85
No. 2
2023 LASER GENERATION OF COLLOIDAL NANOPARTICLES 239 Table 1. Parameters of the generation regimes for gold and silver particles in deionized water
Parameter
Metal
gold
silver
gold
silver
Pulse duration, ns
100
100
0.26 × 10–3
0.26 × 10–3
Speed, mm/s
60
60
100
100
Density, lines/mm
100
100
100
100
Frequency, kHz
20
20
20
20
Particle concentration, g/L
0.25
0.1
0.026
0.05 Analysis of the Stability Mechanisms of Silver and Gold
Nanodispersions Obtained by Laser Ablation
in Deionized Water Using Lasers with Different Pulse
Durations The zeta-potentials were calculated within the
framework of the Hückel approximation for both
deionized water and weak solutions, for which relation
κa 1 was fulfilled (here κ and a are the inverse Debye
length and particle diameter, respectively). For more
concentrated solutions, in which κa
1, the calcula-
tions were carried out using the Smoluchowski
approach. To examine each dispersion by light scatter-
ing, a 1-mL sample was taken from it, and the current
pH value was measured. The pH values were deter-
mined with an ESK 10614 microelectrode (OOO
Izmeritelnye Tekhnologii, Russia) and a Jenco 6230
pH meter operating within a pH range of 1.5–13. ! @ To date, the data on the stability of nanodisper-
sions of metal particles obtained by laser ablation in
deionized water using lasers with different pulse dura-
tions are scarcer in the literature. To fill this gap, the
properties of gold and silver nanoparticles obtained
using femto- and nanosecond lasers were compared in
this work. The regimes of laser treatment of the targets
in deionized water are presented in Table 1. The scanning electron microscopic examinations
of individual particles obtained in the regimes indi-
cated in Table 1 showed that both gold and silver
nanoparticles had a spherical shape and a rather wide
size distribution. Therewith, the diameters of gold
particles obtained by laser ablation at a femtosecond
pulse duration were mainly in a range of 100–120 nm. The diameters of the particles generated by laser abla-
tion with nanosecond duration were distributed over a
size range of 40–60 nm. MAIN COLLOIDAL CHARACTERISTICS
OF NANOPARTICLE DISPERSIONS
PRODUCED BY LASER ABLATION
IN LIQUID MEDIA In silver dispersions, the par-
ticles obtained by nanosecond processing had sizes of
100–150 nm, while after femtosecond laser ablation, a
significant fraction of the particles were characterized
by sizes of 100–140 nm, with a small amount of larger
particles having sizes as large as 350–400 nm. The dispersions of silver and gold nanoparticles
obtained under different ablation regimes had an
intense color: pinkish-violet, for dispersions of gold
nanoparticles, and yellow-gray, for dispersions of sil-
ver nanoparticles, thus being in agreement with the lit-
erature data [103, 112, 115–119]. For subsequent
application of nanoparticles obtained in different laser
ablation regimes, it was of interest to determine the
dependences of the potentials and sizes of the particles
on dispersion medium pH. In this work, to study such
dependences, nanodispersions were, at the first stage,
obtained by ablation in a neutral medium; at the sec-
ond stage, solutions of hydrochloric acid or sodium
hydroxide were added to bring the pH of a dispersion
medium to a required value. After a dispersion was
stored for a half an hour, the potentials and sizes of the
particles were measured. The pH dependences of
nanoparticle zeta-potentials for gold and silver disper-
sions obtained by both nanosecond and femtosecond
treatment of the targets are shown in Figs. 6a and 6b. A Zetasizer Nano ZS device (Malvern Instru-
ments, United Kingdom) was used to study the stabil-
ity of the obtained aqueous dispersions, as well as to
calculate and analyze such characteristics of the dis-
persions as the zeta-potential and particle size. According to the considerations expressed in [115] and
our electron microscopy data on the obtained parti-
cles, a low false peak corresponding to small particles
can appear in the bimodal scattered light intensity dis-
tribution over the hydrodynamic diameters of both sil-
ver and gold nanoparticles. Therefore, to characterize
changes in the state of dispersions, the parameters of
the peak with the maximum intensity were used below. It should be noted here that the particle diameters
determined from the peak with the maximum inten-
sity were in good correlation with the characteristic
sizes obtained by analyzing electron microscopic
images. MAIN COLLOIDAL CHARACTERISTICS
OF NANOPARTICLE DISPERSIONS
PRODUCED BY LASER ABLATION
IN LIQUID MEDIA For the studied range of pH 1.5–13, nanoparticles
of both metals obtained by laser treatment with differ-
ent durations retain a negative potential, while a
reduction in the potential values with decreasing pH
indicates the existence of isoelectric points for both
gold and silver nanoparticles at pH < 1.5. At the same
time, for gold nanoparticles at pH > 8 (see Fig. 6a), a
rather large scatter for the value of each measured
potential does not allow us to speak of any trend in the
changes of the absolute value of the zeta-potential
with a further increase in pH. The data obtained can
be interpreted as the constancy of the potential value COLLOID JOURNAL
Vol. 85
No. 2
2023 COLLOID JOURNAL
Vol. 85
No. 2
2023 NASTULYAVICHUS et al. 240 Fig. 6. Dependences of (1, 2) zeta-potentials and (3) sizes on dispersion pH for (a) gold and (b) silver nanoparticles obtained by
(1, 3) femtosecond and (2) nanosecond laser ablation. Nanoparticle concentrations in dispersions are shown in Table 1. (а)
(b)
0
0
0
1
2
1
2
3
Au
Ag
4
8
pH
12
16
0
4
8
pH
12
-Potential, mV
-Potential, mV
Particle size, nm
–20
–40
–60
–80
–20
–40
–60
–80
–100
120
80
40
0 (b)
0
1
2
Ag
0
4
8
pH
12
-Potential, mV
–20
–40
–60
–80 -Potential, mV Fig. 6. Dependences of (1, 2) zeta-potentials and (3) sizes on dispersion pH for (a) gold and (b) silver nanoparticles obtained by
(1, 3) femtosecond and (2) nanosecond laser ablation. Nanoparticle concentrations in dispersions are shown in Table 1. of gold at a level slightly exceeding –60 mV in a wide
range of pH > 8. media by the methods described above are in good
agreement with the data obtained earlier in a neutral
medium for smaller particles also produced by laser
methods [117–119]. In addition, the potentials of the
dispersions of gold nanoparticles obtained in this work
at different durations and powers of laser radiation
(Fig. 6) are in good agreement with each other within
the measurement error. For silver dispersions, the dif-
ference between the potentials is larger, while the
higher absolute values of the potential correspond to
nanoparticles generated with the femtosecond laser. For silver nanoparticles at high pH values, the
dependence is more pronounced, thereby indicating
an increase in the absolute value of the negative zeta-
potential. MAIN COLLOIDAL CHARACTERISTICS
OF NANOPARTICLE DISPERSIONS
PRODUCED BY LASER ABLATION
IN LIQUID MEDIA In recent years, the high negative surface charge of
inert metallic gold has been widely discussed in the lit-
erature [120–122]. The obvious mechanism of nega-
tive charging of such surfaces in aqueous media is the
physical adsorption of hydroxyl groups present in the
dispersion medium due to the dissociation of water
molecules. Such adsorption is provided by van der
Waals interactions between polarizable OH– ions and
metal particles. Some authors have related the nega-
tive zeta-potentials of aqueous gold dispersions to par-
tial oxidation of the surface atoms of a gold nanoparti-
cle to Au+ and Au3+ [104, 123, 124]. However, recent
detailed reviews [122, 125] have shown that, in many
cases, the existence of oxides on the surfaces of both
gold and silver nanoparticles is not detected in the
presence of a negative surface potential in aqueous dis-
persions. Another mechanism proposed recently in
the literature for negative charging of gold and silver
nanoparticles obtained by laser ablation is associated
with the presence of excess electrons formed in the
plasma during ablation and “captured” by the
nanoparticle surface [120, 122, 126]. Let us now consider in greater detail the stability of
gold and silver dispersions during their long-term stor-
age under room conditions (T = 25°С). In the general case, time variations in the nanopar-
ticle sizes of dispersions may be realized via four
mechanisms. The first mechanism is the dissolution of
particles leading to a decrease in their sizes. The sec-
ond mechanism, which is realized for particles dis-
persed in media with high concentrations of ionic or
molecular forms of the particle substance, is the Ost-
wald ripening or recondensation. This process is asso-
ciated with the dependence of the solubility of parti-
cles on their size and causes the growth of larger parti-
cles at expense of the dissolution of smaller ones. To
start such a process, the concentration of the dissolved
form of the particle substance must be higher and
lower than the saturation concentration for larger and
smaller particles, respectively. The third mechanism,
which is associated with the aggregation of nanoparti-
cles upon their approaching each other, obviously
leads to an increase in the average aggregate size. It
should be recollected here that the van der Waals
forces causing such aggregation are enhanced with the
aggregate sizes [127]. MAIN COLLOIDAL CHARACTERISTICS
OF NANOPARTICLE DISPERSIONS
PRODUCED BY LASER ABLATION
IN LIQUID MEDIA Gold nanoparticles are often
used as inert metal indicators to monitor specific
behavior of nanoparticles in an aqueous medium
[131]. At the same time, it has been shown that such
nanoparticles can be partly dissolved and exhibit a
high migration activity in biologically active media
[131–133]. In connection with all of the aforemen-
tioned, it is necessary to take into account the possible
solubility of the nanoparticles that we have obtained in
deionized water. The analysis of the evolution of the average
nanoparticle size with the time of dispersion aging in
deionized water for 70 days of storage for gold (Fig. 7a)
and silver (7b) particles has indicated a similar charac-
ter of variations in this parameter at the initial stage of
storage. For example, up to 10–14 days of the storage,
a decrease in the particle diameter is observed, with
this decrease being somewhat greater for silver parti-
cles. Such behavior of both dispersions can be
attributed to both the gradual sedimentation of larger
particles after their generation, because the studied
dispersions were not subjected to centrifugation before
the onset of the experiments, and the partial dissolu-
tion of the smallest particles. It is worth noting that,
due to the twice higher density of gold, aggregates with
smaller sizes will be subjected to intense sedimenta-
tion, while the lower solubility of gold must diminish
the effect of solubility on the decrease in the average
particle diameter. Then, a rather long (although hav-
ing different durations for gold and silver) period of
stable average particle diameter is established. How-
ever, at sufficiently long exposure times, t > 30–
35 days, a small but monotonous increase in the aver-
age particle diameter from 110 to 192 nm is observed
for 70 days of storage for silver nanodispersions. At the
same time, average particle diameters for gold remain
on the order of 120 nm up to 70 days of storage. The
significantly higher solubility of silver particles in
water leads us to assume a more significant role of the
Ostwald ripening in the growth of silver nanoparticles
than in the growth of gold ones. Below we shall focus
our attention on the analysis of the contributions from
surface forces of different natures to the aggregation-
type enlargement of particles of both types. MAIN COLLOIDAL CHARACTERISTICS
OF NANOPARTICLE DISPERSIONS
PRODUCED BY LASER ABLATION
IN LIQUID MEDIA Finally, sedimentation of parti- For nanodispersions obtained by the femtosecond
treatment in the aforementioned regimes, the size dis-
tribution determined for the resulting particles by
dynamic light scattering is quite narrow, with the scat-
tered intensity maximum being located in a size range
of 80–120 nm (Fig. 6a). The values of the zeta-poten-
tials for gold particles that we generated in neutral COLLOID JOURNAL
Vol. 85
No. 2
2023 LASER GENERATION OF COLLOIDAL NANOPARTICLES 241 the values average over the distribution (for a Gaussian
distribution, this is the position of the maximum in the
distribution curve) at different aging times of the dis-
persions. The distributions of the zeta-potentials
determined by the contributions of particles with dif-
ferent charges during each measurement cycle are
given in Figs. 7c and 7d. The analysis of the data
obtained has indicated the constancy of the average
values of the zeta-potentials for both gold and silver
nanodispersions throughout the observation period. At the same time, the half-widths of the size distribu-
tions determined at different aging times of the disper-
sions (Fig. 7e) remain almost unchanged for gold
nanoparticles, thus indicating the stability of the par-
ticle distribution in the dispersion and, hence, an
insignificant contribution of solubility to the content
of small particles. On the contrary, for silver nanopar-
ticles, a gradual asymmetric broadening of the size dis-
tribution is observed (Fig. 7f), thereby indicating the
aggregation of particles due to their collision-induced
interaction. The detailed analysis of the distribution in
the region of small sizes has shown a slight decrease in
the smaller sizes at the initial stage of aging. However,
after aging of silver dispersions for 35 days, both wings
of the distribution from the sides of both small and
large particle sizes, shift to the region of larger sizes. cles under the action of gravity will decrease the frac-
tion of large aggregates subjected to the Brownian
motion in a dispersion medium. Of course, when dis-
persing particles in reactive multicomponent media,
particle sizes may also change due to chemical trans-
formations of the particle surface. However, for noble
metal nanoparticles dispersed in deionized water, this
last mechanism does not play any role. The analysis of
the literature has shown a low solubility of silver
nanoparticles and silver oxides in aqueous media, with
the solubility increasing with a reduction in nanopar-
ticle sizes [128–130]. MAIN COLLOIDAL CHARACTERISTICS
OF NANOPARTICLE DISPERSIONS
PRODUCED BY LASER ABLATION
IN LIQUID MEDIA However, the results presented here indicate, as a
whole, a slight increase in the particle size, which can
be considered as a satisfactory kinetic stability of both
studied nanodispersions for 70 days of observation. It was noted above that, for the considered gold and
silver nanoparticles obtained by laser ablation in a liq-
uid without the use of surfactants, the dispersions can
be stabilized due to both the repulsion of double elec-
tric layers and the structural forces. The analysis of the
literature [112, 134, 135] has shown that the role of the
ion-electrostatic component can be revealed by add-
ing dissociating salts to a dispersion medium. Increas-
ing ion concentration in the dispersion medium leads
to the compression of the diffuse part of the double
layer and to a decrease in the ion-electrostatic repul-
sive forces [112, 134, 135]. Therefore, a sharp decrease
in the stability of the dispersion upon the addition of a
salt can be considered as an evidence of the key role of
the ion-electrostatic forces in the stabilization of the
dispersion under consideration. The determination of
the contribution from the structural forces is based on
the strong temperature dependence of these forces
[112, 134]; hence, the study of the evolution of disper-
sion stability with increasing temperature makes it
possible to estimate the role of structural forces. To determine the contributions of the described
mechanisms of the surface forces, we studied the
influence of temperature and the concentration of dis-
solved salts on the potentials and average sizes of dis-
persed particles. Figures 7a and 7b show the evolutions of the aver-
age zeta-potentials determined from the average elec-
trophoretic mobility of the particles. The scatter of the
data in Figs. 7a and 7b suggests an idea of variations in According to the literature data [112, 114, 134],
temperatures above 50°C should contribute to the COLLOID JOURNAL
Vol. 85
No. 2
2023 242
NASTULYAVICHUS et al. (e)
(f)
(а)
Au
Ag
(b)
(c)
0
Au
Storage time, days
Au
Storage time, days
Ag
Storage time, days
Ag
Storage time, days
(d)
0
0
20
40
Time, days
-Potential, mV
-Potential, mV
60
80
0
14
28
Time, days
42
56
70
140
-Potential, mV
Intensity, rel. LASER GENERATION OF COLLOIDAL NANOPARTICLES LASER GENERATION OF COLLOIDAL NANOPARTICLES Fig. 7. Evolution of parameters for dispersions of (a, c, e) gold and (b, d, f) silver nanoparticles in deionized water during 70-day
storage. (a, b) Dependences of average size and ζ-potential of nanoparticles on the storage time of dispersions; (c, d) time evolu-
tions of zeta-potential distributions determined by the contributions of particles with different charges; and (e, f) time evolutions
of nanoparticle size distributions. Dispersions were obtained by ablation with a femtosecond laser. Dispersion concentrations are
0.026 and 0.05 g/L for gold and silver nanoparticles, respectively. Figure 8 shows the data obtained for gold and silver
nanodispersions 10 min after adding the salt to the dis-
persions. The analysis of the data obtained has indi-
cated a significant effect of salt additives on the value
of the zeta-potential. For gold nanoparticles, the
absolute value of the potential decreases by almost
three times upon passing from nanoparticles in deion-
ized water to nanoparticles dispersed in a 0.1 M KCl
solution. For silver, the zeta-potential is almost
halved. The increase in the average diameter of gold
nanoparticles in the salt solutions (Fig. 8b) turns out to
be much larger than that for silver nanoparticles. It is
noteworthy that small additives of ions initiate only
partial aggregation of nanoparticles, which manifests
itself both as the broadening of particle size distribu-
tions in concentrated solutions (inset in Fig. 8b) and as
the discoloration of the dispersions. breakage of both the static and dynamic structures of a
thin liquid interlayer. Therefore, if structural forces
make a significant contribution to the stability of the
dispersions under investigation, heating should lead to
particle aggregation and loss of dispersion stability. In
this study, we compared dispersion parameters at
three temperatures: 25, 50, and 70°C. breakage of both the static and dynamic structures of a
thin liquid interlayer. Therefore, if structural forces
make a significant contribution to the stability of the
dispersions under investigation, heating should lead to
particle aggregation and loss of dispersion stability. In
this study, we compared dispersion parameters at
three temperatures: 25, 50, and 70°C. Table 2 presents the data obtained on the gold and
silver dispersions aged for 14 days before and after
exposure at an elevated temperature for 30 min. The
studied dispersions were prepared by ablation of the
targets with a femtosecond laser in water. LASER GENERATION OF COLLOIDAL NANOPARTICLES Although small changes in the average particle
diameters are observed upon heating, the processes for
gold and silver are differently directed and lead to a
decrease in the average diameter of gold nanoparticles
by 5 nm and an increase in the diameter of silver par-
ticles by 10 nm. In general, the detected changes do
not indicate a marked influence of temperature on the
colloidal characteristics of the dispersions, thus lead-
ing us to conclude that structural forces do not have a
significant effect on their stability. The presented data on the decrease in the zeta-
potential with an increase in the ionic strength of the
solution are in good agreement with the literature data
obtained for diverse surfaces [136, 137]. In this case,
the zeta-potentials at a KCl concentration of 10–1 M
are already too low to stabilize the nanodispersions
due to the ion-electrostatic component of the disjoin-
ing pressure. Thus, the array of the data obtained on
dispersions containing the dissolved salt indicates the
key role of the ionic-electrostatic interactions between
nanoparticles of both metals in the long-term stability
of nanodispersions in deionized water. To determine the role of the repulsion of electrical
double layers in the stability of our dispersions, we
studied the effect of the concentration of KCl added to
a dispersion on the zeta-potential of the particles and
their aggregation in the dispersion. As has been noted
above, a high concentration of ions in a dispersion
medium leads to the enrichment of the near-surface
layer of nanoparticles with counterions, which, being
concentrated in the dense part of the double layer,
screen the field of the charged nanoparticle surface
and, accordingly, lead to a decrease in the thickness of
the diffuse part of the double layer. The latter circum-
stance, in turn, reduces the value of the ion-electro-
static component of the disjoining pressure [112, 134,
135]. For disperse systems, in which the repulsion of
electrical double layers exceeds the van der Waals
attractive forces between nanoparticles, the stability of
dispersions is mainly determined by ion-electrostatic
forces. MAIN COLLOIDAL CHARACTERISTICS
OF NANOPARTICLE DISPERSIONS
PRODUCED BY LASER ABLATION
IN LIQUID MEDIA units
Intensity, %
Particle size, nm
Particle size, nm
–10
–20
–30
–40
–50
–60
0
-Potential, mV
–10
–20
–30
–40
–50
–60
120
100
80
60
200
150
100
50
0
40
20
0
Size
-Potential
Size
-Potential
1000
800
600
400
200
0
12
–150
–100
–50
0
50
0
200
400
600
0
200
400
600
–150
–100
–50
0
50
Intensity, rel. units
1000
800
600
400
200
0
7
14
21
28
35
42
70
0
7
14
21
28
35
42
70
0
7
14
21
28
35
42
70
0
7
14
21
28
35
42
70
10
8
6
4
2
Intensity, %
12
10
8
6
4
2 NASTULYAVICHUS et al. 242 (а)
Au
0
0
20
40
Time days
60
80
140
-Potential, mV
Particle size, nm
–10
–20
–30
–40
–50
–60
120
100
80
60
40
20
0
Size
-Potential (а)
Au
Ag
(b)
0
0
20
40
Time days
60
80
0
14
28
Time days
42
56
70
140
-Potential, mV
Particle size, nm
Particle size, nm
–10
–20
–30
–40
–50
–60
0
-Potential, mV
–10
–20
–30
–40
–50
–60
120
100
80
60
200
150
100
50
0
40
20
0
Size
-Potential
Size
-Potential Ag
(b)
0
14
28
Time, days
42
56
70
Particle size, nm
0
-Potential, mV
–10
–20
–30
–40
–50
–60
200
150
100
50
0
Size
-Potential (b) Time, days (e)
(c)
0
Au
Storage time, days
Time, days
-Potential, mV
Intensity, rel. units
1000
800
600
400
200
0
–150
–100
–50
0
50
7
14
21
28
35
42
70 (f)
Ag
Storage time, days
(d)
-Potential, mV
Time, days
–150
–100
–50
0
50
Intensity, rel. units
1000
800
600
400
200
0
0
7
14
21
28
35
42
70 (d) (c) Intensity, rel. units Intensity, rel. units (f) (e)
Au
Storage time, days
Intensity, %
12
0
200
400
Particle size, nm
600
0
7
14
21
28
35
42
70
10
8
6
4
2 (e) Ag
Storage time, days
0
200
400
Particle size, nm
600
0
7
14
21
28
35
42
70
Intensity, %
12
10
8
6
4
2 Au
Storage time, days Intensity, % Intensity, % Particle size, nm COLLOID JOURNAL
Vol. 85
No. 2
2023 243 CONFLICT OF INTEREST The authors declare that they have no conflicts of
interest. The stability and dimensional and electrochemical
parameters have been studied for gold and silver
nanoparticles generated in deionized water using
femto- and nanosecond lasers. The dependences of
the zeta-potentials of the particles on pH of dispersion
media have been found to be similar for dispersions
obtained by ablation of the same materials using lasers
with different pulse durations. At the same time, the
size of the generated gold nanoparticles has turned out
to be somewhat dependent on the pulse duration. All
obtained dispersions are satisfactorily stable during
long-term storage; however, they show a tendency to
aggregation with an increase in the ionic strength of
the dispersion medium. The analysis of the mecha-
nisms of the aggregative stability of dispersions has
shown the dominance of the contribution of the ion-
electrostatic interparticle interactions over the van der
Waals contribution. The study of the temperature
dependence of the dispersion stability has made it pos-
sible to reveal an insignificant role of the structural FUNDING The work on the characterization of nanoparticle dis-
persions was supported by the Russian Science Foundation,
project no. 21-13-00293, https://rscf.ru/project/21-13-
00293/. interaction forces between nanoparticles in the studied
dispersions. interaction forces between nanoparticles in the studied
dispersions. When comparing subpico-, pico-, and nanosecond
laser generation of gold nanoparticles in a liquid at a
wavelength in the near IR range, a pulse repetition fre-
quency of 20 kHz, and comparable scanning parame-
ters, the highest generation efficiency is observed for
nanosecond ablation, which is limited by the forma-
tion of screening subcritical ablation plasma. At the
same time, the efficiency per unit energy for picosec-
ond generation of nanoparticles free of the influence
of nonlinear effects turns out to be one or two orders
of magnitude higher than that for nanosecond genera-
tion. CONCLUSIONS The product of colloidal solution volume and the
extinction coefficient in the region of interband tran-
sitions recalculated per one pulse and unit radiation
energy has been proposed and verified as a compari-
son test for analyzing the efficiency of laser generation
of gold nanoparticles at comparable parameters of
laser systems with different pulse durations (subpico-,
pico-, and nanosecond ones). COLLOID JOURNAL
Vol. 85
No. 2
2023
Table 2. Changes in the parameters of nanodispersions upon heating
Conditions
Au
Ag
average particle
diameter, nm
average potential, mV
average particle
diameter, nm
average potential, mV
No heating
121 ± 3
–51 ± 4
105 ± 2
–43 ± 1
50°С, 30 min
117 ± 2
–54 ± 2
116 ± 3
–44 ± 2
70°С, 30 min
116 ± 1
–50 ± 2
116 ± 3
–45 ± 2 NASTULYAVICHUS et al. 244 (b)
500
12
Intensity, %
8
4
Particle size, nm
0
500
1000
1500
1
Particle size, nm
KCl concentration, mol/L
0
0.04
0.08
0.12
Au
Ag
400
300
200
100
2
3 Fig. 8. Variations in (a) ζ-potentials and (b) sizes of gold and silver nanoparticles obtained by femtosecond laser ablation in deion-
ized water with the addition of potassium chloride to the dispersion as depending on the concentration of the latter. The inset in
panel (b) shows the size distribution of gold nanoparticles in (1) additive-free dispersion and at KCl concentrations of (2) 0.05
and (3) 0.1 М. (а)
Au
KCl concentration, mol/L
(b)
–10
500
12
Intensity, %
8
4
Particle size, nm
0
500
1000
1500
1
-Potential, mV
Particle size, nm
–20
–30
–40
–50
0
0.04
0.08
0.12
KCl concentration, mol/L
0
0.04
0.08
0.12
Ag
Au
Ag
400
300
200
100
2
3 (а)
Au
KCl concentration, mol/L
–10
-Potential, mV
–20
–30
–40
–50
0
0.04
0.08
0.12
Ag (а) Fig. 8. Variations in (a) ζ-potentials and (b) sizes of gold and silver nanoparticles obtained by femtosecond laser ablation in deion-
ized water with the addition of potassium chloride to the dispersion as depending on the concentration of the latter. The inset in
panel (b) shows the size distribution of gold nanoparticles in (1) additive-free dispersion and at KCl concentrations of (2) 0.05
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https://openalex.org/W2092817189
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Association of hypovitaminosis D with Systemic Lupus Erythematosus and inflammation
|
Brazilian Journal of Nephrology
| 2,014
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cc-by
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1 Universidade Federal de Juiz
de Fora (UFJF).
2 Universidade de Leiden.
3 Universidade Salgado de
Oliveira (UNIVERSO).
4 Fundação Hospitalar do
Estado de Minas Gerais
(FHEMIG). Resumo Introduction: Nowadays it is described a high
prevalence of hypovitaminosis D in Systemic
Lupus Erythematosus (SLE), which is
associated with some clinical manifestations
and
increased
inflammatory
activity. Objective: To evaluate the association
between vitamin D insufficiency with
SLE
and
inflammatory
markers. Methods: Cross-sectional study, in which
have been evaluated 45 SLE patients
and 24 controls without the disease. Levels of 25-hydroxyvitamin D [25(OH)
D] less than 30 ng/mL were considered
inadequate. Disease activity was assessed
by the Systemic Lupus Erythematosus
Disease Activity Index (SLEDAI). High
sensitivity C reactive protein (hsCRP) and
interleukin-6 (IL-6) were evaluated for
verification of the inflammatory status. For assessment of renal involvement,
analysis of abnormal elements and
urinay
sediment
(AES),
quantitative
hematuria and pyuria, proteinuria and
creatinine clearance in 24-hour urine
and serum anti-double stranded DNA
were performed. Results: The prevalence
of 25(OH)D insufficiency was 55% in
SLE patients and 8% in the controls
participants (p = 0.001). The median
of 25(OH)D was lower in patients than
in controls. Patients with insufficient
25(OH)D had higher levels of IL-6 and
higher prevalence of hematuria in the
AES. There was no correlation between
vitamin D and SLEDAI or lupus nephritis. Conclusion: In our study, vitamin D
deficiency was more prevalent in patients
with SLE and was associated with higher
levels of IL-6 and hematuria. Introdução: Atualmente, é descrita ele-
vada prevalência de hipovitaminose D
no Lúpus Eritematoso Sistêmico (LES),
a qual se associa a algumas manifesta-
ções clínicas e maior atividade inflama-
tória. Objetivo: Avaliar a associação
entre
insuficiência
de
vitamina
D
com LES e marcadores inflamatórios. Métodos: Estudo transversal, tendo sido
avaliados 45 pacientes com LES e 24 con-
troles sem a doença. Níveis de 25-hidro-
xivitamina D [25(OH)D] menores que 30
ng/mL foram considerados insuficientes. A atividade da doença foi avaliada pelo Sys-
temic Lupus Erythematosus Disease Activi-
ty Index (SLEDAI). Foram avaliados, ainda,
proteína C reativa ultrassensível (PCRus) e
interleucina-6 (IL-6) para verificação do
status inflamatório. Para avaliação do en-
volvimento renal, foram realizados análise
de elementos anormais e sedimentoscopia
urinárias (EAS), hematúria e piúria quan-
titativas, proteinúria e depuração de creati-
nina em urina de 24 horas e anti-DNA de
dupla hélice sérico. Resultados: A preva-
lência de insuficiência de 25(OH)D foi de
55% nos pacientes lúpicos e 8% nos parti-
cipantes controles (p = 0,001). A mediana
da 25(OH)D foi menor nos pacientes do
que no grupo controle. Correspondência para:
Viviane Angelina de Souza.
Universidade Federal de Juiz
de Fora.
Rua Padre Café, nº 472/801, São
Mateus. Juiz de Fora, MG, Brasil.
CEP: 36016-450.
E-mail: vivi.reumato@gmail.com
Fundação IMEPEN (Instituto
Mineiro de Estudos e Pesquisas
em Nefrologia). Association of hypovitaminosis D with Systemic Lupus Erythematosus
and inflammation Autores
Viviane Angelina de Souza1
Marcus Gomes Bastos1
Natália Maria da Silva
Fernandes1,2
Henrique Novais Mansur3
Nádia Rezende Barbosa
Raposo1
Daniele Maria Knupp de
Souza4
Luiz Carlos Ferreira de
Andrade1 Data de submissão: 16/08/2013.
Data de aprovação: 27/03/2014. Associação de hipovitaminose D com Lúpus Eritematoso
Sistêmico e inflamação Association of hypovitaminosis D with Systemic Lupus Erythematosus
and inflammation Critérios de inclusão Foram incluídos no estudo pacientes com o
diagnóstico de LES (segundo os critérios da American
College of Rheumatology (ACR), propostos em 1982
e revisados em 1997,15,16 que tinham mais de 18
anos e assinaram o Termo de Consentimento Livre e
Esclarecido (TCLE). A deficiência de vitamina D, reconhecida
atualmente como epidêmica, pode ser um fator
ambiental responsável pelo desencadeamento do
LES.5 A função clássica da vitamina D é a regulação
da homeostase óssea;6 entretanto, há evidências de
que ela apresenta efeitos pluripotentes em vários
órgãos e sistemas, destacando-se, neste contexto,
seu papel sobre o sistema imunológico.7 Em relação
ao sistema imune, a vitamina D potencializa a
imunidade inata e suprime a imunidade adaptativa;
afeta indiretamente a polarização dos linfócitos T,
promovendo um desvio da resposta imune no sentido
de tolerância.8 Seu papel sobre as células B consiste
em inibir a secreção de anticorpos e a produção de
autoanticorpos.9 Introdução Universidade Federal de Juiz de Fora (CAS/HU-
UFJF). Identificaram-se 126 pacientes elegíveis, os
quais foram convidados a participar do estudo. Destes, 45 pacientes concordaram em participar e
foram incluídos no estudo. Estabeleceu-se um grupo
controle constituído por 24 indivíduos saudáveis (sem
quaisquer patologias detectáveis aos exames clínico e
laboratorial), pareados por sexo e idade e residentes
na mesma localização geográfica, estudantes dos
cursos de Medicina da UFJF e de Enfermagem da
Universidade Estácio de Sá, de Juiz de Fora. O Lúpus Eritematoso Sistêmico (LES) é uma
doença inflamatória crônica, multissistêmica, que
acomete principalmente mulheres jovens em idade
reprodutiva, em uma proporção de nove mulheres
para cada homem.1 A prevalência varia de 20 a
150 casos/100.000 indivíduos.2 Sua etiologia ainda
é obscura, e parece haver a interação de fatores
genéticos, hormonais, ambientais e imunológicos
para o desenvolvimento da doença.3 O envolvimento renal ainda constitui-se num dos
principais determinantes da morbimortalidade de
pacientes com LES. Manifesta-se clinicamente em
50% a 70% dos casos, mas 100% deles têm doença
renal à microscopia eletrônica (ME). Em geral, as
manifestações renais surgem nos primeiros dois a
cinco anos da doença.4 Critérios de não inclusão Gravidez e/ou presença de doenças sistêmicas que
levam a comprometimento renal, como diabetes
mellitus, vasculites, doenças infecciosas agudas,
hepatites virais B e C e Síndrome da Imunodeficiência
Adquirida (SIDA) foram considerados critérios de
não inclusão no estudo. Metodologia Os dados foram coletados de maio de 2010 a março
de 2011. O estudo foi aprovado pelo Comitê de Ética
em Pesquisa do Hospital Universitário da UFJF. Os
pacientes e controles que concordaram em participar
do estudo responderam um questionário estruturado
abordando as seguintes variáveis clínicas: sexo, idade,
raça (avaliada por autorrelato), exposição ao sol (em
horas/semana), estação do ano em que foi realizada a
avaliação, uso de protetor solar e tabagismo. Vários estudos demonstram elevada prevalência de
hipovitaminose D em doenças autoimunes, entre elas,
o LES.10-13 Além disso, no LES, é descrita a associação
entre deficiência de 25(OH)D e ocorrência de nefrite,
assim como associação com gravidade da doença.14 No Brasil, ainda são poucos os estudos abordando
o tema LES e vitamina D. O presente estudo teve
como objetivo avaliar a associação entre insuficiência
de vitamina D, LES e inflamação. Os pacientes que estavam em uso de cálcio e
vitamina D foram submetidos a um período de wash-
out de seis semanas (equivalente a três vezes a meia
vida da droga) antes da inclusão no estudo. Resumo Os pacientes com
insuficiência de 25(OH)D apresentaram
níveis mais elevados de IL-6 e maior pre-
valência de hematúria ao EAS. Não houve
correlação entre vitamina D, nefrite lúpica
e SLEDAI. Conclusão: Em nosso estudo, a
insuficiência de vitamina D foi mais preva-
lente em pacientes com LES e se associou
com níveis mais elevados de IL-6 e presença
de hematúria. 1 Universidade Federal de Juiz
de Fora (UFJF). 2 Universidade de Leiden. 3 Universidade Salgado de
Oliveira (UNIVERSO). 4 Fundação Hospitalar do
Estado de Minas Gerais
(FHEMIG). Data de submissão: 16/08/2013. Data de aprovação: 27/03/2014. Palavras-chave: inflamação; lúpus eritema-
toso sistêmico; nefrite lúpica; vitamina D. Keywords: inflammation; lupus nephritis;
lupus erythematosus, systemic; vitamin D. 430 Lúpus Eritematoso Sistêmico e vitamina D Nefrite lúpica A definição de nefrite lúpica obedeceu aos critérios
do ACR.16 Caso o paciente tivesse sido submetido
à biópsia renal, foi respeitada a classificação de
glomerulonefrite lúpica da OMS proposta em 1989
e revisada em 2004.17 Para avaliação laboratorial do
envolvimento renal, foram realizados EAS, hematúria
e piúria quantitativas, proteinúria e depuração de
creatinina em urina de 24 horas, além do anti-DNA
nativo sérico. Vinte pacientes (44,4%) foram classificados como
apresentando nefrite lúpica, de acordo com os critérios
do ACR. Destes, 8 (44%) foram submetidos à biópsia
renal, predominando a classe IV da OMS em 5 casos
(62,5%). No subgrupo de pacientes com nefrite,
a média de idade foi de 34,9 ± 7,3 (22-50 anos), a
média do SLEDAI foi de 10 (0-24), apresentaram
níveis de IL-6 variando de 0,9-13,5, com mediana
de 5,0 pg/mL e PCRus entre 0,5-36,2, mediana 4,8
mg/L. A creatinina sérica apresentou média de 0,8
(0,5-2,6) mg/dL e a média da depuração da creatinina
foi de 43,9 (35,6-220) mL/min/1,73 m2. Com relação
à proteinúria, a mesma variou entre 112 a 4000, com
mediana de 946 mg/24 horas. Seus níveis de vitamina
D variaram entre 20,8-44,2 com mediana de 29,5 ng/
mL. Não houve diferença entre os níveis de vitamina
D entre pacientes com LES e nefrite vs. LES sem
nefrite. Análise estatística A amostra do estudo foi estabelecida por
conveniência. A normalidade das variáveis foi
avaliada pelo teste de Shapiro-Wilk e a estatística
descritiva foi realizada por meio da verificação da
média ou mediana para as variáveis contínuas. A
frequência absoluta e relativa foi utilizada para as
variáveis categóricas. Quanto às diferenças entre
os grupos de pacientes e controles, estas foram
avaliadas pelos testes não paramétricos de Mann-
Whitney quando as variáveis foram ordinais ou
intervalares e qui-quadrado quando as variáveis
foram nominais. A correlação entre a 25(OH)D e as
variáveis utilizadas para avaliar nefrite, atividade
da doença e inflamação estudadas foi calculada
pelo coeficiente de correlação de Pearson. Comparando os pacientes lúpicos (amostra total)
com os controles, estes apresentaram prevalência
significativamente superior de proteinúria avaliada
pela fita de imersão comparados aos controles
[16 (35,6%) e 1 (4,0%), p = 0,012]. A mediana
da proteinúria de 24 horas no grupo de pacientes
lúpicos foi estatisticamente superior à encontrada nos
controles [234 (1-4.000) mg/24 horas e 105,5 (47,5-
189,5) mg/24 horas, p = 0,003]. Não houve diferença
estatisticamente significante da creatinina sérica [0,7
(0,5-2,6) mg/dL e 0,7 (0,5-1,0) mg/dL, p = 0,182]
e da depuração da creatinina [101,1 (34-220) mL/
min/1,73 m2 e 107,6 (60-232,7) mL/min/1,73 m2,
(p = 0,258)] entre os grupos doente e controle. A comparação entre os subgrupos de pacientes
com insuficiência e suficiência de vitamina D e o grupo
controle foi realizada pelo teste não paramétrico de
Kruskal-Wallis. Os pacientes com insuficiência de vitamina D
apresentaram maior prevalência de hematúria ao EAS
comparados aos pacientes com suficiência e controles
[10 (40,0%), 3 (15,0%) e 3 (12,5%), (p = 0,043)],
respectivamente. Foi considerado significante p < 0,05. Analisaram-se
os dados por meio do programa SPSS (Statistical Package
for Social Sciences) Inc, Chicago, IL, EUA, versão 19.0. Vitamina D A determinação da 25(OH)D total sérica foi
realizada pelo método de cromatografia líquida de
alta performance (HPLC), utilizando-se sistema da
Shimadzu (Tóquio, Japão). Os níveis de 25(OH)
D foram considerados suficientes se ≥ 30 ng/mL,
insuficientes se entre 15 e 29 ng/mL e deficientes se <
15 ng/mL.7 Amostra A atividade da doença foi avaliada pelo Systemic Lupus
Erythematosus Disease Activity Index (SLEDAI). O
status inflamatório foi avaliado pelas análises séricas da
PCRus (pela técnica de turbidimetria) e IL-6 (por meio
de imunoensaio enzimático competitivo - ELISA). O estudo foi do tipo transversal. Foi realizada
revisão de prontuários médicos dos pacientes com
LES do ambulatório de Reumatologia do Centro
de Atenção à Saúde do Hospital Universitário da J Bras Nefrol 2014;36(4):430-436 431 Lúpus Eritematoso Sistêmico e vitamina D Nefrite lúpica A mediana dos níveis séricos da 25(OH)
D foi menor nos pacientes com LES (29,48 ng/
mL, variação de 20,83-44,23 ng/mL) do que nos
participantes controles (37,68 ng/mL, variação de
22,91-44,07 ng/mL) (p = 0,001). A prevalência de
insuficiência de 25(OH)D foi maior no grupo de
pacientes com LES (55%) do que no grupo controle
(8%) (p = 0,001). Resultados Insuficiência de vitamina D e IL-6 no LES - Box-plots da
variável IL-6 (pg/mL) para os pacientes com insuficiência [4,464 pg/
mL (1,021- 52,049)] e suficiência de vitamina D [3,292 pg/mL (0,898-
10,447)]; e controles [1,386 pg/mL (0,820- 6,934)] (p < 0,0001). Teste
de Kruskal-Wallis, p < 0,05. Tabela 2
Características clínicas basais dos
pacientes
Análise descritiva: mediana (mínimo-máximo) para as variáveis
contínuas e n (%) para as variáveis categóricas. Pacientes (n = 45)
Tempo de duração (meses)
72 (3-264)
SLEDAI
10 (0-24)
SLEDAI
29 (64,4%)
Atividade (≥ 6)
Uso de corticoide
42 (93,3%)
Dose de corticoide (mg de prednisona)
20 (0-60)
Cálcio/vitamina D
8 (12,0%)
Antimaláricos
34 (75,55%)
Difosfato de cloroquina 250 mg/dia
22 (48,9%)
Hidroxicloroquina 400 mg/dia
12 (26,7%)
Imunossupressores
25 (55,55%)
Azatioprina
17 (37,8%)
Micofenolato Mofetil
1 (2,2%)
Ciclofosfamida
3 (6,7%)
Metilprednisolona + ciclofosfamida
1 (2,2%)
Rituximabe
1 (2,2%)
Metilprednisolona
1 (2,2%)
Metotrexate
2 (4,4%) Figura 1. Insuficiência de vitamina D e IL-6 no LES - Box-plots da
variável IL-6 (pg/mL) para os pacientes com insuficiência [4,464 pg/
mL (1,021- 52,049)] e suficiência de vitamina D [3,292 pg/mL (0,898-
10,447)]; e controles [1,386 pg/mL (0,820- 6,934)] (p < 0,0001). Teste
de Kruskal-Wallis, p < 0,05. Figura 1. Insuficiência de vitamina D e IL-6 no LES - Box-plots da
variável IL-6 (pg/mL) para os pacientes com insuficiência [4,464 pg/
mL (1,021- 52,049)] e suficiência de vitamina D [3,292 pg/mL (0,898-
10,447)]; e controles [1,386 pg/mL (0,820- 6,934)] (p < 0,0001). Teste
de Kruskal-Wallis, p < 0,05. 21°45’’ ao sul, demonstrou elevada prevalência de
insuficiência de vitamina D em pacientes portadores
de LES comparados aos controles, em concordância
com os resultados de Fragoso et al.,10 que observaram
insuficiência de vitamina D em 57,7% de 78
pacientes portadores de LES em estudo realizado em
Pernambuco. Outros três estudos brasileiros também
reforçam os nossos achados.11-13 Os níveis de IL-6 foram superiores no grupo com
insuficiência de vitamina D [4,464 pg/mL (1,021-
52,049)], comparado ao grupo com suficiência [3,292
pg/mL (0,898-10,447)], e ao grupo controle [1,386
pg/mL (0,820-6,934)] (p < 0,0001) (Figura 1). Com
relação aos níveis de PCRus, não foram observadas
diferenças. Nosso estudo não observou correlação entre
vitamina D e as variáveis utilizadas para avaliar
nefrite lúpica. Observamos maior prevalência de
hematúria nos pacientes com insuficiência de vitamina
D quando comparados aos grupos com suficiência
dessa vitamina e controles. Resultados Os pacientes avaliados
apresentaram-se com doença renal controlada na
maioria dos casos, o que pode ter influenciado a análise
dos resultados. A nefrite lúpica constitui-se numa das
principais causas de morbimortalidade no LES e a
associação entre hipovitaminose D e nefrite lúpica
foi avaliada em estudos clínicos, como o de Kamen
et al.,22 que evidenciaram associação entre deficiência
de vitamina D e nefrite. Avaliando uma população de
LES juvenil, Robinson et al.23 observaram associação
inversa entre níveis de 25(OH)D e relação proteína/
creatinina, além de níveis mais baixos de vitamina D
em pacientes com proteinúria. A análise bivariada mostrou fraca evidência de
correlação inversa entre vitamina D e IL-6 (r = -0,276;
p = 0,066). Não se observou correlação entre vitamina
D e as outras variáveis utilizadas para avaliação de
atividade da doença e nefrite lúpica. Resultados Com relação à atividade da doença avaliada pelo
SLEDAI, 64,4% (n = 29) dos pacientes apresentavam
doença ativa (SLEDAI ≥ 6). A mediana do SLEDAI
foi de 10 (0-24) (Tabela 2). As
características
clínicas
basais
dos
grupos
de pacientes e controles, assim como os dados
laboratoriais estão expostas nas Tabelas 1 e 2. J Bras Nefrol 2014;36(4):430-436 432 Lúpus Eritematoso Sistêmico e vitamina D Tabela 1
Características clínicas, demográficas e laboratoriais dos grupos
Pacientes
(n = 45)
Controles
(n = 24)
p valor
Idade (anos)
35,3 (20-52)
26 (18-53)
NS
Sexo
NS
Masculino
1 (2,2%)
1 (4,0%)
Feminino
44 (97,8%)
24 (96,0%)
Raça
< 0,0001
Branca
21 (46,7%)
22 (88,0%)
Não Branca
24 (53,3%)
3 (12,0%)
Estação do ano
13 (28,88%)
1 (4,0%)
NS
Outono
20 (44,44%)
0 (0,0%)
Inverno
7 (15,56%)
10 (40,0%)
Primavera
5 (11,11%)
14 (56,0%)
Verão
Exposição ao sol (horas/semana)
0,002
< 1
16 (35,5%)
2 (8,0%)
1 a 2
12 (26,7%)
2 (8,0%)
3 a 4
6 (13,3%)
4 (16,0%)
4 a 5
1 (2,2%)
5 (20,0%)
> 5
10 (22,2%)
12 (48,0%)
Uso de protetor solar
NS
Não
7 (15,66%)
9 (36,0%)
Sim
38 (84,44%)
16 (64,0%)
Número de aplicações
NS
0
7 (15,6%)
9 (36,0%)
1
22 (48,9%)
9 (36,0%)
2
8 (17,8%)
5 (20,0%)
> 2
8 (17,8%)
2 (8,0%)
Tabagismo
0,048
Não
38 (84,4%)
24 (96,0%)
Sim
7 (15,6%)
1 (4,0%)
VHS 1ª hora (mm)
36 (2-173)
15 (3-42)
< 0,0001
PCRus (mg/L)
4,9 (0,4-67,9)
2,5 (0,1-11,3)
0,002
C3 (mg/dL)
156,9 (80,8-301,7)
NA
NA
C4 (mg/dL)
25,8 (10,2-67,0)
NA
NA
IL-6 (pg/mL)
3,81 (0,898-52,049)
1,38 (0,820-6,934)
< 0,0001
Cálcio Total (mg/dL)
9,6 (8,1-11,3)
10,4 (8,5-11,1)
0,015
Fósforo (mg/dL)
3,9 (2,4-5,3)
3,8 (2,8-4,8)
NS
PTH intacto (pg/mL)
44,9 (6,5-545,9)
35,4 (19,0-80,7)
NS
25(OH)D (ng/mL)
29,48 (20,83-44,23)
37,68 (22,91-44,07)
< 0,0001
Depuração de creatinina (mL/min/1,73 m2)
101,1 (34-220)
107,6 (60-232,7)
NS
Proteinúria (mg/24 horas)
234 (1-4.000)
105,5 (47,5-189,5)
0,003
Presença de Hematuria
13 (28,9%)
3 (12,5%)
NS
Dados expressos em mediana (mínimo e máximo) ou n (%); NA: Não aplivável; NS: Não significativo. Características clínicas, demográficas e laboratoriais dos grupos J Bras Nefrol 2014;36(4):430-436 433 Lúpus Eritematoso Sistêmico e vitamina D Figura 1. Discussão Neste estudo, observamos maior prevalência de insu-
ficiência de vitamina D em pacientes com LES, assim
como associação desta com níveis mais elevados de
IL-6. Atualmente,
é
descrita
elevada
prevalência
mundial de hipovitaminose D no LES, em diversas
localizações geográficas.18,19 Dados da população
brasileira, em geral, também refletem os baixos níveis
de vitamina D, mesmo em indivíduos saudáveis.20,21
Os resultados do presente estudo, realizado na
cidade de Juiz de Fora, localizada a uma latitude de A despeito de haver estudos mostrando correlação
entre vitamina D e marcadores de atividade renal, nossos
resultados não os corroboram. Em um estudo iraniano
que avaliou pacientes com LES, aqueles com os níveis J Bras Nefrol 2014;36(4):430-436 434 Lúpus Eritematoso Sistêmico e vitamina D de 25(OH)D menores que 5 ng/mL apresentaram títulos
mais elevados de anti-DNA nativo.24 Uma associação
inversa entre 25(OH)D e os níveis de anti-DNA nativo
(r = -0,13; p = 0,02) e anti-C1q (r = -0,14; p = 0,02)
também foi observada em estudo recente de Mok et al.25 Conclusão Nas pacientes lúpicas estudadas, a insuficiência de
vitamina D foi prevalente e se associou com níveis mais
elevados de IL-6 e com presença de hematúria. Não
foi observada correlação significativa entre os níveis
de vitamina D, nefrite lúpica e SLEDAI. Entretanto,
mais estudos clínicos randomizados são necessários
para avaliar a influência da vitamina D no LES, bem
como estabelecer os níveis de vitamina D necessários
para demonstrarmos seus efeitos imunomoduladores
nesses pacientes. Estudo recente publicado por Petri et al.14
evidenciou melhora significativa da relação proteína/
creatinina após suplementação com vitamina D em
pacientes com níveis insuficientes de 25(OH)D. O LES é, por definição, uma doença inflamatória
autoimune,
e
é
descrito
status
inflamatório
exacerbado nesses pacientes. No presente estudo,
foi observado aumento significativo de PCRus e
de IL-6 nos pacientes quando comparados aos
controles. Analisando especificamente a relação entre
a 25(OH)D e IL-6, essa citocina apresentou níveis
significativamente superiores nos pacientes com
insuficiência de vitamina D, quando comparados
aos pacientes com suficiência e grupo controle. Foi
observada fraca evidência de associação inversa entre
vitamina D e essa citocina nos pacientes avaliados
(r = -0,276; p = 0,066). Conforme já mencionado
previamente, tais resultados podem refletir o baixo
índice de atividade da doença dos pacientes avaliados,
nos quais houve predomínio de atividade leve. Referências 1. Lanna CCD, Ferreira GA, Telles RW. Lúpus Eritematoso Sis-
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199905000-00002 Concordando com nossos achados, Amezcua-
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SLEDAI e VHS/PCR no LES; associação também
encontrada por Chun et al.27 Entretanto, Firooz et al.28
não demonstraram associação desses marcadores
inflamatórios com atividade da doença. A IL-6 é uma
citocina que exerce influência sobre a regulação do
sistema imune e inflamação, atuando na diferenciação
de linfócitos B e T.29 No LES, ocorre aumento dos
níveis de várias citocinas inflamatórias, como IL-6, IL-
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trev.2011.08.003 O
nosso
estudo
apresenta
limitações. Primeiramente, por se tratar de avaliação de natureza
transversal,
não
podemos
sugerir
causalidade
entre as associações encontradas e a ocorrência de
hipovitaminose D no LES. O número de pacientes
avaliados (n = 45) também pode ter influenciado
os resultados, assim como o fato de os pacientes
apresentaram baixos índices de atividade da doença. Uma análise posterior, incluindo um maior número de
pacientes incidentes e com doença mais grave, poderia
nos proporcionar resultados mais robustos no que diz
respeito à associação entre insuficiência de vitamina
D, nefrite lúpica, atividade da doença e inflamação. Referências The 1982 revised criteria for the classification of systemic
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guez V, Rivera-Martinez E, Castillo-Martinez D, et al. C-reactive
protein and complement components but not other acute-phase
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nosso
estudo
apresenta
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transversal,
não
podemos
sugerir
causalidade
entre as associações encontradas e a ocorrência de
hipovitaminose D no LES. O número de pacientes
avaliados (n = 45) também pode ter influenciado
os resultados, assim como o fato de os pacientes
apresentaram baixos índices de atividade da doença. O
nosso
estudo
apresenta
limitações. Primeiramente, por se tratar de avaliação de natureza
transversal,
não
podemos
sugerir
causalidade
entre as associações encontradas e a ocorrência de
hipovitaminose D no LES. O número de pacientes
avaliados (n = 45) também pode ter influenciado
os resultados, assim como o fato de os pacientes
apresentaram baixos índices de atividade da doença. Uma análise posterior, incluindo um maior número de
pacientes incidentes e com doença mais grave, poderia
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http://dx.doi.org/10.1016/j.autrev.2005.05.009 J Bras Nefrol 2014;36(4):430-436 436 J Bras Nefrol 2014;36(4):430-436
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Complete Genome of Hepatitis E Virus from Laboratory Ferrets
|
Emerging infectious diseases
| 2,014
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cc-by
| 2,733
|
Author affiliations: National Institute of Infectious Diseases, Tokyo,
Japan (T.-C. Li, Y. Ami, Y, Suzaki, M. Shirakura, N. Kishida, H.
Asanuma, W. Takaji); Affiliated Hospital of Qingdao University
Medical College, Qingdao, China (T. Yang); and Osaka University,
Osaka, Japan (N. Takeda) Complete Genome
of Hepatitis E Virus
from Laboratory
Ferrets the antigenicity, pathogenicity, and epidemiology of ferret
HEV remain unclear. Ferret HEV was also recently detected in the United
States in serum (11), suggesting that ferret HEV infection
is not restricted to the Netherlands and might be distrib-
uted in ferrets worldwide. Because ferrets are susceptible
to several respiratory viruses, including human and avian
influenza viruses, and severe acute respiratory syndrome
coronavirus (12,13), ferrets have been used as a small-ani-
mal model for these viruses. Ferrets are also kept as pets in
many countries. Thus, information about ferret HEV epi-
demiology, distribution, transmission, and pathogenesis is
urgently needed. The complete genome of hepatitis E virus (HEV) from
laboratory ferrets imported from the United States was iden-
tified. This virus shared only 82.4%–82.5% nt sequence
identities with strains from the Netherlands, which indicated
that the ferret HEV genome is genetically diverse. Some
laboratory ferrets were contaminated with HEV. In this study, we amplified and analyzed the complete
genome of the US strains of ferret HEV to confirm whether
US strains are new ferret HEV genotypes. Phylogenetic
analysis demonstrated that HEVs detected in laboratory
ferrets from the United States are genetically different from
those detected in the Netherlands, suggesting that the ferret
HEV genome is genetically diverse. H H
epatitis E virus (HEV) is a single-stranded positive-
sense RNA virus that belongs to the family Hepeviri-
dae, genus Hepevirus, and is the causative agent of hepa-
titis E (1,2). Because the transmission of HEV from deer,
swine, and wild boars to humans is well known, hepatitis
E is recognized as a zoonosis. Zoonotic hepatitis E is as-
sociated mainly with genotype 3 (G3) and G4 HEV infec-
tion (3,4). In addition to deer, swine, and wild boars, other
animals, including monkeys, rats, ferrets, chickens, and
bats, harbor HEV or HEV-like viruses (5–9). The genus
Hepevirus might include 3 additional species (avian HEV,
bat HEV, and rat/ferret HEV) (10). However, whether
HEV from these animals is transmitted to humans is
not clear. The Study Oligonucleotides used to amplify ferret hepatitis E viruses
Primer (nucleotide positions)*
Sequence, 53
Product length, bp†
Forward FF1 (1–21)
GGCAGACCCCTAATGGAGACA
Reverse FR628 (628–648)
GTTGCGTGCGACATAGGCCTT
626
Forward FF541 (541–561)
AGCAATGTATCGCCATGGCAT
Reverse FR1535 (1535–1554)
ATCTGCATCAGTCGGGCACA
1,014
Forward FF1518 (1518–1538)
AGGATCTGACAGTAGACCTGT
Reverse FR2555 (2555–2577)
TGCAATGCCAAATTAGCTGTGT
1,060
Forward FF2401 (2401–2421)
GGCGATGAGTTGTACCTGTTA
Reverse FR3424 (3432–3445)
GAGCAGCCGGTAACATACTCAA
1,045
Forward FF3336 (3336–3355)
GCACAATTTCTATCTCACCA
Reverse FR4210 (4210–4230)
ACTCCGAATCAGATGATACA
985
Forward FF4181 (4181–4202)
GGCTGGTGCACCTGAATGGCT
Reverse FR5800 (5800–5821)
TCAGGCAGACGGCGTATCTTAT
1,641
Forward FF4812 (4812–4831)
ATGGAGCATGTGTACAAGAT
Reverse TX30SXN
GACTAGTTCTAGATCGCGAGCGGCCGCCC
TTTTTTTTTTTTTTTTTTTTTTTTTTTTTT
2,050
Reverse FR451‡
ACACCGTGTGAATCCCTCCGT
Abridged amplification
GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG
Reverse FR279 (279–300)
ATAGATCTAGGATGCGCACCAA
§
Abridged universal amplification
GGCCACGCGTCGACTAGTAC
Reverse FR191 (191–211)
CGGATGCGACCAAACAACAGA
240
*Values in parentheses indicate positions of the primer corresponding to the entire genome of hepatitis E virus (JN998607) isolated from ferret. †Blank cell indicates that 1 primer pair produced 1 product. ‡Used only for cDNA synthesis. §PCR product was not detected. Table 1. Oligonucleotides used to amplify ferret hepatitis E viruses from the Netherlands. In addition, the ferret HEV strains
from the United States have 2 aa insertions between amino
acid residues 596–597 (Thr) and 631–632 (Ile) and 9 aa
deletions in amino acid residues 645–653 (Cys-Leu-Arg-
Ser-Ser-Pro-Lys-Pro-Pro), which corresponds to those of
strains from the Netherlands. Similar to ferret HEV from
the Netherlands, an additional putative 183-aa ORF 4 (nt
30–581) was found in strains from the United States. Anal-
ysis of 5 other entire ORF2 sequences (GenBank accession
nos. AB890375–AB890379) showed that nucleotide iden-
tities among them were 98.9%–99.5%, which indicated
that genetically similar ferret HEV strains had circulated at
the ferret farm in the United States. manufacturer’s instructions. All PCR products were puri-
fied by using the QIAquick PCR Purification Kit (QIAGEN,
Valencia, CA, USA) and cloned into the TA cloning vec-
tor pCR2.1 (Invitrogen). Nucleotide sequencing was con-
ducted by using an ABI 3130 Genetic Analyzer Automated
Aequencer (Applied Biosystems, Foster City, CA, USA). Both ferret HEV genomes consisted of 6,820 nt and
a poly (A) tail (GenBank accession nos. AB890001 and
AB890374), and nucleotide sequence identity was 99.6%. Genomic structure of strains from the United States was
similar to that of 2 strains from the Netherlands. The amino
acid alignment of ORF2 indicated that ferret HEV ORF2
has an additional 6 amino acids at the N terminus. Howev-
er, because the seventh codon is AUG, it is unclear which
codon was used for the ORF2 translation initiation. The Study Sixty-three fecal samples were collected from labora-
tory ferrets (Mustela putorius furo) at the National Institute
of Infectious Diseases, Tokyo, Japan, on May 24, 2013. These ferrets had been imported from a farm in the United
States for influenza research 7 days before sample collec-
tion. Fecal specimens were diluted with 10 mmol/L phos-
phate-buffered saline to prepare a 10% suspension, shaken
at 4°C for 1 h, and clarified by centrifugation at 10,000 ×
g for 30 min. The supernatant was passed through a 0.45-
µm membrane filter (Millipore, Bedford, MA, USA), and
stored at –80°C until use. RNA was extracted by using the MagNA Pure LC
Total Nucleic Acid Isolation Kit (Roche Applied Science,
Mannheim, Germany) according to the manufacturer’s
recommendations. Reverse transcription was performed
by using the Superscript II RNase H– reverse transcription
procedure (Invitrogen, Carlsbad, CA, USA) and primer
TX30SXN as described (14). Ferret HEV RNA was detect-
ed by using a nested, broad-spectrum reverse transcription
PCR (15). Forty (63.5%) of 63 fecal specimens were posi-
tive for ferret HEV RNA. Sequences were similar to those
detected in ferret serum samples in the United States (11),
which suggested that the laboratory ferrets were infected in
the United States and then transported to Japan. HEV has been detected in ferrets (Mustela putorius)
in the Netherlands (6). The ferret HEV genome contains
3 open reading frames (ORFs 1–3). ORF1 encodes a non-
structural protein of 1,596; ORF2 encodes a capsid pro-
tein of 654 aa, and ORF3 encodes a functionally unknown
phosphoprotein of 108 aa. A putative ORF4 observed in the
ferret HEV genome was also found in the rat HEV genome. Nucleotide sequence analyses indicated that the ferret HEV
genome shares the highest nucleotide sequence identity
(72.3%) with rat HEV. The nucleotide sequence identity
between the ferret HEV and G1–4 HEV, rabbit HEV, and
avian HEV ranges from 54.5% to 60.5% (6). However, RNA from 2 ferret HEVs was randomly selected, and
the full-length genome was amplified by using reverse tran-
scription PCR with primers based on nucleotide sequenc-
es derived from strains from the Netherlands and United
States (Table 1). Sequence of the 5′-terminal noncoding
regions of the genome was determined by using Rapid Am-
plification of cDNA Ends Kits (Invitrogen) according to the DOI: http://dx.doi.org/10.3201/eid2004.131815 709 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 20, No. 4, April 2014 DISPATCHES Table 1. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 20, No. 4, April 2014
*HEV, hepatitis E virus; ORF, open reading frame. The Study Nucleotide and deduced amino acid sequence identi-
ties between ferret HEV from the United States and other
HEVs are shown in Table 2. The entire genome of strains
from the United States shared relatively high nucleotide The ORF1 of strains from the United States encodes
1,589 aa, which is 7 aa shorter than ORF1 of both strains Table 2. Nucleotide and deduced amino acid sequence identities between ferret HEV fro
Ferret HEV Table 2. Nucleotide and deduced amino acid sequence identities between ferret HEV from the Unite
Ferret HEV (AB89037
N
l
id
% 710
Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 20, No. 4, April 2014
Table 2. Nucleotide and deduced amino acid sequence identities between ferret HEV from the United States and other HEVs*
HEV strain
(GenBank accession no.)
Full-length
genome, %
Ferret HEV (AB890374)
Nucleotides, %
Amino acids, %
ORF1
ORF2
ORF3
ORF1
ORF2
ORF3
Genotype 1 (NC-001434)
53.6
51.4
58.9
50.3
51.4
56.9
22.2
Genotype 2 (M74506)
53.8
51.7
59.3
49.4
56.0
57.2
25.2
Genotype 3 (AF060668)
55.2
53.6
59.4
47.9
54.5
58.1
22.9
Genotype 4 (AJ272108)
53.9
51.5
59.5
49.4
53.5
57.5
28.8
Wild boar HEV
(AB573435)
54.4
51.9
60.5
49.1
57.2
57.3
24.3
Wild boar HEV
(AB602441)
54.0
51.9
59.1
50.9
57.4
57.0
31.5
Rabbit HEV (FJ906895)
54.8
57.0
60.5
51.2
55.2
57.7
23.3
Rat HEV (GU345042)
61.2
57.0
71.3
63.3
70.1
79.4
40.4
Rat HEV (JX120573)
62.6
58.1
72.8
64.9
74.1
80.0
43.5
Ferret HEV (998606)
82.4
81.4
84.9
85.9
91.5
94.2
73.1
Ferret HEV (998607)
82.4
81.3
85.1
85.9
91.3
94.8
73.1
Ferret HEV (AB890001)
99.6
99.7
99.5
100.0
99.7
99.8
100.0
Avian HEV (AY535004)
50.8
50.5
54.2
47.0
43.1
47.9
24.2
Bat HEV (JQ001749)
46.8
49.7
54.7
48.8
44.6
54.3
42.9
*HEV, hepatitis E virus; ORF, open reading frame. 710 Complete Genome of HEV from Laboratory Ferrets F
a
b
fe
N
w
X
G
b
m
1
g
jo
n
b
a
It
is
a
v
n Figure. Phylogenetic relationships
among genotypes 1–4 and wild
boar, rabbit, rat, avian, bat, and
ferret isolates of hepatitis E virus. Nucleic acid sequence alignment
was performed by using Clustal
X 1.81 (www.clustal.org/clustal2/). Genetic distance was calculated
by using Kimura’s 2-parameter
method. Phylogenetic trees with
1,000 bootstrap replicates were
generated by using the neighbor-
joining method (Njplot 2.3, http://
njplot.sharewarejunction.com/)
based on A) the entire genome
and B) open reading frame 2. Conclusionsi We amplified the entire genome of 2 ferret HEV
strains isolated from laboratory ferrets imported from the
United States. Nucleotide sequence comparisons showed
that 2 ferret HEV strains from the United States had high
(99.6%) identity and shared 98.6%–100% identities with
partial sequences of ORF1 that were detected in the United
States (11), which indicated that genetically similar ferret
HEV was circulating in laboratory ferrets. The Study Items in boldface indicate strains
isolated in this study. Numbers
along branches indicate bootstrap
values. Scale
bars
indicate
nucleotide substitutions per site. sequence identities (82.4%–82.5%) with strains from the
Netherlands. We generated phylogenetic trees based on
ORF2 or the entire genome. These trees showed that al-
though strains from the United States were closely related
to strains from the Netherlands, they formed a new and
distinct cluster (Figure). We observed similar phyloge-
netic clustering when we analyzed nucleotide sequenc-
es of ORF1 and ORF3 separately. Although we cannot
conclude whether ferret HEV from the United States is a
new genotype, these results indicated that there is genetic
variety in ferret HEV. Researchers should also bear in
mind that some laboratory ferrets are contaminated with
ferret HEV. sequence identities (82.4%–82.5%) with strains from the
Netherlands. We generated phylogenetic trees based on
ORF2 or the entire genome. These trees showed that al-
though strains from the United States were closely related
to strains from the Netherlands, they formed a new and
distinct cluster (Figure). We observed similar phyloge-
netic clustering when we analyzed nucleotide sequenc-
es of ORF1 and ORF3 separately. Although we cannot
conclude whether ferret HEV from the United States is a
new genotype, these results indicated that there is genetic
variety in ferret HEV. Researchers should also bear in
mind that some laboratory ferrets are contaminated with
ferret HEV. In conclusion, we isolated and identified 2 ferret HEV
strains from laboratory ferrets imported from the United
States. These strains were genetically distinct from ferret
HEV isolates from the Netherlands. Some laboratory fer-
rets were contaminated with ferret HEV. Further studies
are needed to confirm the pathogenicity and zoonotic po-
tential of ferret HEV. This study was supported in part by grants for Research
on Emerging and Re-emerging Infectious Diseases, Research
on Hepatitis, and Research on Food Safety from the Ministry of
Health, Labor, and Welfare, Japan. Dr Li is a senior researcher at the National Institute of Infec-
tious Diseases, Tokyo, Japan. His research interests are epidemi-
ology, expression of viral proteins, and 3-dimensional structure
of HEV. References 1. Meng XJ, Anderson DA, Arankalle VA, Emerson SU, Harrison TJ,
Jameel S, et al. Hepeviridae. In: King AM, Adams MJ, Carstens, EB,
Lefkowitz EJ, editors. Virus taxonomy: ninth report of the ICTV. London: Elsevier/Academic Press; 2012. p. 1021–8. 2. Emerson SU, Purcell RH. Hepatitis E virus. Rev Med Virol. 2003;13:145–54. http://dx.doi.org/10.1002/rmv.384 Although nucleotide sequence identities of the entire
genome for strains from the United States and the Neth-
erlands was 82.4%–82.5%, ORF2 showed relatively high
amino acid identities (94.2%–94.8%), which suggested that
isolated from the United States and the Netherlands share
similar antigenicity. Ferret HEV–like particles derived
from 1 of the isolates from the Netherlands were cross-re-
active with serum from HEV-infected laboratory ferrets in
the United States (11). 3. Li TC, Chijiwa K, Sera N, Ishibashi T, Etoh Y, Shinohara Y,
et al. Hepatitis E virus transmission from wild boar meat. Emerg Infect Dis. 2005;11:1958–60. http://dx.doi.org/10.3201/
eid1112.051041 3. Li TC, Chijiwa K, Sera N, Ishibashi T, Etoh Y, Shinohara Y,
et al. Hepatitis E virus transmission from wild boar meat. Emerg Infect Dis. 2005;11:1958–60. http://dx.doi.org/10.3201/
eid1112.051041 4. Tei S, Kitajima N, Takahashi K, Mishiro S. Zoonotic transmission of
hepatitis E virus from deer to human beings. Lancet. 2003;362:371–
3. http://dx.doi.org/10.1016/S0140-6736(03)14025-1 5. Meng XJ. Hepatitis E virus: animal reservoirs and zoonotic risk. Vet
Microbiol. 2010;140:256–65. http://dx.doi.org/10.1016/
j.vetmic.2009.03.017 711 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 20, No. 4, April 2014 DISPATCHES DISPATCHES 6. Raj VS, Smits SL, Pas SD, Provacia LB, Moorman-Roest H,
Osterhaus AD, et al. Novel hepatitis E virus in ferrets, the Neth-
erlands. Emerg Infect Dis. 2012;18:1369–70. http://dx.doi. org/10.3201/eid1808.111659 12. Zhang Q, Shi J, Deng G, Guo J, Zeng X, He X, et al. H7N9 influenza
viruses are transmissible in ferrets by respiratory droplet. Science. 2013;341:410–4. http://dx.doi.org/10.1126/science.1240532 13. Martina
BE,
Haagmans
BL,
Kuiken
T,
Fouchier
RA,
Rimmelzwaan GF, Van Amerongen G, et al. Virology: SARS virus
infection of cats and ferrets. Nature. 2003;425:915. http://dx.doi. org/10.1038/425915a 7. Yamamoto H, Suzuki J, Matsuda A, Ishida T, Ami Y, Suzaki Y, et al. Hepatitis E virus outbreak in monkey facility, Japan. Emerg Infect
Dis. 2012;18:2032–4. http://dx.doi.org/10.3201/eid1812.120884 g
14. Li TC, Yoshimatsu K, Yasuda SP, Arikawa J, Koma T, Kataoka M,
et al. Characterization of self-assembled virus-like particles
of rat hepatitis E virus generated by recombinant baculovi-
ruses. J Gen Virol. 2011;92:2830–7. http://dx.doi.org/10.1099/
vir.0.034835-0 8. Drexler JF, Seelen A, Corman VM, Fumie Tateno A, Cottontail V,
Melim Zerbinati R, et al. Bats worldwide carry hepatitis E virus–related
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9. Johne R, Heckel G, Plenge-Bönig A, Kindler E, Maresch C, Reetz J,
et al. Novel hepatitis E virus genotype in Norway rats, Germany. Emerg
Infect Dis. 2010;16:1452–5. http://dx.doi.org/10.3201/eid1609.100444
10. Smith DB, Purdy MA, Simmonds P. Genetic variability and the
classification of hepatitis E virus. J Virol. 2013;87:4161–9. http://
dx.doi.org/10.1128/JVI.02762-12 9. Johne R, Heckel G, Plenge-Bönig A, Kindler E, Maresch C, Reetz J,
et al. Novel hepatitis E virus genotype in Norway rats, Germany. Emerg
Infect Dis. 2010;16:1452–5. http://dx.doi.org/10.3201/eid1609.100444 15. Johne R, Plenge-Bonig A, Hess M, Ulrich RG, Reetz J, Schielke A. Detection of a novel hepatitis E–like virus in faeces of wild rats
using a nested broad-spectrum RT-PCR. J Gen Virol. 2010;91:
750–8. http://dx.doi.org/10.1099/vir.0.016584-0 10. Smith DB, Purdy MA, Simmonds P. Genetic variability and the
classification of hepatitis E virus. J Virol. 2013;87:4161–9. http://
dx.doi.org/10.1128/JVI.02762-12 11. Yang T, Kataoka M, Ami Y, Suzaki Y, Kishida N, Shirakura M,
et al. Characterization of self-assembled virus-like particles of fer-
ret hepatitis E virus generated by recombinant baculoviruses. J Gen
Virol. 2013;94:2647–56. http://dx.doi.org/10.1099/vir.0.056671-0 11. Yang T, Kataoka M, Ami Y, Suzaki Y, Kishida N, Shirakura M,
et al. Characterization of self-assembled virus-like particles of fer-
ret hepatitis E virus generated by recombinant baculoviruses. J Gen
Virol. 2013;94:2647–56. Address for correspondence: Tian-Cheng Li, Department of Virology
II, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashi-
murayama, Tokyo 208-0011, Japan; email: litc@nih.go.jp DISPATCHES http://dx.doi.org/10.1099/vir.0.056671-0 Address for correspondence: Tian-Cheng Li, Department of Virology
II, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashi-
murayama, Tokyo 208-0011, Japan; email: litc@nih.go.jp Address for correspondence: Tian-Cheng Li, Department of Virology
II, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashi-
murayama, Tokyo 208-0011, Japan; email: litc@nih.go.jp 712
Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 20, No. 4, April 2014 712 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 20, No. 4, April 2014
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Inhaled Carbon Monoxide Provides Cerebral Cytoprotection in Pigs
|
PloS one
| 2,012
|
cc-by
| 10,408
|
Introduction Inhaled CO is an important therapeutic option and has entered
clinical trials (www.clinicaltrials.gov). These include Carbon
Monoxide Therapy for Severe Pulmonary Arterial Hypertension,
Study of Inhaling Carbon Monoxide to Treat Patients with
Intestinal Paralysis after Colon Surgery, and Study of Inhaled
Carbon Monoxide to Treat Idiopathic Pulmonary Fibrosis. At low
concentrations (15 to 250 ppm), inhaled CO has beneficial effects,
mimicking those observed with HO-1 in animal models of
ischemia/reperfusion injury, organ transplantation, ileus, necro-
tizing enterocolitis and airway disease. More recently, Vieira and
colleagues showed that CO prevented neuronal apoptosis induced
by excitotoxicity and oxidative stress in a primary culture of mouse
cerebellar granule cells. [35] In a mouse model, Zeynalov and
colleagues evaluated the role of inhaled CO following 90-minutes
of transient focal brain ischemia. The authors found that
inhalation of 125 ppm or 250 ppm CO begun immediately at
the time of onset of reperfusion resulted in reduction in
hemispheric infarct volume, improved neurological deficit scores,
and limited brain edema. Inhalation of 250 ppm CO begun 1 to 3
hours after ischemia resulted in reduction of infarct volume and
improved neurological deficit scores. [36] Wang et al exposed
male wild-type and Nrf2-knockout mice to 250 ppm CO or air
control for 18 hours immediately after permanent middle cerebral
artery occlusion. CO neuroprotection was completely abolished in
Nrf2-knockout mice suggesting that the beneficial effect of inhaled Neurologic abnormalities occur in neonates undergoing cardio-
pulmonary bypass (CPB) and deep hypothermic circulatory arrest
(DHCA) for correction of congenital heart defects. Neuroprotec-
tive strategies have focused on perioperative management with
very few innovative options to reduce injury. Preconditioning as
a preoperative therapy to protect the brain prior to DHCA has not
been studied, but has otherwise been shown to be useful in other
clinical indications [1–7] such as organ transplantation to improve
function post transplant [8–13]. The preconditioning agent does
not have to be the same as the potentially lethal insult. In organ
transplantation, a brief ischemic time elicits a stress response,
which leads to altered metabolism, less inflammation, better tissue
perfusion and protective gene expression such as the anti-oxidants
and heat shock proteins [14–21]. Among a host of protective genes is heme oxygenase-1 (HO-1),
which is increased dramatically in response to stress and
importantly, when elevated, imparts potent salutary effects [22–
34]. Two isoforms of heme oxygenase exist and are principally
responsible for the catalysis of heme into bilirubin. Inhaled Carbon Monoxide Provides Cerebral
Cytoprotection in Pigs Vicki L. Mahan2,3*, David Zurakowski1, Leo E. Otterbein2,3., Frank A. Pigula1. 1 Department of Pediatric Cardiothoracic Surgery, Boston Children’s Hospital, Boston, Massachusetts, United States of America, 2 Department of Surgery, Beth Israel
Deaconess Medical Center, Boston, Massachusetts, United States of America, 3 Harvard Medical School, Boston, Massachusetts, United States of America 1 Department of Pediatric Cardiothoracic Surgery, Boston Children’s Hospital, Boston, Massachusetts, United States of America,
Deaconess Medical Center, Boston, Massachusetts, United States of America, 3 Harvard Medical School, Boston, Massachusetts, Un Abstract Carbon monoxide (CO) at low concentrations imparts protective effects in numerous preclinical small animal models of
brain injury. Evidence of protection in large animal models of cerebral injury, however, has not been tested. Neurologic
deficits following open heart surgery are likely related in part to ischemia reperfusion injury that occurs during
cardiopulmonary bypass surgery. Using a model of deep hypothermic circulatory arrest (DHCA) in piglets, we evaluated the
effects of CO to reduce cerebral injury. DHCA and cardiopulmonary bypass (CPB) induced significant alterations in metabolic
demands, including a decrease in the oxygen/glucose index (OGI), an increase in lactate/glucose index (LGI) and a rise in
cerebral blood pressure that ultimately resulted in increased cell death in the neocortex and hippocampus that was
completely abrogated in piglets preconditioned with a low, safe dose of CO. Moreover CO-treated animals maintained
normal, pre-CPB OGI and LGI and corresponding cerebral sinus pressures with no change in systemic hemodynamics or
metabolic intermediates. Collectively, our data demonstrate that inhaled CO may be beneficial in preventing cerebral injury
resulting from DHCA and offer important therapeutic options in newborns undergoing DHCA for open heart surgery. Citation: Mahan VL, Zurakowski D, Otterbein LE, Pigula FA (2012) Inhaled Carbon Monoxide Provides Cerebral Cytoprotection in Pigs. PLoS ONE 7(8): e41982. doi:10.1371/journal.pone.0041982 akowski D, Otterbein LE, Pigula FA (2012) Inhaled Carbon Monoxide Provides Cerebral Cytoprotection in Pigs. PLoS ONE 7(8): e41982
0041982 Editor: Andreas Meisel, Charite´ Universitaetsmedizin Berlin, Germany Received April 2, 2012; Accepted June 27, 2012; Published August 7, 2012 Copyright: 2012 Mahan 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: The authors are grateful to the Julie Henry Fund at the Transplant Center of the BIDMC (Beth Israel Deaconess Medical Center) for their support. LEO is
supported by National Institutes of Health grants 5R01GM088666 and R56AI092272. 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: vmahan@bidmc.harvard.edu . These authors contributed equally to this work. PLoS ONE | www.plosone.org August 2012 | Volume 7 | Issue 8 | e41982 Animals The study was performed at the Animal Research Laboratory
Children’s Hospital Boston/Harvard Medical School in Boston
and approved by the Institutional Animal Care and Use
Committee (IACUC) at Children’s Hospital Boston. All animals
received humane care in compliance with the ‘‘Principles of
Laboratory Animal Care’’ formulated by the National Society for
Medical Research and the ‘‘Guide for the Care and Use of
Laboratory Animals’’ published by the National Institutes of
Health (NIH Publication No. 88-23, revised 1985). A Stockert double roller pump was used to generate non-
pulsatile pump flow at an initial rate of 100 ml/kg body weight in
all experiments. The oxygenator gas mixture consisted of 5%
carbon dioxide and 95% oxygen in all CPB groups. The CPB
bypass circuit consisted of the D 901 Lilliput 1 Newborn Hollow
Fiber Oxygenator with hardshell venous cardiotomy reservoir
(COBE Cardiovascular Inc., Arvada CO), Dideco D736 40
micron Arterial Line Filter (COBE Cardiovascular, Inc., Arvada
CO), Custom Smart Tubing Perfusion Pack (COBE Cardiovas-
cular Inc., Arvada CO), Terumo CDI 500 (Terumo Cardiovas-
cular Systems Corp., Tustin CA), and Hemocor HPH 400
(Minntech Corporation, Minneapolis MN). Venous drainage was
by gravity. The blood used for priming of the CPB circuit was
drawn on the morning of the experiment from an adult donor pig. The CPB circuit was initially primed with Plasmalyte A, sodium
bicarbonate 7.4%, 2500 units heparin, and fresh heparinized
Yorkshire pig whole blood to achieve a hematocrit greater than or
equal to 30%. Twelve Yorkshire female piglets (Parsons Farms, Providence
RI), 12 to 19 days old (mean 12.9 days) and weighing 2.2 kg to
4.7 kg (mean 3.4 kg ), were allowed to acclimate 2 days before
experimentation with food and water ad libitum. The piglets were
randomly assigned to Group I (no preconditioning before CPB/
DHCA –6 piglets) or Group II (preconditioned with CO the day
before CPB/DHCA –6 piglets). The Group II animals were preconditioned with inhaled CO
(280 ppm CO/balance air – Airgas East, Cambridge MA) 1 day
prior to surgery. Piglets were placed in a closed chamber that
contained a CO monitoring device (T40 Rattler Single Gas
Monitor, Industrial Scientific Corporation, Oakdale PA). The CO
gas mixture was pumped into the chamber until a steady state CO
concentration of 250 ppm was reached. The piglets were exposed
continuously for 3 hours. For safety reasons, the chamber was then
flushed with 100% oxygen over 1 minute. Animals The animals were
removed from the chamber and returned to their respective pens. Identical exposures were done with controls except the pigs were
exposed to normal air. Through a median sternotomy, the thymus was reflected
superiorly or excised and the pericardium opened. Two hundred
U/kg of heparin was given intravenously and the activated clotting
time maintained greater than 400 seconds until the piglet was
weaned from the circuit. A left atrial catheter for radioisotope
injection was positioned through the left atrial appendage into the
left atrium. The ascending aorta was cannulated with an 8F
Paediatric Arterial Cannula (Polystan A/S, Denmark) and the
right atrium was cannulated with a single 12F or 14F venous
cannula (Medtronic DLP, Minneapolis MN). A pulmonary artery
catheter for monitoring pulmonary artery pressure was placed. CPB was initiated and the flow adjusted to maintain a perfusion
pressure of 40 to 50 mmHg. pH-stat strategy (natural alkaline shift
in response to the decreasing temperature is corrected by adding
carbon dioxide) was used during cooling below 32 degrees
Centigrade and for rewarming up to 32 degrees Centigrade
nasopharyngeal temperature. All animals were cooled using
surface cooling and the heater/cooler to a nasopharyngeal
temperature of 16 degrees Centigrade and then subjected to 100
minutes of circulatory arrest. Cardiac arrest was achieved after
placing a cross-clamp distal to the aortic cannula followed by
20 cc/kg of blood cardioplegia containing 1 part oxygenated
pump blood with 4 parts cardioplegia solution (1000 cc plasma-
lyte, 16.3 cc of 20% mannitol, 4.0 cc of 50% magnesium sulfate,
13.0 cc of 1 mEq/ml sodium bicarbonate,
13.0 cc of 1%
lidocaine, and 13.0 cc of 2 mEq/ml potassium chloride) which
was administered through a side port of the aortic cannula. Blood Sampling Baseline blood samples (,1 cc) for all studies was drawn using
a blood gas syringe after insertion of the femoral artery catheter
and superior sagittal sinus catheter. Repeat samples were drawn
during cooling and rewarming on CPB, immediately after weaning
from CPB, every 30 minutes after weaning from CPB until death,
and when clinically indicated. Blood samples were immediately
analyzed for pH, oxygen, carbon dioxide, glucose, lactate,
hematocrit, sodium, and potassium using the blood gas analyzer. Inhaled CO Provides Cerebral Cytoprotection in Pigs Inhaled CO Provides Cerebral Cytoprotection in Pigs CO would at least partially be mediated through the Nrf2
pathway. [37]. surgical stimulation. They were maintained on positive-pressure
ventilation (inspired oxygen greater than 40% and arterial carbon
dioxide tension 35 to 45 mm Hg). An intravenous catheter was
placed in an ear vein. A nasopharyngeal temperature probe was
placed in the esophagus. Femoral artery and femoral venous
catheters were placed for monitoring and blood drawing purposes. The animals were placed on a cooling/warming blanket which
was initially used to surface cool the animals before DHCA and
later to rewarm the animals and to help maintain normal body
temperature after DHCA. Cefazolin (25 mg/kg) and methylpred-
nisolone (30 mg/kg) were given intravenously. Animals were
surface cooled and the head and groins were packed in ice. However, the role of inhaled CO as a preconditioning
neuroprotective agent during DHCA has not been studied. Neuroprotective studies by Mezrow et al suggest that disturbances
in cerebral blood flow and cerebral vascular resistance correlate
with clinical findings of neurologic injury after hypothermic
circulatory arrest and that cerebral metabolism is maintained by
increases in oxygen and glucose extraction, a relationship viewed
as potentially very important. [38,39] In order to define effects of
inhaled CO on neuroprotection during DHCA, this study
investigated differences in cerebral blood flow, cerebral hemody-
namics, cerebral metabolism, and cerebral pathology in piglets
preconditioned with inhaled CO before undergoing CPB and
DHCA. A superior sagittal sinus (sss) cannulation was done prior to
cannulation for CPB. A midline scalp incision was made and
carried down to the periosteum. The periosteum was removed and
a 3 mm cutting burr was used to remove bone over the sinus using
3.5X magnification. A 24 gauge catheter was placed into the
sagittal sinus for cerebral venous blood sampling and monitoring
of cerebral venous pressure. PLoS ONE | www.plosone.org Introduction During the
breakdown of heme, a molecule of carbon monoxide (CO),
biliverdin and iron are released. Biliverdin is rapidly converted to
bilirubin by biliverdin reductase while the iron is sequestered into
ferritin. The CO generated is exhaled unmodified. Recent efforts
have identified CO as the primary mechanism by which HO-1
imparts its beneficial effects in animals. 1 PLoS ONE | www.plosone.org August 2012 | Volume 7 | Issue 8 | e41982 TUNEL Staining and Quantitation Piglets were reheparinized with 200 units/kg of heparin 2
minutes before exsanguination. Three liters of chilled normal
saline were infused at a pressure of 150 mm Hg through the side
port of the clamped aortic cannula followed by 3 liters of 10%
formalin. The piglets were decapitated and the head was placed in
chilled 10% formalin and refrigerated. Brains were removed in toto
no sooner than 1 week after death. Tissue sections from the right frontal neocortex/striatum and
hippocampuswerecutfromparaffinblocksatathicknessof5 mmand
mounted. Sections were stained by terminal nick-end labeling of
cleaved DNA with fluorescein-conjugated nucleotides (ApopTag
Fluorescein In Situ Apoptosis Detection Kit; Chemicon Internation-
al, Billerica, Massachusetts). The sections were also stained for nuclei
(DAPI;MolecularProbes, Eugene,Oregon). TUNEL-positive and –
negative nuclei were counted using Metamorph software (Version
6.2; Molecular Devices, Downingtown, Pennsylvania) on 5 to 10
random frontal neocortex/striatum and hippocampus fields per
section,read atlow magnification.Results areexpressedasapoptotic
nuclei (AN) per 1,000 total nuclei. Blood Flow Determination Fixed brains were bisected in the sagittal plane. Tissue blocks
from the left hemisphere were cut to encompass brain regions
known for their vulnerability to hypoxia/ischemia. Two specimens
were taken from 3 areas of the fixed brain of each animal: frontal
neocortex, striatum, and hippocampus. Analyses of these samples
to determine blood flow were carried out by computer solution of
multiple simultaneous linear equations (Compusphere, Packard
instruments). Blood flow was calculated using the following
equation: Cerebral blood flow (ml/100 g/min) = (cerebral tissue
counts6rate of withdrawal)6100/(counts in reference sample6
weight of brain sample). Cerebral vascular resistance (CVR) was
calculated using the equation: CVR (mm Hg/ml/100 g/min) =
[MAP – MSSSP (mm Hg)]/CBF (ml/100 gm/min) where MAP is
mean arterial pressure and MSSSP is mean superior sagittal sinus
pressure. Induction of Circulatory Arrest On the day of surgery, piglets were premedicated with telazol
(6.6 mg/kg), xylazine (2.2 mg/kg), and atropine (0.04 mg/kg)
intramuscularly, intubated (non-cuffed 3.0 tube) and placed on
isoflourane and the animal’s depth of anesthesia was determined
by evaluating reflexes, muscle tone, and response of vital signs to PLoS ONE | www.plosone.org August 2012 | Volume 7 | Issue 8 | e41982 2 Inhaled CO Provides Cerebral Cytoprotection in Pigs were examined for each animal by bright-field microscopy. Counts
for each area were averaged for a final score. were examined for each animal by bright-field microscopy. Counts
for each area were averaged for a final score. After 100 minutes of circulatory arrest, ice was removed from
the head and groins and whole body perfusion was reestablished
using the CPB circuit. The animals were rewarmed to a nasopha-
ryngeal temperature of 36 degrees centigrade using the heater/
cooler and the cooling/warming blanket. Weaning cardiac
support was provided with epinephrine (0.01 to 0.03 mg/kg/
minute) and was discontinued in all animals within the first 30
minutes. Weaning from bypass occurred approximately 34
minutes after the start of rewarming. Heparin was reversed with
2 mg/kg intravenous protamine. Animals were maintained on
isoflurane anesthesia until they were exsanguinated 6 hours after
the termination of CPB. Caspase-3 Immunohistochemistry Formalin-fixed, paraffin-embedded brain tissue sections of
hippocampus were deparaffinized with xylene, rehydrated grad-
ually with graded alcohol solutions (100%, 95%, and 80%), and
then washed with deionized water. For antigen unmasking,
sections were treated in trypsin solution for 10 minutes at 37uC. Sections were then washed with deionized water and incubated
with 3% H2O2 for 5 minutes. Sections were then incubated with
a 1:300 dilution of the rabbit polyclonal anti-active caspase-3
(Promega, Madison, WI) overnight at 4uC. After three PBS
washes, sections were incubated with the secondary antibody,
a biotinylated goat anti-rabbit IgG, at 37uC for 30 minutes, and
with peroxidase-conjugated strepavidin-biotin complex (Santa
Cruz Biotechnology, Santa Cruz, CA) at 37uC for 30 minutes. Diaminobenzidine (DAB) substrate (Zymed, South San Francisco,
CA) was applied as the chromogen, giving a brown reaction
product, and the sections were counterstained with Mayer’s
hematoxylin. Serum Measurements Blood samples from the femoral artery and superior sagittal
sinus were obtained simultaneously for calculation of cerebral
oxygen extraction (arteriovenous difference in content of oxygen),
cerebral glucose extraction (arteriovenous difference in glucose),
cerebral lactate extraction (arteriovenous difference in lactate),
oxygen/glucose index (OGI), and lactate/glucose index (LGI). Statistical Analysis The Kolmogorov-Smirnov goodness-of-fit test was used to
assess for normality of the data and no gross departures were
detected. Therefore, baseline values were compared by one-way
analysis of variance (ANOVA) with means and standard deviations
reported. Mean differences between the treatment groups at
various time points were assessed using two-way repeated-
measures ANOVA (group and time as factors) with a mixed-
model approach and a compound symmetry covariance structure
to account for the within-animal correlation. [41] The Green-
house-Geisser F-test for small samples was chosen for assessing
significance group and time effects. [42] TUNEL results were
compared between the two groups using a Poisson regression
model for analyzing count data. Analysis of the data was
performed with use of the PROC MIXED and GEE procedures
in the SPSS software package (version 16.0, SPSS Inc./IBM,
Chicago, IL). Two-tailed values of P,.05 were regarded as
statistically significant. Measurements of Cerebral Blood Flow and Cerebral
Vascular Resistance Cerebral blood flow (CBF) was measured with radionuclide-
labeled microspheres as originally described by Rudolph and
Heymann. [40] Approximately 0.5 to 1.56106 microspheres
1660.5 mm in diameter labeled with Ce141, Cr51, Ru103, Nb95
or Sc46 (PerkinElmer Life and Analytical Sciences, Billerica, MA)
were injected and flushed with 5 ml of saline into the left atrial
catheter i.) before CPB, ii.) immediately after weaning from CPB,
iii.) 1 hour after weaning from CPB, iv.) 3 hours after weaning
from CPB, and v.) 6 hours after weaning from CPB. Samples of
blood were taken from the femoral artery line at a constant rate
(2.00 ml/min) with a Harvard withdrawal pump beginning 15
seconds before injection of microspheres and ending 75 seconds
after injection. PLoS ONE | www.plosone.org Effects of Inhaled CO on Systemic and Cerebral
Hemodynamics In the control group the OGI decreased with CPB and cooling. This is attributed to a reduction in A-VDO2 to a greater extent
than
the
reduction
in
A-VDGLUCOSE
(Figure
3). During
rewarming, the OGI returned to baseline immediately after
weaning from CPB. OGI then decreased to below baseline values
for the duration of the study. (Figure 4). Mean pulmonary artery pressure (MPAP) and mean superior
sagittal sinus pressure (MSSSP) were higher in CO precondi-
tioned
animals
at
all
time
points
(Figure
1). Statistical
significance was observed for MPAP immediately after CPB
and for MSSSP 6 hours after CPB. Although cerebral perfusion
pressure (MAP – MSSSP) was slightly higher in the control
group during the study, this difference did not reach statistical
significance. Cerebral blood flow (CBF) was similar in both air
and CO-treated piglets at all time points (Figure 2). Immedi-
ately after weaning from CPB, CBF increased markedly from
baseline and then decreased to below baseline at 1 hour and 3
hours after weaning from CPB. Six hours after weaning from
CPB, CBF was higher than baseline in all groups and was
similar in air and CO-treated piglets. In the CO-treated group, the OGI increased over baseline in
the presence of CPB and cooling. (Figure 4) A-VDO2, superior
sagittal sinus saturation and oxygen extraction ratios were in close
agreement between the groups, but A-VDGLUCOSE was lower in
the CO group before, during and immediately after CPB
(Figure 3) suggesting that glucose was being metabolized at
a lower rate in the CO-treated animals. Both A-VDO2 and A-
VDGLUCOSE returned to baseline by the end of the study. The
calculated OGI, likewise, returned to baseline. A negative A-VDLACTATE is consistent with lactate being
produced in the brain. Assessment of A-VDLACTATE levels trended
toward being more negative in the control group basally, but did
not achieve statistical significance (Figure 5). During cooling on
CPB, A-VDLACTATE levels were similar. While A-VDLACTATE
was negative in air and CO-treated animals during cooling, A-
VDLACTATE became positive in the control group and remained
negative in CO-treated animals during rewarming on CPB. CO Influences Metabolism in the Brain Following CPB CO Influences Metabolism in the Brain Following CPB
Differences between arteriovenous oxygen tension (A-VDO2)
were greater than differences between arteriovenous glucose (A-
VDGLUCOSE) at baseline. No significant differences between A-
VDO2 and A-VDGLUCOSE values between air and CO-treated
animals were observed. (Figure 3). The lactate/glucose index (LGI) is a quantitative expression
describing the amount of glucose consumption that appears as
lactate production. If all extracted glucose is metabolized to
lactate, A-VDLACTATE/A-VDGLUCOSE is equal to the absolute
value 2. The equation for LGI defines this biochemical relation-
ship: The oxygen/glucose index (OGI) describes the relationship
between A-VDO2 and A-VDGLUCOSE and is independent of
cerebral blood flow. Stoichiometrically, 6 moles of oxygen are
required to metabolize 1 mole of glucose. If all glucose is oxidized
and no other substrate is consumed, A-VDO2/A-VDGLUCOSE is
equal to 6. The ratio is less than 6 if all the glucose extracted is not
oxidized and greater than 6 if other substrates (i.e. lactate,
pyruvate, acetoacetate, b-hydroxybutyrate, glutamate) are used as
a source of carbon. The equation for OGI defines this biochemical
relationship: Hemodynamic and Hematologic Parameters Remained
Stable Throughout CPB Preoperative data (age and weight) were similar. Cooling and
rewarming times during CPB were similar. There were no
statistically significant differences at baseline between the groups
regarding temperature, mean arterial pressure, arterial blood
gases, sss blood gases, sss saturation values, oxygen extraction
ratio, hematocrit values, arterial glucose values, and arterial
lactate values, which were compared using 2-way repeated-
measures
ANOVA. (Table
1)
No
significant
differences
between the 2 groups were found for any of these variables
throughout the time period from baseline to 6 hours after
weaning from CPB. If all of the glucose extracted is oxidized and no other substrate
is consumed, OGI(%) is 100%. Values less than 100% indicate
that not all of the glucose extracted is oxidized (anaerobic
metabolism) and values greater than 100% indicate that other
substrates are used as a source of carbon. Prebypass mean OGI (%) was comparable in both groups and
determined to be 63% (642) in the control group and 63% (647)
in the CO group. The OGI was higher in the CO-treated animals
at all other time points (p = 0.006; Figure 4, * indicates
statistical significance). Effects of Inhaled CO on Systemic and Cerebral
Hemodynamics Assessment of Cell Death Damaged
neurons
(DN)
included
nuclear
pyknosis
with
wrinkled nuclear outlines and minimal cytoplasmic change,
nuclear pyknosis with eosinophilic cytoplasm, ghost neurons, or
apoptotic bodies. Counts were scored as follows: 1, 0 to 5 DN seen
per high-power field; 2, 6 to 15 DN seen per high-power field; 3,
16 to 25 DN seen per high-power field; and 4, greater than 25 DN
seen per high-power field. Twenty randomly selected 4006 fields
of H&E stained frontal neocortex, striatum, and hippocampus August 2012 | Volume 7 | Issue 8 | e41982 PLoS ONE | www.plosone.org 3 Inhaled CO Provides Cerebral Cytoprotection in Pigs Inhaled CO Provides Cerebral Cytoprotection in Pigs Effects of Inhaled CO on Systemic and Cerebral
Hemodynamics After
weaning from CPB, however, A-VDLACTATE became negative in
controls and remained low in contrast to CO-treated animals
which recovered and were positive after CPB at 1 and 3 hr (1 hr
post CPB: air at 20.02 mml/ml versus CO at +0.03 mmol/ml,
p,0.05; 3 hr post CPB: air at 20.01 mmol/ml versus CO at
+0.04 mmol/ml, p,0.05) at which point the A-VDLACTATE in
both groups became positive (Figure 5, * indicates statistical
significance). Cerebral vascular resistance (CVR) was elevated over baseline
in both groups 1 hour and 3 hours after weaning from CPB and
then dropped below baseline 6 hours after weaning. This was
expected based on CBF (Figure 2). CVR immediately after
weaning did not change significantly from baseline with no
statistical difference between air or CO-treated animals. Given the reports that CO can impart vasodilatory effects, we
expected that cerebral perfusion pressures and CBF would be
higher and that CVR would be lower than what was observed
in
the
air
treated
animals. These
results
suggest
that
preconditioning with CO was ineffective at modulating cerebral
vasodilation and improved cerebral blood flow immediately
before or after CPB. LGI(%)~(arteriovenous lactate difference½mmol=ml)=
(arteriovenous glucosedifference ½mmol=ml
|2)|100: If all of the extracted glucose is metabolized to lactate the LGI
(%) is 100%. A negative LGI results if glucose is metabolized to PLoS ONE | www.plosone.org August 2012 | Volume 7 | Issue 8 | e41982 4 Inhaled CO Provides Cerebral Cytoprotection in Pigs Table 1. Hemodynamic and Hematologic Parameters. PRE CPB
AFTER CPB
1 HR AFTER CPB
3 HRS AFTER CPB
6 HRS AFTER CPB
TEMP
CONTROL
32.4+/21.6
34.6+/21.4
32.3+/22.0
34.2+/21.1
35.9+/21.3
CO-TREATED
33.0+/22.1
34.0+/22.6
33.1+/21.8
35.9+/21.8
36.2+/21.2
MAP (mm Hg)
CONTROL
55+/210
74+/214
66+/26
65+/28
60+/210
CO-TREATED
54+/28
71+/213
62+/26
59+/24
58+/24
pHa
CONTROL
7.49+/20.10
7.36+/20.11
7.43+/20.04
7.45+/20.06
7.43+/20.06
CO-TREATED
7.50+/20.06
7.40+/20.06
7.40+/20.04
7.44+/20.06
7.42+/20.08
PaO2 (mm Hg)
CONTROL
619+/267
314+/2184
451+/2199
444+/2142
441+/2171
CO-TREATED
600+/2106
372+/2279
463+/2162
419+/2142
480+/2183
PaCO2 (mm Hg)
CONTROL
37.7+/29.7
46.3+/213.9
40.2+/24.1
41.9+/24.8
43.5+/23.3
CO-TREATED
34.6+/28.5
38.3+/210.7
40.1+/24.8
39.1+/20.9
42.1+/25.0
pHsss
CONTROL
7.42+/20.08
7.30+/20.10
7.34+/20.01
7.38+/20.04
7.35+/20.06
CO-TREATED
7.43+/20.05
7.36+/20.03
7.31+/20.05
7.35+/20.06
7.38+/20.03
PsssO2 (mm Hg)
CONTROL
44.1+/211.8
50.6+/210.5
37.9+/26.6
46.3+/28.8
47.4+/215.8
CO-TREATED
45.3+/213.4
43.4+/218.6
44.3+/29.9
39.3+/29.9
43.4+/212.2
PsssCO2 (mm Hg)
CONTROL
48.2+/212.4
56.6+/213.6
60.0+/29.8
55.7+/26.1
58.4+/26.5
CO-TREATED
46.9+/25.4
47.4+/27.2
55.5+/26.7
57.2+/26.7
53.3+/25.2
sss Saturation (%)
CONTROL
60+/210
64+/214
50+/212
59+/212
60+/222
CO-TREATED
69+/219
61+/227
56+/213
51+/214
60+/215
OER
CONTROL
70+/26
53+/211
66+/218
62+/214
63+/216
CO-TREATED
63+/210
58+/218
65+/210
66+/214
67+/211
HEMATOCRIT
CONTROL
35.2+/24.5
36.7+/25.8
34.7+/25.6
35.3+/28.1
34.2+/27.6
CO-TREATED
37.3+/28.3
38.2+/24.3
35.7+/22.4
37.8+/25.0
33.0+/24.2
Arterial Glucose
CONTROL
161+/258
138+/236
142+/251
181+/253
201+/258
CO-TREATED
143+/2122
136+/281
173+/278
174+/283
184+/257
Arterial Lactate
CONTROL
4.0+/22.8
9.9+/24.3
7.4+/21.7
5.2+/21.7
4.6+/23.9
CO-TREATED
3.5+/21.3
8.2+/21.3
7.5+/20.9
5.7+/21.8
5.4+/21.7
doi:10 1371/journal pone 0041982 t001 doi:10.1371/journal.pone.0041982.t001 lactate (sss lactate is higher than arterial lactate.) A positive LGI
suggests that lactate is further metabolized by the cell and tissue. animals immediately after weaning from CPB (0.69) Differences
between air treated and CO-treated groups reached statistical
significance 1 hour post CPB (air treated group 0.15 and CO-treated
group 2.50, p,0.05) and 3 hours post CPB (air treated group 20.48
and CO-treated group 1.92, p,0.05). lactate (sss lactate is higher than arterial lactate.) A positive LGI
suggests that lactate is further metabolized by the cell and tissue. The LGI basally before surgery were 0.03 and 20.25 for the
control and CO-treated animals respectively with both air and CO
being negative during cooling with no statistical difference between
groups observed. The LGI became positive in control animals (1.25)
duringrewarmingonCPBsuggestingthatlactatewasmetabolizedby
the brain during this time (Figure 5, * indicates statistical
significance). LGI(%)~(arteriovenous lactate difference½mmol=ml)=
(arteriovenous glucosedifference ½mmol=ml
|2)|100: The LGI in the CO-treated animals was negative
(24.80) during rewarming, but became positive in CO-treated The LGI basally before surgery were 0.03 and 20.25 for the
control and CO-treated animals respectively with both air and CO
being negative during cooling with no statistical difference between
groups observed. The LGI became positive in control animals (1.25)
duringrewarmingonCPBsuggestingthatlactatewasmetabolizedby
the brain during this time (Figure 5, * indicates statistical
significance). The LGI in the CO-treated animals was negative
(24.80) during rewarming, but became positive in CO-treated Taken together these data suggest that CO induces a metabolic
change in the brain of piglets undergoing CPB/DHCA. OGI was
higher in CO-treated animals except at baseline and was statistically
significant during cooling and warming on CPB and at 3 hours after
weaning from CPB. One hour after weaning from CPB, LGI PLoS ONE | www.plosone.org August 2012 | Volume 7 | Issue 8 | e41982 5 Inhaled CO Provides Cerebral Cytoprotection in Pigs Figure 1. Mean pulmonary artery pressure (MPAP) and mean superior sagittal sinus pressure (MSSSP) were higher in the CO
preconditioned piglets at all time points. Data represent the means 6 SD of 6 animals/group, MPAP p,0.05 immediately after CPB and MSSSP
p,0.05 6 hours after CPB. doi:10.1371/journal.pone.0041982.g001 Figure 1. Mean pulmonary artery pressure (MPAP) and mean superior sagittal sinus pressure (MSSSP) were higher in the CO
preconditioned piglets at all time points. Data represent the means 6 SD of 6 animals/group, MPAP p,0.05 immediately after CPB and MSSSP
p,0.05 6 hours after CPB. doi:10.1371/journal.pone.0041982.g001 remained higher in CO-treated animals and was statistically
significant at 1 hour and 3 hours after weaning from bypass. an effect of CO that results in altered bioenergetics and
metabolism in the brain. Cerebroprotective effects have been well-described with HO-1
induction. [43–45] The protective effects of HO-1 induction, and
therein the endogenous generation of CO, can be mimicked in
nearly all instances using inhaled CO or administration of carbon
monoxide releasing molecules (CO-RMs). [46] Activation of HO-
1 or administration of CO has been shown to regulate a number of
specific cell signaling pathways including MAP kinases, BKCa
channels, guanylyl cyclase, PPARc, and HIF1a. [47] Modulation
of
these
pathways
is
highly
dependent
on
when
CO
is
administered, either pre or post stressor. Paradoxically, CO
increases ROS generation when administered as a pretreatment
primarily through binding to mitochondrial oxidase complexes. CO Abrogated Apoptosis of Neurons Cellular damage in the frontal neocortex, striatum, and
hippocampus was determined by TUNEL staining. Cell death
likely comprised a combination of necrosis and apoptosis in both
groups (Figure 6). TUNEL staining showed a significant increase
in positive apoptotic counts in the neocortex/striatum versus naı¨ve
which was abrogated in CO-treated piglets versus controls (0.5/
1000 cells versus 21.6/1000 cells), but did not achieve significance
in this cohort. In the hippocampus, however, there was a greater
number of TUNEL positive cells in the air-treated animals which
was significantly reduced counts in CO-preconditioned piglets
(4.4/1000 cells versus 1.1/1000 cells respectively, p,0.03). Caspase-3 staining of the hippocampus showed a greater number
of caspase-3 positive staining, indicative of cell death in air-treated
animals undergoing –CPB and DHCA. In contrast, animals
treated with CO and then subjected to CPB and DHCA showed
a
marked
reduction
in
caspase-3
positive
staining
in
the
hippocampus (Figure 6) thereby indicating that CO offers
salutary effects in limiting cell death in the brain in piglets
undergoing CPB and DHCA. Our study suggests that a change in metabolic substrate
modulation may be key in neuroprotection. Functions of the
central nervous system are mainly excitation and conduction,
electrical energy being derived from chemical processes. Stoichio-
metric modeling of brain metabolism in response to tissue hypoxia
involves central metabolism (glycolysis, pentose phosphate path-
way, TCA cyle), lipid metabolism, reactive oxygen species
detoxification, amino acid metabolism, the glutamate-glutamine
cycle and neurotransmitter metabolism. [48] There has been
continued debate as to the primary fuel of brain cells during basal
and stressed conditions. [49] Lactate may be the preferred energy
substrate of activated neurons. Our findings are consistent with
a change to lactate as the metabolic substrate for neurons in
a piglet model of DHCA/CPB. A-VDLACTATE and LGI became
positive and statistically significant at 1 and 3 hours after weaning
from CPB. Changes in bioenergetics in response to CO may LGI(%)~(arteriovenous lactate difference½mmol=ml)=
(arteriovenous glucosedifference ½mmol=ml
|2)|100: The transient ROS generation leads to preconditioning of the cell
towards an anti-inflammatory and anti-apoptotic phenotype via
upregulating cytoprotective molecules including HO-1. PLoS ONE | www.plosone.org August 2012 | Volume 7 | Issue 8 | e41982 Discussion The data presented here continue to support the concept that
low concentrations of inhaled CO are beneficial in models of
ischemic injury. A clinically relevant CO preconditioning regimen
imparted potent cerebroprotection evidenced by less cell death in
the cortex and hippocampus in response to CPB and DHCA. While the exact molecular target remains unknown, there is clearly PLoS ONE | www.plosone.org August 2012 | Volume 7 | Issue 8 | e41982 6 Inhaled CO Provides Cerebral Cytoprotection in Pigs likewise reflect the observed cytoprotective effects. Historically,
this would corroborate the fact that CO targets the mitochondrial
whole cell preparations or tissues. CO also ac
cyclase to increase cGMP levels in neurons
Figure 2. (CBF). Data represent the means 6 SD of 6 animals/group. Cerebral blood flow (CBF) was similar in air and CO-t
time points. After weaning from CPB, CBF increased from baseline and then decreased to below baseline at 1 and 3 hours after w
after weaning, CBF was above baseline. There were no statistical differences between the Air and CO groups. 2 (CVR). Data repr
SD of 6 animals/group. Cerebral vascular resistance (CVR) reflected changes seen with CBF and were similar in air and CO-treated
points. CVR was higher than baseline in both groups 1 hour and 3 hours after weaning from CPB and below baseline 6 hours af
were no statistical differences between the Air and CO groups. doi:10.1371/journal.pone.0041982.g002 Figure 2. (CBF). Data represent the means 6 SD of 6 animals/group. Cerebral blood flow (CBF) was similar in air and CO-treated piglets at all
time points. After weaning from CPB, CBF increased from baseline and then decreased to below baseline at 1 and 3 hours after weaning. At 6 hours
after weaning, CBF was above baseline. There were no statistical differences between the Air and CO groups. 2 (CVR). Data represent the means 6
SD of 6 animals/group. Cerebral vascular resistance (CVR) reflected changes seen with CBF and were similar in air and CO-treated piglets at all time
points. CVR was higher than baseline in both groups 1 hour and 3 hours after weaning from CPB and below baseline 6 hours after weaning. There
were no statistical differences between the Air and CO groups. doi:10.1371/journal.pone.0041982.g002 likewise reflect the observed cytoprotective effects. Discussion doi:10.1371/journal.pone.0041982.g004 August 2012 | Volume 7 | Issue 8 | e41982 PLoS ONE | www.plosone.org 8 Inhaled CO Provides Cerebral Cytoprotection in Pigs Figure 5. (A-VDLACTATE). In the air group, A-VDLACTATE was more negative at baseline than the CO-treated g
during warming on CPB, and was negative immediately after weaning from CPB, 1 hour after CPB, and 3 hou
A-VDLACTATE was negative during CPB and become positive after weaning from CPB. At 6 hours after CPB, A-VDLACTATE wa
Data represent the means 6 SD of 6 animals/group, p,0.05 at 1 hour after CPB and 3 hours after CPB. * indicates statistic
LGI% was similar at baseline and negative in both groups during cooling on CPB. In the air group, LGI% became positive
LGI% was negative in the CO_treated animals during warming on CPB but was positive thereafter. Data represent the m
group, p,0.05 at 1 hour after CPB and 3 hours after CPB. * indicates statistical significance. doi:10.1371/journal.pone.0041982.g005 Figure 5. (A-VDLACTATE). In the air group, A-VDLACTATE was more negative at baseline than the CO-treated group, became positive
during warming on CPB, and was negative immediately after weaning from CPB, 1 hour after CPB, and 3 hours after CPB. In contrast,
A-VDLACTATE was negative during CPB and become positive after weaning from CPB. At 6 hours after CPB, A-VDLACTATE was positive in both groups. Data represent the means 6 SD of 6 animals/group, p,0.05 at 1 hour after CPB and 3 hours after CPB. * indicates statistical significance. 5 (LGI%). LGI% was similar at baseline and negative in both groups during cooling on CPB. In the air group, LGI% became positive during warming on CPB. LGI% was negative in the CO_treated animals during warming on CPB but was positive thereafter. Data represent the means 6 SD of 6 animals/
group, p,0.05 at 1 hour after CPB and 3 hours after CPB. * indicates statistical significance. doi:10.1371/journal.pone.0041982.g005 Figure 5. (A-VDLACTATE). In the air group, A-VDLACTATE was more negative at baseline than the CO-treated group, became positive
during warming on CPB, and was negative immediately after weaning from CPB, 1 hour after CPB, and 3 hours after CPB. In contrast,
A-VDLACTATE was negative during CPB and become positive after weaning from CPB. At 6 hours after CPB, A-VDLACTATE was positive in both groups. Data represent the means 6 SD of 6 animals/group, p,0.05 at 1 hour after CPB and 3 hours after CPB. Discussion Historically,
this would corroborate the fact that CO targets the mitochondrial
hemoproteins to modulate ATP generation and O2 consumption;
however this was performed in isolated mitochondria and not whole cell preparations or tissues. CO also activates guanylate
cyclase to increase cGMP levels in neurons and subsequent
opening of mitochondrial K channels, critical for CO inhibition of
apoptosis. [35] Queiroga and colleagues studied the ability of CO August 2012 | Volume 7 | Issue 8 | e41982 PLoS ONE | www.plosone.org August 2012 | Volume 7 | Issue 8 | e41982 7 Inhaled CO Provides Cerebral Cytoprotection in Pigs Figure 3. Arteriovenous difference in oxygen tension (A-VDO2) was greater than arteriovenous difference in glucose (A-VDGLUCOSE)
except during cooling when A-VDGLUCOSE of the control animals was greater than A-VDO2. A-VDO2 in the CO-treated group was higher at
3 and 6 hours after CPB. A-VDGLUCOSE was higher in the air group at baseline, during cooling and rewarming on CPB and immediately after CPB. While
there were trends in A-VDO2 and A-VDGLUCOSE values between air and CO-treated animals, no significant differences were observed. Data represent
the means 6 SD of 6 animals/group. doi:10.1371/journal.pone.0041982.g003 Figure 3. Arteriovenous difference in oxygen tension (A-VDO2) was greater than arteriovenous difference in glucose (A-VDGLUCOSE)
except during cooling when A-VDGLUCOSE of the control animals was greater than A-VDO2. A-VDO2 in the CO-treated group was higher at
3 and 6 hours after CPB. A-VDGLUCOSE was higher in the air group at baseline, during cooling and rewarming on CPB and immediately after CPB. While
there were trends in A-VDO2 and A-VDGLUCOSE values between air and CO-treated animals, no significant differences were observed. Data represent
the means 6 SD of 6 animals/group. doi:10.1371/journal.pone.0041982.g003 Figure 4. OGI(%) was similar at baseline in both groups, but was above baseline in the CO-treated group thereafter. In the air group,
OGI(%) was below baseline at all time points. Data represent the means 6 SD of 6 animals/group, p = 0.006 repeated measure ANOVA. * indicates
statistical significance. doi:10.1371/journal.pone.0041982.g004 Figure 4. OGI(%) was similar at baseline in both groups, but was above baseline in the CO-treated group thereafter. In the air group,
OGI(%) was below baseline at all time points. Data represent the means 6 SD of 6 animals/group, p = 0.006 repeated measure ANOVA. * indicates
statistical significance. Discussion * indicates statistical significance. 5 (LGI%). LGI% was similar at baseline and negative in both groups during cooling on CPB. In the air group, LGI% became positive during warming on CPB. LGI% was negative in the CO_treated animals during warming on CPB but was positive thereafter. Data represent the means 6 SD of 6 animals/
group, p,0.05 at 1 hour after CPB and 3 hours after CPB. * indicates statistical significance. doi:10.1371/journal.pone.0041982.g005 PLoS ONE | www.plosone.org August 2012 | Volume 7 | Issue 8 | e41982 9 Figure 6. (TUNEL). Quantitation of TUNEL staining in indicated brain sections from neonatal piglet after CPB 6 CO showed more
apoptosis in the control group versus CO in the hippocampus and trending sections from the neocortex and striatum. Data represent
mean 6 SD of 6 animals/group, *p,0.03. Untreated pigs showed ,0.1 positively stained cell/field of view (FOV). (Caspase-3). Representative tissue
sections stained for activated Caspase-3 in the hippocampus from neonatal piglets after CPB 6 CO. Note the intense positive brown staining
indicating activated caspase-3 localized to the hippocampus in the control, air-treated pigs versus nearly no positive staining in the CO treated pigs. Images are representative of n = 6–8/group. doi:10.1371/journal.pone.0041982.g006
Inhaled CO Provides Cerebral Cytoprotection in Pigs Inhaled CO Provides Cerebral Cytoprotection in Pigs Figure 6. (TUNEL). Quantitation of TUNEL staining in indicated brain sections from neonatal piglet after CPB 6 CO showed more
apoptosis in the control group versus CO in the hippocampus and trending sections from the neocortex and striatum. Data represent
mean 6 SD of 6 animals/group, *p,0.03. Untreated pigs showed ,0.1 positively stained cell/field of view (FOV). (Caspase-3). Representative tissue
sections stained for activated Caspase-3 in the hippocampus from neonatal piglets after CPB 6 CO. Note the intense positive brown staining
indicating activated caspase-3 localized to the hippocampus in the control, air-treated pigs versus nearly no positive staining in the CO treated pigs. Images are representative of n = 6–8/group. d
l Figure 6. (TUNEL). Quantitation of TUNEL staining in indicated brain sections from neonatal piglet after CPB 6 CO showed more
apoptosis in the control group versus CO in the hippocampus and trending sections from the neocortex and striatum. Data represent
mean 6 SD of 6 animals/group, *p,0.03. Untreated pigs showed ,0.1 positively stained cell/field of view (FOV). (Caspase-3). Representative tissue
sections stained for activated Caspase-3 in the hippocampus from neonatal piglets after CPB 6 CO. Inhaled CO Provides Cerebral Cytoprotection in Pigs These results suggest that
preconditioning with CO modulates cerebral injury following
a prolonged period of DHCA despite an increase in MSSSP and
might improve cerebral outcome after a prolonged period of
DHCA despite an increased MSSSP. The observed cerebropro-
tective effects may result from changes in cerebral metabolism or
modulation of inflammation and apoptosis which is the focus of
future experiments [57]. Additionally, in a pig model of kidney
transplant and ischemic-reperfusion injury, CO was also protective
when
administered intraoperatively
[58]. CO treatment
of
recipients only led to increased repair of the damaged kidney
through induction of proliferative genes. This is unlikely the mode
of action of CO here since CO was administered as a pretreatment,
but remains to be explored. More likely is that the preconditioning
results in a state of tolerance to the subsequent stress. During hypoxia and ischemia, astrocytes provide both structural
and metabolic support and exhibit a high glycolytic rate
generating primarily lactate. Vega and colleagues concluded that
there is a shift in the metabolic response to energy conservation
and a change in cerebral metabolism [63]. Bouzier and colleagues
showed that 13C-labeled lactate was a major substrate for oxidative
metabolism in C6 glioma cells and hypoxic conditions were found
to lead to the accumulation of lactate as a rich energy source. [64]
In a study in newborn piglets with intrauterine growth restriction,
Moxon-Lester et al concluded that increased cerebral lactate is
neuroprotective. [65] In our study, A-VDO2 and A-VDGLUCOSE
showed no differences between air- and CO-treated animals, but
statistically significant differences in OGI (statistically significant
during cooling and warming and 3 hours after weaning from
CPB), A-VDLACTATE (statististically significant 1 hour and 3 hours
after weaning from CPB), and LGI (statistically significant 1 hour
and 3 hours after weaning from CPB) between groups. The
change in glucose and lactate metabolism in the brains of piglets
preconditioned with CO undergoing CPB/DHCA showed less
lactate production and/or higher lactate consumption. Figure 4
suggests that CO increases oxidative metabolism which would
result
in
less
lactate
production. However,
higher
lactate
consumption by neurons induced by CO as an alternative source
of carbon cannot be overlooked. Schurr et al and Occhipinti et al studied the role of lactate
during glycolysis and hypothesized that lactate is the primary
energy substrate for neurons under aerobic conditions and that
interference with neuronal lactate utilization could result in
damage [66,67]. Inhaled CO Provides Cerebral Cytoprotection in Pigs Inhaled CO Provides Cerebral Cytoprotection in Pigs metabolic substrate as a neuroprotective agent. We speculate that
lowering cellular dependency on oxidative phosphorylation, even
transiently, would lead to reduced ROS and be reflected as
abrogated cell death. suggest increased lactate production as active neurons release large
amounts of lactate [59–61]. In a seizure model in rats, Schridde et
al showed that increases in hemodynamic, metabolic, and
neuronal activity are dependent on the interaction between
hemodynamics and metabolism. [62]. CO also acts to influence vasomotor tone, albeit rather small
potency when compared to nitric oxide, but has been shown
regulates cerebral vasodilation. [51] Exogenous administration of
CO dilates pial arteries of baby and juvenile pigs acting as an
autocrine/paracrine messenger in the regulation of cerebrovascu-
lar hemodynamics and can prevent epileptic seizures in newborn
piglets with no loss of cerebrovascular reactivity 2 days after
administration of CO. Zimmermann et al concluded that pre-
treatment of the piglets elicits vasodilator properties on the
cerebral circulation protecting the brain from cerebrovascular
injury caused by seizures. [52] Immediately following DHCA, an
elevated intracranial pressure (ICP) may result in reduction of
cerebral blood flow and it is speculated that neurophysiological
recovery and behavior are impaired in animals with a higher ICP
and that suppression of cerebral metabolism is associated with
a lower ICP. [53] Daley and colleagues showed that this
preconditioning effect results in increased ICP caused by cerebral
vasodilation in the piglet model. [54] In hypoxia/ischemia injury
seen with DHCA, elevation of ICP is associated with brain
pathology [55,56]. In our study, superior sagittal sinus saturation
and superior sagittal sinus oxygen content were similar in Groups I
and II (air vs CO respectively). Cerebral perfusion pressures and
CBF and CVR in the neocortex, striatum, and hippocampus were
also similar between treatment groups. CO-treated animals
exhibited higher MSSSP with less apoptosis which did not
correlate with decreased oxygen delivery, changes in CBF or
neuropathology. Mean pulmonary artery pressures (MPAP) were
higher in the CO-treated group and statistically significant
immediately after CPB. The higher MPAP seen in the CO-
treated animals may explain the elevated MSSSP observed in the
CO treated animals; however, cerebral perfusion pressures and
CBF and CVR in the neocortex, striatum, and hippocampus were
similar between treatment groups. PLoS ONE | www.plosone.org Inhaled CO Provides Cerebral Cytoprotection in Pigs Gordon et al showed that the ability of astrocytes
to induce vasodilation relies on the metabolic state of rat brain
tissue and that lactate attenuates transporter-mediated uptake of
prostaglandin E2 resulting in vasodilation, a preconditioning effect
[68]. Won and colleagues have investigated whether intraperito-
neal injection of lactate after hypoglycemia reduced neuronal
death. Lactate
administration with glucose
supplementation
reduced neuronal death by 80% in the hippocampus and reduced
superoxide production and microglia activation. The authors
hypothesize that increasing brain lactate following hypoglycemia
offsets the decrease in NAD(+). This was found to be due to
overactivation of PARP-1 acting as an alternative energy substrate
that bypassed glycolysis and be fed directly to the citric acid cycle
to maintain cellular ATP levels. [69] In our study, A-VDLACTATE
and LGI became positive in both groups, but was more striking in
the CO preconditioned piglets. This suggests a change in cerebral
metabolism of lactate related to CO preconditioning prior to
DHCA that may be important in neuroprotection. Cerebral metabolism with DHCA is highly complex and
incompletely understood. Metabolic compartmentalization in the
intact brain reflects metabolic alterations between different
neuronal
components
and
even
among
different
neuronal
populations. Under normal physiological conditions, glucose is
the primary source of fuel for the brain. The distribution of energy
substrates from the systemic circulation into neurons is principally
determined by astrocytes and the dependence of cerebral function
on blood glucose as a fuel does not exclude lactate or other
substrates as an energy source. The concept of ‘‘coupling’’ of
oxidative metabolism and functional activity in the brain has,
however, been challenged. A greater increase in CBF and
CMRGLUCOSE with little change in CMRO2 during stimulation In summary, preconditioning of the neonatal piglet with inhaled
CO prior to CPB/DHCA did not result in differences in CBF and
CVR between groups. MSSSP was increased in the precondi-
tioned animals, but A-VDO2 was not changed. There were
significant differences in glucose and lactate metabolism between
the groups and corresponding histopathology clearly showed that
apoptosis was significantly less in the CO-treated animals. These
results suggest that preconditioning with CO results in a change in
cerebral metabolism of glucose and lactate that corresponds with
neuroprotection. Whether the change in tissue bioenergetics is
a direct effect of CO or indirectly through improved tissue
protection remains to be determined. Discussion Note the intense positive brown staining
indicating activated caspase-3 localized to the hippocampus in the control, air-treated pigs versus nearly no positive staining in the CO treated pigs. Images are representative of n = 6–8/group. doi:10.1371/journal.pone.0041982.g006 to prevent apoptosis in primary astrocytes and observed that the
antiapoptotic effects of CO is due to the inhibition of mitochon-
drial membrane permeabilization, a major factor in the intrinsic to prevent apoptosis in primary astrocytes and observed that the
antiapoptotic effects of CO is due to the inhibition of mitochon-
drial membrane permeabilization, a major factor in the intrinsic apoptotic pathway. [50] Precisely how metabolism of lactate
relates to the changes observed in mitochondria and subsequent
cerebral cytoprotection will help to further define the role of this August 2012 | Volume 7 | Issue 8 | e41982 PLoS ONE | www.plosone.org August 2012 | Volume 7 | Issue 8 | e41982 10 References Deshane J, Wright M, Agarwal A (2005) Heme oxygenase-1 expression in
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ing. Anesteziol Intens Ter 42(4): 206–212. 31. Acknowledgments Conceived and designed the experiments: VLM FAP. Performed the
experiments: VLM. Analyzed the data: VLM DZ LEO FAP. Wrote the
paper: VLM DZ LEO FAP. Statistical analysis: DZ LEO. Conceived and designed the experiments: VLM FAP. Performed the
experiments: VLM. Analyzed the data: VLM DZ LEO FAP. Wrote the
paper: VLM DZ LEO FAP. Statistical analysis: DZ LEO. Conceived and designed the experiments: VLM FAP. Performed the
experiments: VLM. Analyzed the data: VLM DZ LEO FAP. Wrote the
paper: VLM DZ LEO FAP. Statistical analysis: DZ LEO. We greatly appreciate the help of Dr. Arthur Nedder, DVD and his
technical staff for their invaluable assistance with the piglets. We also thank
Patricia Dunning for her help with the radioisotopes and determining
cerebral blood flow, Hart Lidov MD, PhD for helping set-up the brain We greatly appreciate the help of Dr. Arthur Nedder, DVD and his
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https://europepmc.org/articles/pmc7375078?pdf=render
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English
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Functional differentiation in the human ventromedial frontal lobe: A data‐driven parcellation
|
Human brain mapping
| 2,020
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cc-by
| 15,376
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Received: 27 November 2019
Revised: 6 March 2020
Accepted: 7 April 2020 Received: 27 November 2019
Revised: 6 March 2020
Accepted: 7 April 2020 Received: 27 November 2019
Revised: 6 March 2020
Accepted: 7 April 2020
DOI: 10.1002/hbm.25014 Henry W. Chase1
|
Anthony A. Grace1,2
|
Peter T. Fox3,4,5,6
|
Mary L. Phillips1
|
Simon B. Eickhoff7,8 1Department of Psychiatry, University of
Pittsburgh School of Medicine, Pittsburgh,
Pennsylvania
2Department of Neuroscience and Psychology,
University of Pittsburgh, Pittsburgh,
Pennsylvania
3Research Imaging Institute, University of
Texas Health Science Center, San Antonio,
Texas
4Department of Radiology, University of Texas
Health Science Center, San Antonio, Texas
5Department of Psychiatry, University of
Texas Health Science Center, San Antonio,
Texas
6Research and Development Service, South
Texas Veterans Health Care System, San
Antonio, Texas
7Institute of Systems Neuroscience, Medical
Faculty, Heinrich Heine University Düsseldorf,
Düsseldorf, Germany
8Institute of Neuroscience and Medicine,
Brain & Behaviour (INM-7), Research Centre
Jülich, Jülich, Germany
Correspondence
Henry W. Chase, Western Psychiatric Institute
and Clinic, Loeffler Building, 121 Meyran
Avenue, Pittsburgh, PA 15213. Email: chaseh@upmc.edu
Simon Eickhoff, Institute for Systems
NeuroscienceHeinrich-Heine University
Düsseldorf
Email: s.eickhoff@fz-juelich.de 1Department of Psychiatry, University of
Pittsburgh School of Medicine, Pittsburgh,
Pennsylvania 1Department of Psychiatry, University of
Pittsburgh School of Medicine, Pittsburgh,
Pennsylvania
2Department of Neuroscience and Psychology,
University of Pittsburgh, Pittsburgh,
Pennsylvania
3Research Imaging Institute, University of
Texas Health Science Center, San Antonio,
Texas
4Department of Radiology, University of Texas
Health Science Center, San Antonio, Texas
5Department of Psychiatry, University of
Texas Health Science Center, San Antonio,
Texas
6Research and Development Service, South
Texas Veterans Health Care System, San
Antonio, Texas
7Institute of Systems Neuroscience, Medical
Faculty, Heinrich Heine University Düsseldorf,
Düsseldorf, Germany
8Institute of Neuroscience and Medicine,
Brain & Behaviour (INM-7), Research Centre
Jülich, Jülich, Germany
Correspondence
Henry W. Chase, Western Psychiatric Institute
and Clinic, Loeffler Building, 121 Meyran
Avenue, Pittsburgh, PA 15213. R E S E A R C H A R T I C L E R E S E A R C H A R T I C L E Functional differentiation in the human ventromedial
frontal lobe: A data-driven parcellation Henry W. Chase1
|
Anthony A. Grace1,2
|
Peter T. Fox3,4,5,6
|
Mary L. Phillips1
|
Simon B. Eickhoff7,8 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. Abstract Abstract
Ventromedial regions of the frontal lobe (vmFL) are thought to play a key role in
decision-making and emotional regulation. However, aspects of this area's functional
organization, including the presence of a multiple subregions, their functional and
anatomical connectivity, and the cross-species homologies of these subregions with
those of other species, remain poorly understood. To address this uncertainty, we
employed a two-stage parcellation of the region to identify six distinct structures
within the region on the basis of data-driven classification of functional connectivity
patterns obtained using the meta-analytic connectivity modeling (MACM) approach. From anterior to posterior, the derived subregions included two lateralized posterior
regions, an intermediate posterior region, a dorsal and ventral central region, and a
single anterior region. The regions were characterized further by functional connec-
tivity derived using resting-state fMRI and functional decoding using the Brain Map
database. In general, the regions could be differentiated on the basis of different pat-
terns of functional connectivity with canonical “default mode network” regions
and/or subcortical regions such as the striatum. Together, the findings suggest the
presence of functionally distinct neural structures within vmFL, consistent with data
from experimental animals as well prior demonstrations of anatomical differences
within the region. Detailed correspondence with the anterior cingulate, medial
orbitofrontal cortex, and rostroventral prefrontal cortex, as well as specific animal
homologs are discussed. The findings may suggest future directions for resolving
potential functional and structural correspondence of subregions within the frontal
lobe across behavioral contexts, and across mammalian species. Correspondence
Henry W. Chase, Western Psychiatric Institute
and Clinic, Loeffler Building, 121 Meyran
Avenue, Pittsburgh, PA 15213. Email: chaseh@upmc.edu
Simon Eickhoff, Institute for Systems
NeuroscienceHeinrich-Heine University
Düsseldorf
Email: s.eickhoff@fz-juelich.de Correspondence
Henry W. Chase, Western Psychiatric Institute
and Clinic, Loeffler Building, 121 Meyran
Avenue, Pittsburgh, PA 15213. Email: chaseh@upmc.edu
Simon Eickhoff, Institute for Systems
NeuroscienceHeinrich-Heine University
Düsseldorf
Email: s.eickhoff@fz-juelich.de 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.
© 2020 The Authors. Human Brain Mapping published by Wiley Periodicals, Inc. 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.
© 2020 The Authors. Human Brain Mapping published by Wiley Periodicals, Inc.
3266
Hum Brain Mapp. 2020;41:3266–3283.
wileyonlinelibrary.com/journal/hbm 1
|
INTRODUCTION In the case of the vmFL, there is already
some evidence that such a parcellation of distinct regions is possible
using co-activation maps: Clithero and Rangel found evidence for
three distinct clusters of activation within the vmFL when examining
fMRI
literature
on
value
encoding
in
this
region
(Clithero
&
Rangel, 2014): see also (Hiser & Koenigs, 2018) for another meta-
analytic approach). In contrast to these previous parcellation studies, a
novel aspect of the present work, from a methodological point of
view, is the use of a two-stage parcellation to address the indetermi-
nacy of the boundaries of the vmFL—the lateral and dorsal extents in
particular. First, a rather general vmFL region of interest was drawn
by hand which was used for the initial parcellation. A second
parcellation was performed on the best-resolved subregions within
the ventral and medial regions of the frontal lobe, with ambiguous,
peripheral regions being omitted from this analysis. It was hoped that
this would lead to a parcellation which effectively balanced complete-
ness and focus on the vmFL. Nevertheless, differentiation of function within the vmFL is antic-
ipated by at least two separate lines of evidence. First, confined
lesions of this region in rodents and monkeys can reveal distinct pat-
terns of deficit on flexible decision-making tasks, depending on the
precise location of the lesion and the task employed (Chudasama &
Robbins, 2003; Killcross & Coutureau, 2003; Rudebeck, Bannerman, &
Rushworth, 2008; Rudebeck & Murray, 2011). Consequently, the con-
cept of the vmFL as a unitary neuroanatomical region is challenged by
these experiments, and may refer to several independent but inter-
acting subregions. Further evidence that the vmFL is not a single
entity is a result of neuroanatomical evidence delineating granular and
agranular subregions within the vmFL (Barbas & Garcia-Cabezas, 2016;
Henssen et al., 2016; Wise, 2008). Presupposing that functional dif-
ferences may be carried by of anatomical differences (Eickhoff, Con-
stable,
&
Yeo,
2018),
there
are
significant
cytoarchitectonic
differences that might subserve the functional differences described
earlier. For example, Mackey and Petrides (Mackey & Petrides, 2014)
describe a gradient of increasingly developed, granular cortex from
posterior (i.e., subgenual and pregenual regions of the cingulate cor-
tex) to anterior (i.e., rostromedial prefrontal cortex) regions, with more
posterior
regions
showing
a
more
primitive,
agranular
cytoarchitecture. 1
|
INTRODUCTION Moreover, cross-species homologies are potentially more distant in
the frontal lobe compared to other neural structures (Wise, 2008),
underscoring the need for supporting evidence in humans to supple-
ment work in experimental animals. The famous case of Phineas Gage (Van Horn et al., 2012), as well as
more recent neuropsychological studies of patients with damage to
the ventromedial regions of the frontal lobe
(vmFL: Bechara,
Damasio, & Damasio, 2000; Clark et al., 2008; Fellows, 2011) have
helped to establish the vmFL as playing a critical role in adaptive eco-
nomic and social decision-making in humans (Delgado et al., 2016). Studies of human lesion patients have, in general, only limited capacity
to provide finely resolved anatomical detail regarding the precise
organization within this structure that underlies the profound deficits
that can be observed (Mah, Husain, Rees, & Nachev, 2014, but see
Glascher et al., 2012; Manohar & Husain, 2016). Thus, the extent to
which different syndromes and dimensions of psychiatric symptom-
atology are associated with alteration of particular subregions of the
vmFL remains uncertain. In the present study, we employed a parcellation algorithm which
has been applied to distinguishing clusters of similar brain (co-)activa-
tion patterns stored within the BrainMap database (P. T. Fox &
Lancaster, 2002). Examining patterns of co-activation across the brain
is a well-established approach to assessing functional connectivity
(Eickhoff et al., 2011). Thus, the combination of co-activation maps
with data-driven clustering algorithms offers the possibility of dis-
tinguishing subregions within a larger region of interest based on
interaction with different distal regions and/or networks. This method
has been applied to identify distinct regions within the dorsolateral
prefrontal cortex (Cieslik et al., 2013), the posterior cingulate (Bzdok
et al., 2015), the amygdala (Bzdok, Laird, Zilles, Fox, & Eickhoff, 2013)
and the subiculum (Chase et al., 2015), for example. In several cases,
the subregions that have been identified are regions with known ana-
tomical and functional differences: for example, Bzdok et al. found a
parcellation of the amygdala that corresponded well with the classic
tripartite (superficial, basolateral, and centromedian) model of the
region (Bzdok et al., 2013). 3267 CHASE ET AL. K E Y W O R D S connectivity-based parcellation, functional decoding, meta-analytic connectivity modeling,
resting-state fMRI, ventromedial frontal lobe 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 © 2020 The Authors. Human Brain Mapping published by Wiley Periodicals, Inc. Hum Brain Mapp. 2020;41:3266–3283. 2.2
|
Meta-analytic connectivity mapping (MACM)
and connectivity-based parcellation Parcellation involved the MACM-based approach employed in previ-
ous studies (Bzdok et al., 2013; Cieslik et al., 2013). Briefly, the
MACM approach involves computing the co-occurrence of significant
activations across studies within each voxel within the vmFL volume
of interest (VOI). Data from the BrainMap database were used (www. brainmap.org; P. T. Fox & Lancaster, 2002; Laird et al., 2011), regis-
tered into Montreal Neurological Institute (MNI) space. Given the
sparsity of voxel-wise activation, experiments were pooled within the
vicinity of each seed voxel using a spatial filter of different sizes. A
subsequent coordinate-based meta-analysis was performed on the
retrieved experiments, generating a brain-wide co-occurrence of acti-
vation profile of a given seed voxel, for each filter size (37). The brain-
wide pattern of co-occurrence for each individual seed voxel was
computed by activation likelihood estimation (ALE; Eickhoff, Bzdok,
Laird, Kurth, & Fox, 2012; Turkeltaub, Eden, Jones, & Zeffiro, 2002)
meta-analysis over the experiments that were associated with that
particular voxel by the pooling procedure outlined earlier. However,
no thresholding was performed at this stage, with the goal to obtain
whole-brain map of co-occurrence probabilities for each seed voxel,
to be used as a basis for parcellation of each VOI. MACM was used to estimate functional connectivity, and was
used as the basic dependent measure for the parcellation. Seed-based
connectivity using resting-state fMRI (rsfMRI) was used to character-
ize the functional connectivity of the resulting subregions further. RsfMRI contributes an important inferential component to the present
work: the MACM maps for each subregion were used to define the
subregion via the parcellation, and thus estimates of connectivity are
thus somewhat circularly defined. RsfMRI can be used to provide con-
firmation that similar functional connectivity can also be observed
using an independent data set and methodology. Finally, we aimed to characterize the neurofunctional properties
of the regions. To the best of our knowledge, a comprehensive exami-
nation of the functional profile of the activation properties of the
region has not been conducted across a variety of psychological tasks
(i.e., the types of paradigm and psychological domain that activate the
region). Using the BrainMap database, we examined the paradigms
and psychological domains that activated the identified subregions of
the vmFL, and also confirmed the capacity of activation within the
vmFL to be decoded in terms of a particular paradigm or domain. 2.1
|
ROI definition and methods overview The demarcation of the vmFL ROI used for clustering was centered on a
roughly defined region (Figure S4), based largely on the medial OFC
regions defined within the automated anatomical labeling template
(Tzourio-Mazoyer et al., 2002). However, this region was expanded con-
siderably by drawing a wider volume around the predefined OFC regions
using the Marina tool (Walter et al., 2003). This expanded region com-
prised the entire anterior–posterior dimension of the ventral frontal cor-
tex, that is, from just anterior of the ventral striatum to the entire frontal
pole, and extended dorsally just above the genu. We took this approach
in order to avoid dependence on any one particular anatomical scheme,
and to be as inclusive as possible. As this definition was relatively arbi-
trary, this region formed the basis for an initial parcellation which would
define a focus for a subsequent parcellation, mostly by removing the lat-
eral, anterior (i.e., frontal polar) and dorsal edges of the region (Figure S5). The second parcellation would then be conducted on the clusters derived
from this initial parcellation which most clearly reflected the ventromedial CHASE ET AL. aspect of the frontal lobe. This second parcellation is the main focus of
the analysis: we characterized the subregions derived from this second
parcellation in terms of their co-activation patterns, resting-state func-
tional connectivity, and BrainMap-database derived activation properties. empirical data from rodents and nonhuman primates, the precise loca-
tion and functional characteristics of the hypothesized regions has
not, to the best of our knowledge, been confirmed in humans despite
the fact that in vivo evidence of functional and anatomical differentia-
tion has been obtained (Clithero & Rangel, 2014; Samara et al., 2017). Furthermore, recent work suggests that different brain states may
influence the nature of parcellation, and thus different methods can-
not be assumed to yield identical subregions (Kahnt & Tobler, 2017;
Salehi et al., 2020). The Meta-analytic connectivity modeling (MACM)
parcellation approach using the BrainMap database might therefore
provide
complementary
insights
into
the
context-specificity
of
parcellation within the vmFL. 2.2
|
Meta-analytic connectivity mapping (MACM)
and connectivity-based parcellation Following our previous studies (Bzdok et al., 2013; Cieslik
et al., 2013), we used K-means clustering (MATLAB, Mathworks) to
parcellate each VOI, using K = 2–10 for the first and K = 2–11 for the
second. K-means clustering was performed on the unthresholded
brain-wide co-occurrence profiles for all seed voxels: specifically, the
NS × NT co-occurrence matrix, where NS denotes the number of seed
voxels in each VOI and NT the number of target voxels in the (down-
sampled) reference brain volume. The distance measure used for the
K-means clustering was one minus the correlation between the co-
occurrence patterns of seed voxels defined earlier (correlation dis-
tance). Importantly, maps of co-occurrence of activations were com-
puted for each of the 37 different spatial filter sizes, and the K-means
parcellation was performed for each filter size independently, yielding
(K clusters) × 37 (filter size) independent cluster solutions (Clos,
Amunts,
Laird,
Fox,
&
Eickhoff,
2013). Replications
of
each
parcellation were performed for each VOI using random initial condi-
tions (centroids), to avoid local minima. 1
|
INTRODUCTION The vmFL also shows differential patterns of ana-
tomical connectivity, across its structure, with distal regions including
the striatum, insula, thalamus, hypothalamus, hippocampus, amygdala,
and
other
temporal
lobe
structures
(Heilbronner,
Rodriguez-
Romaguera,
Quirk,
Groenewegen,
&
Haber,
2016;
Hoover
&
Vertes, 2007; Reppucci & Petrovich, 2016; Saleem, Kondo, &
Price, 2008; Vertes, 2004). We expected to distinguish regions corresponding to the human
homologs of the infralimbic (IL) and prelimbic (PL) cortices, which are
likely to correspond to Brodmann area 25 and 32 in humans
(Azuma & Chiba, 1996; Heilbronner et al., 2016; Room, Russchen,
Groenewegen, & Lohman, 1985; Takagishi & Chiba, 1991). We also
expected to identify regions corresponding to the medial OFC (11, 14)
and
posterior
regions
of
Brodmann
Area
10
(Mackey
&
Petrides, 2014; Ongur et al., 2003; Wise, 2008). The frontal pole itself
was not a primary focus of the study, and has been previously exam-
ined in the context of other anatomical studies (Bludau et al., 2014;
Ray et al., 2015). The functional connectivity of the emerging subre-
gions was expected to correspond to known anatomical relationships
with distal neural regions, including the posterior cingulate cortex
(PCC), amygdala, hippocampus, temporal lobe, midbrain, ventral stria-
tum, and thalamus. Although these expectations were grounded in These separate lines of evidence thus appear to favor separate
functional and anatomical subregions within the human vmFL. Briefly,
these subregions may tentatively include subgenual and pregenual
regions of the anterior cingulate cortex (ACC), the medial orbitofrontal
cortex (OFC), and parts of the rostromedial prefrontal cortex
corresponding to Brodmann Area 10 (Ongur, Ferry, & Price, 2003). There remains relatively little in vivo evidence for functional differen-
tiation along these lines in humans, however, and functional similari-
ties among these regions can be emphasized (Delgado et al., 2016). 3268 2.5
|
Functional connectivity analysis: Task-based
and resting-state Further analyses were conducted to characterize the subregions
resulting from the second vmFL parcellation, which reflected the core
region of interest for the present work. First, meta-analytic connectiv-
ity modeling (MACM) was employed. For this, all experiments in the
BrainMap database that featured at least one focus of activation in a
particular subregion were compiled, and convolved with a 3D Gauss-
ian as described in Section 2.2. However, now conventional inference
was performed with reference to a null distribution reflecting a ran-
dom spatial association between experiments with a fixed within-
experiment distribution of foci (Eickhoff et al., 2009). A nonparametric
p-value based on the proportion of equal or higher random values
than the null distribution was thereby obtained for each voxel
(Eickhoff et al., 2012), and these were transformed into Z-scores and
thresholded at a cluster-level family-wise error (FWE) rate-corrected
threshold
of
p < .05
(cluster-forming
threshold
at
voxel-level
p < .001). The second step was to determine the optimal solution of
K within the restricted filter range of filter sizes. We considered five
criteria representing the characteristics of the cluster solutions,
reflecting topological, information-theoretic, and cluster separation
properties (Figures S2 and S3). First, misclassified voxels (deviants)
were examined as a topological criterion, with optimal K parcellations
being those in which the percentage of deviants was not significantly
increased compared to the K −1 solution and but where the K + 1
was
associated
with
significantly
increased
deviants
(Bzdok
et al., 2015). Second, the variation of information (VI) metric was
employed as an information-theoretic criterion to assess the similarity
of cluster assignments for each filter size between the current solu-
tion and the neighboring (K −1 and K + 1) solutions (Meila, 2007). Third, the silhouette value averaged across voxels for each filter size
was considered a cluster separation criterion. Fourth, we assessed the
percentage of voxels not related to the dominant parent cluster com-
pared with the K −1 solution as a second topological criterion. This
measure corresponds to the percentage voxels that are not present in
hierarchy, K, compared with the previous K −1 solution, and is related
to the hierarchy index (Kahnt, Chang, Park, Heinzle, & Haynes, 2012). Finally, the change in inter- versus intra-cluster distance ratio was
computed (Chang, Kenney, Loucks, Poletto, & Ludlow, 2009) as a sec-
ond cluster separation criterion. Finally, the locations of the clusters were
mapped back on the brain, taking the mode across filter sizes. 2.5
|
Functional connectivity analysis: Task-based
and resting-state This ratio is the first derivative of the
ratio between the average distance of a given voxel to its own cluster
center and the average distance between the cluster centers. We tested for differences in co-occurrence patterns between all
pairs of clusters by performing MACM separately on the experiments
associated with either cluster, and then computing the voxel-wise dif-
ference between the ensuing ALE maps. All experiments contributing
to the two contrasted clusters were pooled and randomly divided into
two groups of the same size as the two original sets of experiments
defined by activation in the first or second cluster (Eickhoff
et al., 2011). ALE-scores for these two randomly assembled groups
were calculated and the difference between these ALE-scores was
recorded for each voxel in the brain. Repeating this process 10,000
times then yielded a null distribution of differences in ALE-scores
between the MACM analyses of the two clusters. The “true” differ-
ence in ALE scores was then tested against this null distribution yield-
ing a posterior probability that the true difference was not due to
random noise in an exchangeable set of labels based on the propor-
tion of lower differences in the random exchange. The resulting prob-
ability values were then thresholded at p > .95 (95% chance for true
difference) and inclusively masked by the respective main effects, that
is, the significant effects in the MACM for the particular cluster. To
simplify the analysis and reduce the number of comparisons, we com-
puted a conjunction map of the contrasts of a given subregion with all
others (e.g., Clusters 1 vs. 2–6). This would identify regions which
were preferentially connected to the subregion compared to all other
subregions. 3269 CHASE ET AL. the most stable range of filter sizes. This prevented a combinatorial
expansion of possible solutions, and avoided the requirement of aver-
aging across filter sizes (Bzdok et al., 2013; Cieslik et al., 2013). We
implemented a two-step procedure that involved a decision on those
filter sizes to be included in the final analysis and subsequently a deci-
sion on the optimal cluster solution. In the first step, we examined the
consistency of the cluster assignment for the individual voxels across
the cluster solutions of the co-occurrence maps performed at differ-
ent filter sizes. We selected a filter range with the lowest number of
deviants, that is, number of voxels that were assigned differently com-
pared with the solution from the majority of filters. In other words,
we identified those filter sizes which produced solutions most similar
to the consensus-solution across all filter sizes. For example, the pro-
portion of deviants for the second parcellation is illustrated in
Figure S1; this shows the borders of the filter range to be used for
subsequent steps was based on the Z-scores of the number of
deviants. the most stable range of filter sizes. This prevented a combinatorial
expansion of possible solutions, and avoided the requirement of aver-
aging across filter sizes (Bzdok et al., 2013; Cieslik et al., 2013). We
implemented a two-step procedure that involved a decision on those
filter sizes to be included in the final analysis and subsequently a deci-
sion on the optimal cluster solution. In the first step, we examined the
consistency of the cluster assignment for the individual voxels across
the cluster solutions of the co-occurrence maps performed at differ-
ent filter sizes. We selected a filter range with the lowest number of
deviants, that is, number of voxels that were assigned differently com-
pared with the solution from the majority of filters. In other words,
we identified those filter sizes which produced solutions most similar
to the consensus-solution across all filter sizes. For example, the pro-
portion of deviants for the second parcellation is illustrated in
Figure S1; this shows the borders of the filter range to be used for
subsequent steps was based on the Z-scores of the number of
deviants. the goodness-of-fit criterion. Finally, the locations of the clusters were
mapped back on the brain, taking the mode across filter sizes. the goodness-of-fit criterion. 2.3
|
Selection of optimal filter range and number
of clusters Our approach to selecting the optimal solution of K-means clustering
was to examine the properties of these various solutions and establish CHASE ET AL. CHASE ET AL. a psychological process being present given knowledge of activation
in a particular brain region. In the forward inference approach, a clus-
ter's functional profile was determined by identifying taxonomic
labels, for which the probability of finding activation in the respective
cluster was significantly higher than the overall chance (across the
entire database) of finding activation in that particular cluster. Signifi-
cance was established using a binomial test (p < .05, corrected for
multiple comparisons with reference to the false discovery rate
[FDR]). Thus, we tested whether the conditional probability of activa-
tion given a particular label (P[Activation|Task]) was higher than the
base rate probability of activating a given subregion per se (P[Activa-
tion]). In the reverse inference approach, a cluster's functional profile
was determined by identifying the most likely behavioral domains and
paradigm classes given activation in a particular subregion. This likeli-
hood P(Task|Activation) can be derived from P(Activation|Task) as well
as P(Task) and P(Activation) using Bayes' rule. Significance was then
assessed by means of a chi-square test (p < .05, FDR corrected). Acquisition parameters and preprocessing of these images has been
described in previous work (Chase et al., 2015). Briefly, 404 echo-
planar images (EPIs) were acquired on a Siemens Trio 3T scanner,
using a TR of 1.4 s. Preprocessing involved realignment and normali-
zation using the “unified segmentation” approach into MNI space,
followed by spatial smoothing with a 5 mm Gaussian kernel. Nuisance
correction was then performed using motion parameters and their
first derivatives, and mean cerebrospinal fluid, gray, white matter time
series. Following nuisance correction, the time series were band pass
filtered
between
0.01
and
0.08 Hz. The
connectivity-based
parcellation (CBP)-derived clusters from the second parcellation were
used as seeds for the resting-state analysis. Linear (Pearson) correla-
tion coefficients between the time series of the seed regions and all
other gray matter voxels in the brain were computed to quantify
rsfMRI connectivity. These voxel-wise correlation coefficients were
then transformed into Fisher's Z-scores and tested for consistency in
a flexible factorial model across subjects. We used these maps for two analyses. First, we investigated the
similarity between the MACM and resting-state analyses: rsfMRI Z-
score maps were masked using the thresholded maps from the
MACM analysis: inference was performed only within the regions
identified
as
co-activated
by
a
MACM
analysis
using
the
corresponding subregion as a seed. 3.1
|
Connectivity-based parcellation The clustering analysis proceeded in two stages. The initial vmFL ROI
was deliberately drawn to be large (Figure S4), and had relatively few
anatomical constraints defining its shape. After the first parcellation, a
nine-cluster solution was chosen (Figure S5). This was supported in
the following ways: a significant increase in VI from 9 to 10, but not
8 to 9; a significant increase in silhouette from 8 to 9, but not 9 to 10;
VI across clusters being very low at 9 but high going to 10, indicating
that from 8 to 9 there is good consistency, but little consistency from
9 to 10; change in inter/intra-cluster distance identified up to 9, which
is a local maximum, that is, separation becomes much better when
going to 9 but improves little going up to 10; a decline in misclassified
voxels from 8 to 9 but a significant increase from 9 to 10. Of the nine subregions, we focused on four as reflecting the
vmFL (numbers 2, 4, 7, and 8) and excluded the remaining five. The
selected clusters occupied ventral and medial regions of the frontal
lobe: in particular, the medial width of selected regions was similar to
anatomical definitions of the medial OFC (Henssen et al., 2016). Of
the excluded clusters, Regions 1 and 3 were not fully resolved, and
corresponded to regions that might be distinct from the vmFL (ante-
rior cingulate and frontal pole). Interesting, the frontal pole cluster
overlapped clearly with an anatomically defined ROI of the frontal
pole (Bludau et al., 2014). The remaining clusters (5, 6, and 9) were
also not fully resolved, appearing on the edges of the initial vmFL ROI
(Figure S5). A cluster was reported as signifi-
cant in Table 2 (positive/negative: far right-hand column) if an FWE-
corrected peak threshold of p < .05 was reached (corrected for voxels
within the MACM mask rather than the whole brain). Second, we
examined whole-brain connectivity for each subregion using a similar
strategy for the MACM contrast maps, that is, calculate a given subre-
gion's positive functional connectivity compared to all others. The
contrast was weighted so that the target region was coded as 5 and
the other regions were coded as −1. Post hoc analyses were per-
formed to distinguish subregions with similar connectivity. The stan-
dard
SPM8
implementations
were
used
including
appropriate
nonsphericity correction, and correction for age and gender. These
analyses were thresholded at p < .05 (FWE cluster-corrected; cluster-
forming threshold at voxel-level p < .001). 2.4
|
Visualization of the best cluster solution Voxels which were located in the gray matter, and were hierarchically
and spatially consistent, were considered for subsequent analyses. Multidimensional
scaling
(MDS)
was
used
to
visualize
the
2-dimensional cluster separation. We computed the NS × NS correla-
tion distance matrix (Section 2.3) for each of the filter sizes. Next,
MDS was performed on the eigenimage of the resulting correlation
distance matrixes, with Sammon's nonlinear mapping being used as In addition, we also delineated the task independent resting-state
functional connectivity pattern of each cluster from the second
parcellation. RsfMRI images of 196 healthy volunteers (mean age 39.8
± 15.1 years; 76 males) from the enhanced Nathan Kline Institute
(NKI)/Rockland sample were obtained through the 1,000 Functional
Connectomes
Project
(www.nitrc.org/projects/fcon_1000/). 3270 3270 3270 2.6
|
Functional characterization: Meta-data The functional characterization of the CBP-derived clusters was based
on the “Behavioral Domain” and “Paradigm Class” meta-data catego-
ries available for each neuroimaging experiment included in the
BrainMap database. Behavioral domains include the main categories
cognition, action, perception, emotion, and interoception, as well as
their related sub-categories. Paradigm classes categorize the specific
task employed (see http://brainmap.org/scribe/ for the complete
BrainMap taxonomy). We then performed a second parcellation on the four vmFL sub-
regions from the initial parcellation, which yielded a six-cluster solu-
tion (Figure 1). This parcellation was assessed using the same
measures as before, and supported in the following ways: silhouette—
a significant increase from 5 to 6 but a decrease from 6 to 7; VI across
clusters—very low up to 6 but high from 7 onward, indicating that In a first step, we determined the individual functional profile of
the six CBP-derived clusters by using forward and reverse inference
(Bzdok et al., 2013; Cieslik et al., 2013). Forward inference is the prob-
ability of observing activity in a brain region given knowledge of the
psychological process, whereas reverse inference is the probability of 3271 3271 CHASE ET AL. FIGURE 1
Locations of the six-cluster solution within the vmFL (clusters color-coded as follows: 1 = blue, 4 = cyan, 6 = green; 2 = yellow,
3 = magenta, 5 = red). vmFL, ventromedial regions of the frontal lobe FIGURE 1
Locations of the six-cluster solution within the vmFL (clusters color-coded as follows: 1 = blue, 4 = cyan, 6 = green; 2 = yellow,
3 = magenta, 5 = red). vmFL, ventromedial regions of the frontal lobe he six-cluster solution within the vmFL (clusters color-coded as follows: 1 = blue, 4 = cyan, 6 = green; 2 = yellow,
ventromedial regions of the frontal lobe strong functional connectivity. Specifically, the most posterior, sub-
genual Clusters 1 and 6 were connected to proximal regions of the
vmFL, and the ventral striatum and amygdala, the right insula
(Cluster 1 only), the thalamus (Cluster 6 only). The other posterior
cluster (4) was connected to a number of DMN regions including the
PCC and the superior frontal gyrus (SFG), and also the insula, left
OFC, and the ventral striatum, amygdala, and hippocampus. The two
central clusters (3 and 5) were similarly connected to the DMN, ven-
tral striatum, amygdala, hippocampus, the thalamus (Cluster 3 only),
and left OFC (Cluster 5 only). 2.6
|
Functional characterization: Meta-data Finally, the most anterior cluster
showed connectivity with the DMN, the left ventrolateral prefrontal
cortex (VLPFC), the amygdala and hippocampus, and the left middle
temporal gyrus. In general, patterns of functional connectivity as
estimated using MACM overlapped well with those using rsfMRI:
Table 2 describes the present of significant (small volume corrected)
rsfMRI functional connectivity within the regions specified by
MACM. There was only one clear contradiction to this pattern, aside
from a few null findings: Cluster 4 showed (positive) functional con-
nectivity with the right dorsal insula using MACM, but anti-
correlation using rsfMRI. from 5 to 6 there is good consistency, but little from 6 to 7; change in
inter/intra-cluster distance shows that 6 is a local maximum, that is,
separation becomes much better when going to 6 but improves little
going to 7. VI across filter sizes was uninformative, as it increased sim-
ilarly across cluster solutions. However, misclassified voxels did not
support the six-cluster solution, since there was an increase from 5 to
6 but none from 6 to 7. Thus, on balance, a six-cluster solution was
preferred, if less decisively than the first parcellation. For comparison
purposes, we have included figures of the nonpreferred cluster solu-
tions in supplementary information (Clusters 3–5/7–8; Figure S6). The six clusters which were derived are displayed in Figure 1, and
include two subgenual clusters (1 and 6), a posterior cluster (4), two
central clusters (dorsal [3] and ventral [5]), and one anterior cluster (2). We
examined
the
overlap
of
these
clusters
with
anatomical
parcellations of OFC (Henssen et al., 2016; Mackey & Petrides, 2014);
see Table 1). Neither of the anatomical parcellations cleanly mapped
onto the six CBP-derived clusters: rather, the six CBP clusters were
made up of very roughly similar ratios of cingulate (e.g., area 24/32)
and orbitofrontal (e.g., Fo/Fp or areas 11/14) anatomical labels
derived from both templates. Comparison of each cluster's MACM map was performed. First,
in
order
to
identify
crucial
regions
that
contributed
to
the
parcellation, we contrasted each region with all of the other five
regions (finding described in Table 3). Contrasting in a similar way
rsfMRI maps largely corroborated these findings, insofar as all clus-
ters aside from 4 and 6, and Cluster 2's parietal region, showed
greater connectivity in the same region(s) as identified by the MACM
contrasts (p < .001). 2.6
|
Functional characterization: Meta-data In the case of Cluster 6, this effect was some-
what weaker (p < .05), but for Cluster 4, no difference in rsfMRI was
seen in the putative hypothalamic region. Three of the more anterior
regions (central dorsal and ventral, anterior) all showed preferential
connectivity with areas of the DMN, including the PCC and SFG, 3.3 We also interrogated the BrainMap database to characterize the func-
tional properties of the regions (Figure 4: fuller description of cluster
comparisons is included in Tables S1 and S2). Emotion, cognition and
reward were domains/paradigms that were most commonly represen-
ted across all regions, with relatively little preference for one region
over any of the others in these dimensions, and numerous conjunc-
tions between regions. Interestingly, while forward inference likeli-
hoods were relatively well matched across relevant paradigms
(Figure 4), cognition and reward showed much larger posterior proba-
bilities of decoding paradigm/domain from an activation using reverse
inference
(~0.15–0.2)
than
other
types
of
study
(generally
~0.015–0.05). This might be related to a greater frequency of reward/
cognition studies in the BrainMap database, which would be associ-
ated with a greater prior probability for these studies. Gustation and taste paradigms were also quite widely represen-
ted, with perhaps Clusters 3, 4, and 5 showing a slight advantage. Thirst also modulated activity in regions 3 and 5. Evidence for selec-
tivity was seen in other domains/paradigms, including olfaction/olfac-
tory monitoring and discrimination, in which Cluster 2 was relatively
selectively engaged. Together, this might suggest the presence of
regional specialization of gustatory and olfactory systems in the vmFL. Although this preference was mostly qualitative rather than supported
by clear dissociations, Cluster 2 did show significant preference for
olfaction over Clusters 4 and 5. although with slightly different emphasis in each case. In addition,
the anterior region showed preferential connectivity with the left
VLPFC. The three more posterior regions showed preferential con-
nectivity with the insula, as well as subcortical regions such as the
thalamus and basal ganglia. We also employed a similar method to examine rsfMRI functional
connectivity, in which one seed was contrasted with all others to
obtain regions where a given seed region was more positively corre-
lated with target regions compared to the other regions. Cluster 1 rev-
ealed widespread connectivity, predominantly with “task-positive”
regions such as the lateral prefrontal cortex and regions of the parietal
lobe (central executive network), the anterior insula and supplemen-
tary motor area (“salience network”), as well as the visual cortex, cau-
date, premotor cortex and regions of the cerebellum. 3272 3272 CHASE ET AL. CHASE ET AL. TABLE 1
Overlap of the six clusters with the JuBrain template
and the Mackey and Petrides ventromedial atlas (percentages
describe proportions of cluster within given anatomical label; all
overlap >2% reported)
Anatomy toolbox (JuBrain)
Mackey &
Petrides, 2014
Cluster
1
42.8% Fo2; 11.3% Fo3; 5.3% area
25; 3.2% area s24 (all right)
35% right 14c
24.5% right 14r
10.5% right 25
5.7% right 32
Cluster
2
15.1% right Fo1; 14.7% left Fp2;
11.7% left Fo1; 11.6% left Fp1
8.8% left 11 m
13.4% right 11 m
6.5% left 14 m
7.5% right 14 m
Cluster
3
22.5% left area p32; 16.9% right
area p32; 10.1% left area
p24ab; 9.0% left area s32
31.2% left 14 m
27.9% right 14 m
3.3% left 24
3.2% right 24
9.6% left 32
8.0% right 32
Cluster
4
22.3% left area Fo2; 15.1% left
area s24; 15.0% right area Fo2;
11.3% right area s24
3.6% left 14c
2.8% right 14c
19.3% left 14 m
18.0% right 14 m
12.2% left 14r
11.6% right 14r
2.1% left 24
7.8% left 32
6.4% right 32
Cluster
5
32.8% right area Fo1; 29.3% left
area Fo1; 4.6% left area s32;
4.1% right area s32
9.3% left 11 m
10.6% right 11 m
11.6% left 14 m
13.3% right 14 m
3.4% left 14r
5.8% right 14r
12.5% left 14r0
18.8% right 14r0
Cluster
6
36.1% Fo2; 7.9% area 25; 4.6%
area Fo3; 2.8% area 33 (all left)
45.0% left 14c
3.4% left 14r
13.7% left 25
5.5% left 32 TABLE 1
Overlap of the six clusters with the JuBrain template
and the Mackey and Petrides ventromedial atlas (percentages
describe proportions of cluster within given anatomical label; all
overlap >2% reported) prefrontal cortex, posterior cingulate/precuneus, inferior parietal lob-
ule, cerebellum, and temporal lobe. Cluster 3 was also strongly con-
nected to similar DMN regions, but also more ventromedial frontal
regions and, insula, striatum (caudate/putamen), hippocampus (but
more weakly with the amygdala), thalamus and midbrain structures. Cluster 4 was also connected to DMN structures, but also medial tem-
poral lobe regions (amygdala/hippocampus), somatosensory/motor
cortex, posterior insula, and VLPFC. Cluster 5 structures were very
similar to those identified in Cluster 4: direct contrast of Clusters
4 and 5 yielded greater insula connectivity for Clusters 4 than 5, and
greater visual cortex and cerebellum connectivity for Cluster 5 than
4. 3272 Finally, Cluster 6 yielded similar central executive, salience and
visual network regions to Cluster 1. Direct comparison of Clusters
1 and 6 suggested that Cluster 1 was better coupled to “task-positive”
networks than Cluster 6, and vice versa for Cluster 6 and the default
mode regions. 3.2
|
Functional connectivity (MACM/rsfMRI) We examined the connectivity of the six clusters using MACM and
resting fMRI (Figures 2 and 3; Table 2). Several broad patterns
emerged. As the vmFL is part of the default mode network (DMN;
Raichle et al., 2001), many of the clusters showed evidence of func-
tional connectivity with posterior cingulate, temporal and parietal
regions canonically associated with this network. In addition, regions
with strong anatomical connectivity with the vmFL, including the
ventral striatum, amygdala, hippocampus, and thalamus, also showed 3.3 Cluster 2 was
positively connected to DMN regions, particularly the dorsomedial Several candidate cognitive subdomains or paradigms were
highlighted within the overall “cognition” domain: these included
explicit memory (Cluster 2), episodic recall (Cluster 6), orthography
(Cluster 2), social cognition (Cluster 3), face monitoring/discrimination
(Cluster 3), theory of mind (Cluster 6), and music comprehension/pro-
duction (Cluster 5). Finally, as well as reward paradigms, evidence for
the representation of negative affect in the vmFL was obtained, inso-
far as sadness (Cluster 4) and fear (Clusters 3, 4, and 5) domains were
also identified as capable of activating subregions of the vmFL. 3273 CHASE ET AL. 3273 FIGURE 2
MACM plots for each of the posterior subregions. Clusters color-coded as previously—top to bottom: 1 = blue, 4 = cyan,
6 = green. MACM, meta-analytic connectivity modeling FIGURE 2
MACM plots for each of the posterior subregions. Clusters color-coded as previously—top to bottom: 1 = blue, 4 = cyan,
6 = green. MACM, meta-analytic connectivity modeling 4
|
DISCUSSION the human vmFL. Interestingly, all of these studies have yielded some-
what different findings, both from one another and the present work. In general, they have examined larger regions of the ventral or medial
prefrontal cortex, and have parcellated the region at a lower resolu-
tion than we have here. To the best of our knowledge, our study is
the first to employ a two-stage parcellation technique, which may
have provided greater capacity to distinguish particular subregions
within the vmFL. In addition, prior research largely reflects anatomical
or resting differences. However, there is evidence that functional clus-
ters within in the vmFL might change following dopaminergic manipu-
lations (Kahnt & Tobler, 2017), while other evidence suggests that,
due to dynamic shifts in connectivity, functional clustering may differ
based on task state (Iraji, Miller, Adali, & Calhoun, 2020; Salehi
et al., 2020). Thus, our findings add to this literature by examining
across a wide variety of tasks states within the BrainMap database. An exception within the prior vmFL studies is the study of Clithero
and Rangel (2014), which involved a parcellation of coordinate maps—
although these were restricted to studies of value. This study identi-
fied three clusters, one of which roughly reflected a frontal polar
region. The remaining dorsal and ventral regions might approximately
map onto our dorsal central and posterior/subgenual clusters, respec-
tively. These latter regions were characterized by distinct patterns of
functional connectivity, with the dorsal central region showing con-
nectivity with the dorsal PCC, SFG, ventral striatum, and central OFC. Our findings are consistent with these findings insofar as similar In the present study, we aimed to identify distinct functional regions
within the vmFL. We distinguished six subregions within a two-stage
parcellation process, in which a candidate vmFL was initially identified
from surrounding areas (e.g., dorsal ACC, lateral/central OFC, and
rostral prefrontal cortex) before it was subdivided further. The six
clusters can be summarized as follows, moving in order from posterior
to anterior: most posterior were separate left and right subgenual
ACC regions, followed by a posterior region, two central regions (one
dorsal and one ventral), and finally an anterior region (Figure 1). Corre-
spondence between each of these regions with conventional anatomi-
cal labeling is discussed in detail in Section 3.1 Each region was, by
definition, associated with differential MACM-estimated connectivity,
but these differences were also supported by distinct resting fMRI
connectivity for each region. 4
|
DISCUSSION Moreover, some differences in the func-
tional properties of the clusters were also observed, although in many
cases similarities in these properties, particularly with respect to
reward and emotion, were also pronounced. In the present study, we aimed to identify distinct functional regions
within the vmFL. We distinguished six subregions within a two-stage
parcellation process, in which a candidate vmFL was initially identified
from surrounding areas (e.g., dorsal ACC, lateral/central OFC, and
rostral prefrontal cortex) before it was subdivided further. The six
clusters can be summarized as follows, moving in order from posterior
to anterior: most posterior were separate left and right subgenual
ACC regions, followed by a posterior region, two central regions (one
dorsal and one ventral), and finally an anterior region (Figure 1). Corre-
spondence between each of these regions with conventional anatomi-
cal labeling is discussed in detail in Section 3.1 Each region was, by
definition, associated with differential MACM-estimated connectivity,
but these differences were also supported by distinct resting fMRI
connectivity for each region. Moreover, some differences in the func-
tional properties of the clusters were also observed, although in many
cases similarities in these properties, particularly with respect to
reward and emotion, were also pronounced. The findings extend previous investigations using diffusion tensor
imaging (Jackson, Bajada, Lambon Ralph, & Cloutman, 2019; Neubert,
Mars, Sallet, & Rushworth, 2015), resting fMRI (Kahnt et al., 2012;
Samara et al., 2017, Neubert et al., 2015), structural MRI (Liu, Qin, Qi,
Jiang, & Yu, 2015) and value fMRI studies (Clithero & Rangel, 2014)
which suggested that distinct neural regions could be identified within 3274 3274 CHASE ET AL. CHASE ET AL. FIGURE 3
MACM plots for each of the anterior subregions. Clusters color-coded as previously—top to bottom: 2 = yellow, 3 = magenta,
5 = red. MACM, meta-analytic connectivity modeling FIGURE 3
MACM plots for each of the anterior subregions. Clusters color-coded as previously—top to bottom: 2 = yellow, 3 = magenta,
5 = red. MACM, meta-analytic connectivity modeling studies have also shown a good correspondence between a CBP-
based parcellation of the amygdala and cytoarchitectonic definitions
of the region (Bzdok et al., 2013). Thus, given that the CBP method
has been shown to yield distinct functional clusters that are consistent
with anatomical labels, we might consider reasons we did not demon-
strate this in the present study. 4
|
DISCUSSION First, the clustering method is depen-
dent on the functional connectivity of the region and not on other
properties. Thus, it is possible that there are stark differences in func-
tional or anatomical properties across the vmFL which are not
reflected in MACM-based functional connectivity. For example, acti-
vation of the basolateral amygdala may be reflected in activity both in
the
OFC
and
medial
PFC
(Lee
et
al.,
2010;
Logothetis
&
Wandell, 2004), despite the possibility that they decode or process
this
information
input
differently
(Rudebeck,
Mitz,
Chacko,
&
Murray, 2013). Likewise, activity in separate vmFL regions might con-
verge onto a single distal region, or two regions which are below the
resolution where they can be differentiated. Furthermore, recent evi-
dence has suggested finely “interdigitated” networks (Braga &
Buckner, 2017), which may be reflected in a patchwork which might
cut across the boundaries of discrete clusters. regions are identified and play a role in distinguishing the different
vmFL subregions (particularly SFG and PCC). The parcellation of the
medial prefrontal cortex by Samara et al. also shows similarities with
our findings (Samara et al., 2017), yielding around four regions which
approximately overlap with our six subregions. This article is sugges-
tive that a combination of rsfMRI and MACM may yield a “consensus”
parcellation consisting of subregions defined jointly by both metrics,
while examining differences between task states may provide further
insight into the potential for a state-independent parcellation which
maps onto anatomical regions. 4.1
|
Comparison with other parcellation schemes:
Technical considerations The subregions we identified did not map cleanly onto two MNI-
based
anatomical
labeling
templates
of
the
vmFL
(Henssen
et al., 2016; Mackey & Petrides, 2014), with most of the CBP-defined
clusters mapping on to approximately 2–4 anatomical labels. Before
considering reasons why this might be the case, it is important to note
that a very anterior region identified by the initial parcellation
(to obtain the vmFL cluster used for the main parcellation) overlapped
quite clearly with a frontal pole region within JuBrain (Bludau
et al., 2014; a parcellation of the frontal pole and overlap with ana-
tomical regions has been reported in Ray et al., 2015). Previous Second, for a clear structure/function overlap to be seen, the par-
adigms employed must necessarily be highly selective for the process
subserved by the particular subregion. However, the functional
decoding analysis yielded examples of paradigms/domains which were
related to the activation of several subregions. This suggests, at least, TABLE 2
(Continued) TABLE 2
(Continued) TABLE 2
(Continued)
Cluster size
Peaks
RsfMRI
•
Bilateral hippocampus
•
Left central OFC
•
Superior frontal gyrus
−2, 2, –12
14, 26, –10
Precuneus/posterior cingulate
1,061
−4, −56, 20
−2, −52, 30
6, –52, 18
0, –36, 38
10, –56, 28
Positive
Left angular/middle temporal gyrus (PGp/PGa)
329
−48, −68, 32
−50, −66, 20
Positive
Right dorsal insula
238
38, 4, 10
42, –2, 4
Negative
Left putamen/left dorsal insula
148
−28, 4, 8
−42, −6, 8
None
Cluster 5 (central ventral)
Large cluster including:
•
Ventromedial PFC
•
Posterior frontal pole/dorsomedial PFC
•
Bilateral medial ventral striatum
•
Left amygdala
•
Left hippocampus
5,303
−2, 38, –16
−22, −14, −22
−2, 62, 4
8, 10, –10
8, 54, –8
−12, 42, 42
Positive (all regions)
Precuneus/posterior cingulate
1,009
−4, −56, 20
2, –54, 30
4, –56, 40
Positive
Right amygdala (LB/SF)/right hippocampus (CA)
540
22, –4, −20
28, –16, –20
Positive
Left central OFC/ventrolateral PFC
352
−36, 32, –16
−38, 36, –12
Positive
Left angular/middle occipital gyrus (PGp/PGa)
171
−42, −78, 30
−50, −66, 30
−44, −72, 24
Positive
Left middle temporal gyrus
121
−50, 4, –30
Positive
Cluster 6 (left sgACC)
Large cluster including:
•
Posterior ventromedial FL
•
Bilateral caudate
•
Bilateral medial VS
•
Bilateral amygdala (LB)
•
Right hippocampus
3,081
−16, 14, –14
8, 8, –12
14, 10, –14
8, 6, 2
16, –2, −2
24, –10, −20
Positive including:
• vmFL
• Caudate
• VS
• Amyg/HC
Thalamus (prefrontal/temporal-connected region)
454
4, –18, 8
−4, −16, 6
−10, −16, 8
−10, −30, 8
None (positive but n.s.)
Abbreviations: Amyg, Amygdala; CA, cornu ammonis; HC, hippocampus; LB, laterobasal subregion; MACM, meta-analytic connectivity modeling;
OFC, orbitofrontal cortex; PFC, prefrontal cortex; rsfMRI, resting-state fMRI; SF, superficial subregion. Abbreviations: Amyg, Amygdala; CA, cornu ammonis; HC, hippocampus; LB, laterobasal subregion; MACM, meta-analytic connectivity modeling;
OFC, orbitofrontal cortex; PFC, prefrontal cortex; rsfMRI, resting-state fMRI; SF, superficial subregion. CA, cornu ammonis; HC, hippocampus; LB, laterobasal subregion; MACM, meta-analytic connectivity modeling;
efrontal cortex; rsfMRI, resting-state fMRI; SF, superficial subregion. and functional characteristics—to characterize the regions more deci-
sively (Eickhoff et al., 2018; Vogel et al., 2020). that many of the paradigms which make up the BrainMap database
are not selective in this regard. 3275 CHASE ET AL. TABLE 2
MACM coordinates for all six clusters, as well as the presence of overlap with significant rsfMRI connectivity in the cluster
Cluster size
Peaks
RsfMRI
Cluster 1 (right sgACC)
Large cluster including:
•
Ventromedial prefrontal cortex
•
Bilateral amygdala (LB/SF)
•
Bilateral hippocampus (CA)
•
Bilateral medial ventral striatum
•
Left caudate
3,556
12, 12, –14
−20, −6, −16
6, 38, –10
30, –14, –16
−22, −14, −14
Positive including:
• vmFL
• Medial VS
• Right Amyg
• Left HC
Right anterior insula
211
36, 18, 2
Positive
Cluster 2 (anterior medial)
Large cluster including:
•
Anterior vmFL
•
Frontal pole/dorsomedial PFC
•
Left amygdala (LB)
•
Left hippocampus (CA)
•
Bilateral ventral striatum/anterior caudate
5,188
−2, 50, –14
−20, −16, –20
10, 14, –10
−2, 40, 4
−10, 12, –6
−8, 56, 24
Positive including:
• vmFL
• Frontal pole
• HC/Amyg
• Medial VS
Precuneus/posterior cingulate
1,468
2, –52, 26
2, –44, 36
−6, −54, 10
2, –30, 46
Positive
Right hippocampus (CA)/right amygdala (LB/SF)
651
24, –6, −20
28, –18, −16
Positive
Left angular gyrus (PGp/PGa)
521
−48, −70, 28
−42, −78, 32
−50, −58, 26
Positive
Left ventrolateral PFC
396
−41, 34, –8
−42, 30, –18
−48, 26, –6
−48, 26, –12
Positive
Left middle/superior temporal gyrus
284
−58, −6, −18
−56, −2, −6
Positive
Right middle temporal gyrus (PGp/PGa)
145
50, –68, 18
50, 62, 22
Positive
Cluster 3 (central dorsal)
Large cluster including:
•
Ventromedial PFC
•
Bilateral medial ventral striatum/anterior
caudate
•
Bilateral amygdala
•
Bilateral hippocampus
•
Left central OFC, anterior insula
8,617
0, 48, –6
−10, 10, –6
−24, −14, –20
26, –6, −20
12, 10, –6
10, 14, –8
Positive (all regions)
Precuneus/posterior cingulate
2,252
−2, 52, 30
4, –50, 18
Positive
Left angular/middle occipital gyrus
658
−44, −76, 32
−48, −68, 30
Positive
Left superior frontal gyrus
500
−20, 32, 46
Positive
Right anterior insula
269
38, 16, 0
46, 16, –12
48, 14, –10
Positive
Bilateral thalamus
243
−8, −14, 8
10, –16, 6
Positive
Cluster 4 (posterior) r all six clusters, as well as the presence of overlap with significant rsfMRI connectivity in the cluster Cluster 1 (right sgACC) (Continues) (Continues) CHASE ET AL. 3276 CHASE ET AL. Region
Unique clusters Region
Unique clusters
Cluster 1
Right insula (32 voxels; 38, 21, 2)
Cluster 2
Left ventrolateral prefrontal cortex
(13 voxels; −48, 29, −15)
Left middle temporal gyrus (9 voxels: −57, 1, –20)
Frontal pole/Fp2 (3 voxels: 6 64 10)
Left inferior parietal lobule (PGa: 2 voxels; −55, −57, 28)
Posterior cingulate cortex (2 voxels; 4, 47, 27)
Cluster 3
Left superior frontal gyrus (4 voxels; −18, 32, 40)
Posterior cingulate cortex (2 voxels; −5, 47, 30)
Cluster 4
Possible hypothalamus (9 voxels; 4, −7, −8)
Cluster 5
Left superior frontal gyrus (28 voxels; −9, 43, 41)
Left central orbitofrontal gyrus (13 voxels; −39, 39, −15)
Cluster 6
Thalamus (prefrontal/temporal-connected regions: 223
voxels; −4, −18, 7)
Right caudate (95 voxels; 8, 8, 5)
Right pallidum (28 voxels; 20, 0, −6) Region
Unique clusters
Cluster 1
Right insula (32 voxels; 38, 21, 2)
Cluster 2
Left ventrolateral prefrontal cortex
(13 voxels; −48, 29, −15)
Left middle temporal gyrus (9 voxels: −57, 1, –20)
Frontal pole/Fp2 (3 voxels: 6 64 10)
Left inferior parietal lobule (PGa: 2 voxels; −55, −57, 28)
Posterior cingulate cortex (2 voxels; 4, 47, 27)
Cluster 3
Left superior frontal gyrus (4 voxels; −18, 32, 40)
Posterior cingulate cortex (2 voxels; −5, 47, 30)
Cluster 4
Possible hypothalamus (9 voxels; 4, −7, −8)
Cluster 5
Left superior frontal gyrus (28 voxels; −9, 43, 41)
Left central orbitofrontal gyrus (13 voxels; −39, 39, −15)
Cluster 6
Thalamus (prefrontal/temporal-connected regions: 223
voxels; −4, −18, 7)
Right caudate (95 voxels; 8, 8, 5)
Right pallidum (28 voxels; 20, 0, −6) Cluster 1
Right insula (32 voxels; 38, 21, 2) Cluster 2
Left ventrolateral prefrontal cortex
(13 voxels; −48, 29, −15)
Left middle temporal gyrus (9 voxels: −57, 1, –20)
Frontal pole/Fp2 (3 voxels: 6 64 10)
Left inferior parietal lobule (PGa: 2 voxels; −55, −57, 28)
Posterior cingulate cortex (2 voxels; 4, 47, 27) Overall, functional connectivity provides a valuable window into
the cross-species homology of a given brain region as the limitations
of
functional
characterization
(e.g.,
paradigms
which
are
underspecified for a particular construct; regions which act in concert
with others to support a particular process) do not fully apply to
connectivity—given
that
anatomical
connectivity
is
contextually
invariant, and that functional connectivity is constrained by anatomi-
cal connectivity. Region
Unique clusters In addition, considerable information available from
translational studies (discussed in the following) can provide particular
insight into connectivity and inform interpretation of human studies. Note: Clusters 4 and 6, and Cluster 2 parietal were not strongly corrobo-
rated by a similar rsfMRI analysis (p < .001), but other regions were. Note
that the hypothalamus region identified by Cluster 4 is on the very edge
of the raw Cluster 4 MACM image, rather than being a distinct activation. Consequently, this may reflect an artifact of smoothing. Note: Clusters 4 and 6, and Cluster 2 parietal were not strongly corrobo-
rated by a similar rsfMRI analysis (p < .001), but other regions were. Note
that the hypothalamus region identified by Cluster 4 is on the very edge
of the raw Cluster 4 MACM image, rather than being a distinct activation. Consequently, this may reflect an artifact of smoothing. Abbreviations:
MACM,
meta-analytic
connectivity
modeling;
rsfMRI,
resting-state fMRI. TABLE 2
(Continued) Moreover, this may also be a reflection
of the proposed “distributed coding” within the vmFL: specifically, the
proposal that the region represents information via a population code
which may be widely distributed across the region (Kahnt, Heinzle,
Park, & Haynes, 2010; van Duuren et al., 2009). Third, different ana-
tomical
methods
can
provide
different
specifications
of
vmFL
parcellation (Henssen et al., 2016)/(Mackey & Petrides, 2014), and it
may require a combination of methods—including richer anatomical Finally, although different methods may yield compatible infor-
mation about different aspects of the structure, there are still poten-
tial
caveats
of
the
approach
we
used
here. In
particular,
susceptibility artifacts in the vmFL are often present in conventional
(EPI) sequences (but can be addressed: for example, (Weiskopf,
Hutton, Josephs, & Deichmann, 2006). These artifacts would be
expected to lead to both a loss of signal, and consequently false 3277 CHASE ET AL. negatives, and also a misplacement of the signal due to distortion. Although researchers interested in the vmFL may choose an imaging
protocol appropriately, this would potentially lead to some bias
within the BrainMap database. However, this caveat is partly addressed by our use of resting fMRI to confirm the patterns of
functional connectivity. This convergent approach also provides fur-
ther validation of MACM as a method of examining functional con-
nectivity (Smith et al., 2009). One area of future investigation would be the potential for exam-
ining effects of between-participant heterogeneity, particularly with
respect to discontinuous variation (Chiavaras & Petrides, 2000). Our
approach assumes the presence of discrete clusters within the vmFL
which are broadly comparable across individuals, but it may be that
substantial individual differences in the size or reach of an area could
create transition zones which are not easy to classify. In the present
study, Cluster 4 may be an example (Section 3.3). TABLE 3
Regions with unique connectivity with each subregion,
by finding the overlap of each cluster contrasted with all other
clusters CHASE ET AL. CHASE ET AL. ventral striatum (specifically the nucleus accumbens shell) might be
indicative of an IL cortex-like region (Gorelova & Yang, 1997;
Heilbronner et al., 2016). Partially consistent with this, Clusters 1 and
6 both showed strong connectivity with the ventral striatum, with
Cluster 6 being defined by its interactions with this region. Notably,
Cluster 4 also overlapped with the OFC subregions of the anatomical
atlases (area 14/Fo2), suggesting that it might be an OFC/IL transi-
tional zone. Importantly, connectivity of the IL with the amygdala can
be modulated by excitatory input (Patton et al., 2013), and it is nota-
ble that positive functional connectivity between the amygdala and
these posterior subregions was seen using MACM but not strongly
using rsfMRI. regions classically associated with the DMN (e.g., parietal and PCC)
and the left VLPFC, versus the association of the posterior regions
(Clusters 1, 4, and 6) with regions including the insula, thalamus, and
basal ganglia. Within the anterior regions, the most anterior subregion
(Cluster 2) was distinguished with respect to the temporal lobe
(Petrides & Pandya, 2007; Saleem et al., 2008), parietal cortex (Barbas,
1993) and left VLPFC (Barbas & Pandya, 1989). The central clusters
both
connected
to
the
SFG
(Yeterian,
Pandya,
Tomaiuolo,
&
Petrides, 2012), but were differentiated by left OFC (central ventral:
Cluster 3) and PCC (central dorsal: Cluster 5). Posterior regions were divided on the basis of functional connec-
tivity with the insula (right sgACC: Cluster 1), the striatum and thala-
mus (left sgACC: Cluster 6), and possibly the hypothalamus (posterior:
Cluster 4). Although the voxels which distinguish Cluster 4 are
approximately where the hypothalamus should be (Reppucci &
Petrovich, 2016), it is difficult to rule out an artifactual effect of image
smoothness in this case, as the distinguishing voxels were toward the
edge of the MACM cluster centered on the seed region. A notable
feature of these posterior subregions was the presence of hemi-
spheric specialization between Clusters 1 and 6. This may relate to
the hemispheric differentiation of connected subcortical regions. Despite the functionally significant connections between the hippo-
campal
formation and
amygdala
with
the
vmFL
(Price,
1999;
Reppucci & Petrovich, 2016), neither structure played a significant
role in distinguishing any of the subregions using MACM. Indeed, all
clusters showed some evidence of MACM-defined functional connec-
tivity in one or both of the structures. Cluster 3 may be a potential homolog of the PL cortex. A salient
feature of this region was its connectivity with the midbrain, striatum,
and thalamus as assessed with rsfMRI, but not MACM. In addition,
functional connectivity with the thalamus and insula (Vertes, 2004),
and striatum (Gorelova & Yang, 1997; Sesack, Deutch, Roth, &
Bunney, 1989) would support the attribution to the PL. Moreover, the
region overlapped with areas 32 and 24ab in the SPM anatomy tool-
box templates (Henssen et al., 2016; Palomero-Gallagher et al., 2015). The region was also characterized by strong default mode connectiv-
ity, consistent with the implication of the PL in the rodent DMN (Hsu
et al., 2016). Interestingly, rsfMRI analysis did not reveal strong posi-
tive connectivity with the amygdala despite connectivity with other
subcortical structures, consistent with data suggesting that PL activa-
tion inhibits the amygdala (Gomes & Grace, 2017; Rosenkranz &
Grace, 2002). MACM did, however, yield evidence of positive func-
tional connectivity, but MACM might be expected to be less sensitive
to rapid sequential effects. Specifically, the amygdala and vmFL might
be concurrently activated by a paradigm, and while the vmFL might
begin to inhibit the amygdala, the impact of this inhibition might not
be reflected in the BOLD signal right away, allowing the hemodynamic
response function to fit the stimulus-locked BOLD activation in the
amygdala. Notably, this differentiation with respect to posterior and anterior
subregions was somewhat different when using rsfMRI. In this case,
the more posterior regions tended to show greater relative connectiv-
ity with “task-positive” regions including the central executive and
salience networks. Consistent with the MACM analysis, the more
anterior regions showed greater connectivity with the DMN. In addi-
tion, while the posterior subregions had shown strong functional con-
nectivity with subcortical regions using MACM, Cluster 3 (central
dorsal) now showed particularly strong connectivity across striatum
(Heilbronner et al., 2016) and midbrain (Amat et al., 2005; Celada,
Puig, Casanovas, Guillazo, & Artigas, 2001; Jo & Mizumori, 2016; Pat-
ton, Bizup, & Grace, 2013; Price, 1999). Cluster 5 is a potential homolog of the medial OFC due to its rela-
tively anterior and ventral location, primarily overlapping with areas
11/14 (Mackey & Petrides, 2014) and Fo (Henssen et al., 2016). This
region showed distal connectivity with a number of areas that would
be consistent with a medial OFC homolog, including the amygdala,
hippocampal formation, and temporal lobe (Price, 1999; Saleem
et al., 2008). It was also characterized by connectivity with the central
OFC,
suggesting
that
within-OFC
connectivity
(Barbas
&
Pandya, 1989) may be functionally relevant. The region was also char-
acterized by connectivity with primary visual regions, at least in com-
parison with Clusters 3 and 4. It is likely that visual input would reach
the medial OFC indirectly, via the temporal lobe (Rolls, 2004), connec-
tions which are thought to be crucial for visual perception (Bar
et al., 2006). In summary, the capacity of the MACM algorithm to differentiate
subregions of the vmFL appeared to be largely based on different pat-
terns of connectivity within the DMN, but also, for more posterior
regions, the striatum, thalamus, and insula. 4.2
|
Key distal, functionally connected regions
that distinguish the subregions One prominent axis of differentiation, using MACM, was the relation-
ship of the three more anterior regions (Clusters 2, 3, and 5) with Abbreviations:
MACM,
meta-analytic
connectivity
modeling;
rsfMRI,
resting-state fMRI. FIGURE 4
Spider plots describing forward inference (probability of activation given paradigm/domain) for each of the study types identified
as significant for one or more subregion(s) (Table S1). Axes describe likelihood of activation, log-transformed for display purposes FIGURE 4
Spider plots describing forward inference (probability of activation given paradigm/domain) for each of the study types identified
as significant for one or more subregion(s) (Table S1). Axes describe likelihood of activation, log-transformed for display purposes 3278 4.4
|
Clinical relevance and functional decoding The vmFL is a critical locus of interest for understanding the symptoms
of psychiatric disorders, particularly those associated with disturbances
of emotional or social behavior (Hiser & Koenigs, 2018). Recent evi-
dence has extended this to forms of pain, where it may play an inhibi-
tory role (Leaver et al., 2011; Rauschecker, May, Maudoux, &
Ploner, 2015). Our primary interest in differentiation of function within
the vmFL has arisen from studies of mood disorders, which has been
frequently associated with functional abnormalities within the sub-
genual cingulate cortex (Drevets, Price, & Furey, 2008). From a transla-
tional perspective, work by Maier and others has implicated a potential
homolog of the vmFL in rodents as underlying the effects of uncontrol-
lable
stress and mitigating effects of behavioral control (Amat
et al., 2005), a potential rodent model of major depression or post-
traumatic stress disorder. More recent evidence suggested that these
effects might be localized to PL regions (Christianson et al., 2014)—
consistent
with
its
role
in
instrumental
control
(Killcross
&
Coutureau,
2003)—whereas
the
subgenual
region
identified
in
depressed humans is typically thought be homolog of the IL cortex
(Rudebeck et al., 2014). Our findings may contribute to this body of
work by suggesting the presence of differentiation of function within
the vmFL in the human brain (McNamee, Rangel, & O'Doherty, 2013)
at a resolution that is relevant for the precise localization necessary for
designing interventions for mood disorders (Mayberg et al., 2005). For
example, Cluster 3, the potential homolog of PL which we identified
here, was characterized by rsfMRI functional connectivity with the mid-
brain. This finding may be relevant for understanding the effect of
uncontrollable stress on the dorsal raphe nucleus via regulation by the
rodent PL/IL (Amat et al., 2005). In general, examination of vmFL func-
tional connectivity in mood and anxiety disorders has employed a vari-
ety of methods and seed regions of interest, and our findings may help
to provide tools to integrate some of these investigations. For example,
our previous work suggests that seed-based analysis which uses dis-
tinct subregions of larger neural structures can be an effective approach
to identifying relevant individual differences (Chase et al., 2017). CHASE ET AL. 3279 illness and health, as well as across task conditions or mood state,
remains to be determined. vmFL regions we describe (Ongur et al., 2003). It showed functional
connectivity with more advanced cortical areas such as the frontal
pole and left ventrolateral cortex, both of which have shown evolu-
tionary development in humans compared to rodents (Wise, 2008). However, it was partially overlapping with Mackay and Petrides'
(Mackey & Petrides, 2014) areas 11 and 14, but also with Henssen
et al.'s Fp region (Henssen et al., 2016). In the present work, functional decoding of the vmFL provided
independent confirmation of relevance for psychiatric disorders—
particularly disorders involving deficits of emotional regulation and
reward function such as mood disorders, given emotion (Roy,
Shohamy, & Wager, 2012) and reward studies frequently activated
the region. The region was also associated with cognition, perhaps via
the frequent identification of task-related deactivations or contrasts
with reduced attentional demands (K. C. Fox, Spreng, Ellamil,
Andrews-Hanna, & Christoff, 2015). These neural dynamics might also
be relevant for alternations in cognitive function in psychiatric disor-
ders (Farruggia, Laird, & Mattfield, 2020). The capacity to decode par-
adigms/domains
from
vmFL
activations
was
modest,
but
was
markedly larger for reward and cognition than other types of study. This may be due to the larger proportion of these studies in the
BrainMap database, which would make the posterior probabilities
larger. While evidence of functional specialization within the vmFL
was generally not strong, there was a suggestion of a spatial dissocia-
tion of gustatory versus olfactory processing (Rolls & Baylis, 1994),
insofar as the anterior subregion (Cluster 2) was most strongly related
to olfaction, whereas more posterior subregions (including 1, 4, and 5)
were most strongly related to gustation. Together, the findings are
broadly consistent with models of the vmFL which emphasize diver-
sity of function via the representation of predictive models of the
environment (Barrett & Simmons, 2015; Niv, 2019). In summary, the data provide some preliminary suggestions of
potential, discrete homologs of the rodent IL/PL subregions and
medial OFC in the human brain, as well as a more rostral region that
might not be reflected in the rodent frontal lobe. These subregions
might provide a basis for more detailed future investigations, including
characterization of structure/function relationships in this region. 4.3
| The question of cross-species homology within the vmFL is complex
and enduring, due to obvious anatomical differences between mam-
malian species in this region (Wise, 2008). Moreover, approaches to
resolution are often strongly dependent on the method employed, be
it anatomical (Heilbronner et al., 2016) or behavioral (Birrell &
Brown, 2000). For example, the presence of connectivity with the As the most anterior region, Cluster 2 is perhaps most likely not
to have a direct rodent homolog, and perhaps be characterized in
terms of Brodmann area 10 (Ongur et al., 2003). As such, it may also
be most likely to be eulaminate/granular (Barbas & Garcia-Cabezas,-
2016), as opposed to agranular or dysgranular like the more posterior 3280 3280 CHASE ET AL. CHASE ET AL. 4.5 Birrell, J. M., & Brown, V. J. (2000). Medial frontal cortex mediates percep-
tual attentional set shifting in the rat. The Journal of Neuroscience, 20
(11), 4320–4324. In the present work, we aimed to identify separate subregions of
the vmFL, consistent with prior suggestions of differentiation in
experimental animals and potential functional differences seen in
humans. Using MACM-based functional connectivity parcellation in
two steps, we found evidence for a six subregion solution. The sub-
regions included two unilateral subgenual cingulate regions, a pos-
terior region, a ventral and dorsal central region and an anterior
region. These regions were characterized by different patterns of
within-DMN and subcortical functional connectivity (using MACM
and resting fMRI), and showed suggestions of functional differ-
ences. Together, the findings are consistent with the proposal that
the vmFL may be divided into functionally independent subregions. Future work might aim to clarify differences between the derived
functional subregions and previous observations of anatomical dif-
ferentiation within the vmFL. In the present work, we aimed to identify separate subregions of
the vmFL, consistent with prior suggestions of differentiation in
experimental animals and potential functional differences seen in
humans. Using MACM-based functional connectivity parcellation in
two steps, we found evidence for a six subregion solution. The sub-
regions included two unilateral subgenual cingulate regions, a pos-
terior region, a ventral and dorsal central region and an anterior
region. These regions were characterized by different patterns of
within-DMN and subcortical functional connectivity (using MACM
and resting fMRI), and showed suggestions of functional differ-
ences. Together, the findings are consistent with the proposal that
the vmFL may be divided into functionally independent subregions. Future work might aim to clarify differences between the derived
functional subregions and previous observations of anatomical dif-
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https://orcid.org/0000-0001-9780-095X Henry W. Chase
https://orcid.org/0000-0001-9780-095X Chase, H. W., Segreti, A. M., Keller, T. A., Cherkassky, V. L., Just, M. A.,
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|
Clinical relevance and functional decoding Fur-
ther examination of the consistency of functional differentiation across Within the present data set, one notable feature of the func-
tional connectivity profiles of the different regions was that there
was evidence that more posterior regions (Clusters 1, 6, and 4)
showed more discrepant connectivity between MACM and rsfMRI. For example, voxels which uniquely characterized Clusters 4 and
6 using MACM did not show strong functional connectivity with
Clusters 4 and 6 using rsfMRI. In addition, these posterior regions
showed relatively higher rsfMRI functional connectivity with task
positive than DMN regions, which contrasted somewhat with Clus-
ter 4's MACM map. Recent theoretical work suggests that IL and PL
cortices are in competition, with the IL mediating inflexible stimulus–
response learning, and the PL underling flexible use of contextual
cues to optimize task performance (Sharpe & Killcross, 2018). Given
the novelty of the fMRI scanner and the tasks that participants are
often asked to perform, this model might suggest that the PL-
homolog in humans might dominate, and show stronger and perhaps
therefore more consistent functional connectivity patterns. By con-
trast, activation of the IL-homolog might only be evident after over-
training and higher scanner familiarity. Moreover, IL activity might
be (rapidly) followed by more “task-positive” network activity, as
individuals seek to overcome prepotent biases, should they become
engaged. In this case, positive correlations between IL and task-
positive networks might be seen, given the sluggish nature of the
BOLD response (Buxton, Uludag, Dubowitz, & Liu, 2004). Overall,
the present findings suggest the potential to test this model empiri-
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NKI/Rockland sample which are publically accessible. The derived
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Supporting Information section at the end of this article. Supporting Information section at the end of this article. How to cite this article: Chase HW, Grace AA, Fox PT,
Phillips ML, Eickhoff SB. Functional differentiation in the
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EPI parameters for reduction of susceptibility-induced BOLD
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Knowledge audit as a key tool for business research in the information society
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Entrepreneurship and sustainability issues
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Liudmila V. Kashirskaya *1, Alexey A. Sitnov 2, Dilmurod Аsh. Davlatzoda 3, Tatiana M. Vorozheykina 4 Liudmila V. Kashirskaya *1, Alexey A. Sitnov 2, Dilmurod Аsh. Davlatzoda 3, Tatiana M. Vorozheykina 4 1Financial University under the Government of the Russian Federation, Leningradsky Prospekt, 49, 125993, Moscow,
Russian Federation
2 Financial University under the Government of the Russian Federation, Leningradsky Prospekt, 49, 125993, Moscow,
Russian Federation
3Tajik National University 35, Ayni Ave 734025, Dushanbe, Republic of Tajikistan
4Russian State Agrarian University - Moscow Agricultural Academy named after K.A. Timiryazev, 127550
Timiryazevskaya st., 49, Moscow, Russian Federation 3Tajik National University 35, Ayni Ave 734025, Dushanbe, Republic of Tajikistan
ate Agrarian University - Moscow Agricultural Academy named after K.A. Timiryazev, 127550
Timiryazevskaya st., 49, Moscow, Russian Federation 3Tajik National University 35, Ayni Ave 734025, Dushanbe, Republic of Tajikistan
4Russian State Agrarian University - Moscow Agricultural Academy named after K.A. Timiryazev, 127550
Timiryazevskaya st., 49, Moscow, Russian Federation JEL Classifications: M 41, М 42, M 49, G22. Reference to this paper should be made as follows: Kashirskaya, L.V., Sitnov, A.A., Davlatzoda, D.A., Vorozheykina, T.M. (2020).
Knowledge audit as a key tool for business research in the information society. Entrepreneurship and Sustainability Issues, 7(3), 2299-
2312. http://doi.org/10.9770/jesi.2020.7.3(56) ENTREPRENEURSHIP AND SUSTAINABILITY ISSUES
ISSN 2345-0282 (online) http://jssidoi.org/jesi/
2020 Volume 7 Number 3 (March)
http://doi.org/10.9770/jesi.2020.7.3(56) ENTREPRENEURSHIP AND SUSTAINABILITY ISSUES
ISSN 2345-0282 (online) http://jssidoi.org/jesi/
2020 Volume 7 Number 3 (March)
http://doi.org/10.9770/jesi.2020.7.3(56) ENTREPRENEURSHIP AND SUSTAINABILITY ISSUES
ISSN 2345-0282 (online) http://jssidoi.org/jesi/
2020 Volume 7 Number 3 (March)
http://doi.org/10.9770/jesi.2020.7.3(56) ENTREPRENEURSHIP AND SUSTAINABILITY ISSUES
ISSN 2345-0282 (online) http://jssidoi.org/jesi/
2020 Volume 7 Number 3 (March)
http://doi.org/10.9770/jesi.2020.7.3(56) Publisher http://jssidoi.org/esc/home KNOWLEDGE AUDIT AS A KEY TOOL FOR BUSINESS RESEARCH IN
THE INFORMATION SOCIETY KNOWLEDGE AUDIT AS A KEY TOOL FOR BUSINESS RESEARCH IN
THE INFORMATION SOCIETY 1. Introduction In the frames of modern global changes, accompanied by continuous technological changes, which are being
carried out at a very rapid pace, a need of quick search for appropriate and relevant information on an
unprecedented scale arises almost simultaneously with the appearance of this information in the information space
surrounding these systems. The business seeks to improve and strengthen its capabilities by strengthening the
emphasis on a modern and progressive management methodology, the so-called knowledge management
methodology. At the same time, knowledge in this context is understood as a combination of data and information, including
various combinations of new technologies, production experience, emotions, culture, values of indicators, ideas,
intuition, motivation, styles of learning, attitudes, ability to trust and solve complex problem situations, frankness,
the ability to work in a modern information network, sociability, attitude to permanent risk, the presence of an
entrepreneurial spirit (Kashirskaya et al., 2019; Saenko et al., 2019; Voronkova et al., 2019). In the general understanding, knowledge management is a set of conceptual apparatus, subject disciplines and
tools for organizing knowledge that allow managers to take responsibility for corporate knowledge and, based on
this corporate knowledge, effective management decisions. The tendency of managers to use knowledge leads to the accumulation of particularly valuable assets, improves
their ability to make and use in practice effective rather than traditionally rational management decisions. The tendency of managers to use knowledge leads to the accumulation of particularly valuable assets, improves
their ability to make and use in practice effective rather than traditionally rational management decisions. According to J. Gardner, one of the famous modern researchers of management problems, the only possible
stability of the existence of business is “the stability in motion”. And since the movement is generated by
contradictions, their resolution consists in the search for “dynamic balance” between the stability of the business
system and its continuous improvement, that is, in the search for new effective management decisions, as outdated
stereotypes become unacceptable for this subject (Gardner, 1981). The concept of "management decision" is usually considered from three points of view. Firstly, it denotes a
process of a certain sequence of management actions aimed at choosing the optimal path for the activity of this
subject. Secondly, it is the process of choosing an option to solve a particular problem or task. A problem is a
complex theoretical or practical aspect that requires study and resolution. ENTREPRENEURSHIP AND SUSTAINABILITY ISSUES
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http://doi.org/10.9770/jesi.2020.7.3(56) Received 14 August 2019; accepted 10 December 2019; published 30 March 2020 Abstract. The article discusses and systematizes the possibilities of knowledge audit in the frames of the formation of the information
society. The features of developing a methodology, organization and knowledge audit tools that meet the modern needs of the development
of business audit are assessed. The problem of modern times also lies in the fact that the audit of explicit (formalized) knowledge is
fundamentally different from the audit of implicit (non-formalized) knowledge. As one of the main areas of business audit, knowledge
audit is highly professional consulting services in a wide range of subject areas of economic, financial, legal and many other areas of
modern business. A research of the demand for this area of audit in the global community showed that the lack of an agreed methodology
prevents from making the decision about conducting an audit of knowledge. The article examines modern approaches that allow combining
the capabilities of modern business audit in general and information systems audit, operational audit, intellectual capital audit and
knowledge audit, in particular. It is concluded that combining these types of audits into a single audit of a business shows a significant
business effect. The practical significance of the article lies in the fact that conclusions and suggestions aimed at strengthening the role of
modern audit contribute to the real optimization of modern business. A research made by the authors showed that the new reality of the
21st century has changed the attitude towards traditional audit, which requires justification of the business effect when introducing new
information systems and modernizing old ones, moving from complex automation of business processes to specialized solutions. Notwithstanding with it, the demand for business development and IT strategies and feasibility studies is growing steadily. Projects on the
use of corporate knowledge at all levels of management come to the fore. Keywords: knowledge audit; corporate memory; knowledge management system; explicit (formalized) knowledge; implicit (non
formalized) knowledge; reengineering; optimization; intellectual capital; intellectual potential; intangible assets; knowledge assets Reference to this paper should be made as follows: Kashirskaya, L.V., Sitnov, A.A., Davlatzoda, D.A., Vorozheykina, T.M. (2020). Knowledge audit as a key tool for business research in the information society. Entrepreneurship and Sustainability Issues, 7(3), 2299-
2312. http://doi.org/10.9770/jesi.2020.7.3(56) 2299 1. Introduction In his turn, G. Simon argues that, when solving a particular problem, business usually is incined to behavior that
cannot be called as optimization of managerial decisions. In this case, the optimal management solution is not
used at all because of the lack of time and the inability to take into account all relevant information and a
significant number of possible options for such a solution. In this situation, managers may take inappropriate
actions that are subjectively acceptable, but not always the best of all possible (Simon, 1995). In practice, knowledge is divided into explicit (formalized), expressed in objects, words, numbers, graphic forms,
drawings, specifications, textbooks, procedures, and implicit (unformalized), which are theoretical models,
models of behavior and perspectives based on empirical data and experience of the carriers of this knowledge
themselves (Kashirskaya et al., 2019, Lafer and Tarman, 2019; Nonaka and Takeuchi, 1995; Sharafutdinov et al.,
2019; Neizvestnaya et al., 2018; Akhmetshin et al., 2018). However, up to nowadays, all efforts aimed at creating modern systems of knowledge management in business
are reduced, as a rule, only to the promotion of individual information technologies related to the introduction of
modern software products that allow solving only certain aspects of this problem. In a number of cases, in the
most developed business, these technologies are combined into a single information system for a business at
various levels of its development (Korableva et al., 2018). In the era of modern and very intense changes in the 21st century, traditional approaches to management, which
guarantee business success for a long time, cannot stop the gradual decline of its resistance to both internal
contradictions and external threats from a very aggressive and sometimes very unpredictable competitive
environment. A static, non-developing, corporate memory that serves as the basis for making managerial
decisions becomes an increasingly distorted view of the future for business, and the rules and procedures
established in business begin to lose their significance over time (Gibbert et al., 2002). Corporate memory in this context is an implicit or explicit interpretation of business processes or manufactured
products, goods or services that does not subject to business. It should be borne in mind that “companies, like
people, remember the past, including old processes and procedures, as well as corporate traditions and values.”
(Koulopoulos and Frappaolo, 1999, p. 114). 1. Introduction Usually “a problem means a mismatch
between the desired (normative) and actual levels of achieving goals” (Golubkov, 2005, p. 45). And, finally,
thirdly, it is a specific managerial action (Trofimova et al., 2019; Luzina et al., 2019; Suryono et al., 2019;
Prodanova et al., 2019). Any control process is a definite and continuous in time sequence of actions, combined into appropriate stages
according to the quality content and uniformity of those operations that are necessary for their implementation. The making of managerial decisions, as mentioned earlier, is preceded by a study of the current situation and the
choice of options for these decisions related to a particular problem. Therefore, a situation is understood as “a
combination of conditions and circumstances in which a problem arose” (Golubkov, 2005, p. 45). Ideally, it is
desirable to have all possible options of action that could eliminate the causes of the problematic disturbances
and, thus, provide the business with the opportunity to achieve the stated goals. Identification and description of a
problem situation provides an initial information base for evaluating the time available for making a decision and
the amount of resources needed for this. However, in practice, managers most often do not have sufficient
relevant information and, moreover, time to identify and evaluate each alternative solution. At the same time, a
significant number of alternative solutions are more likely to interfere than to help management. Therefore, as a
rule, managers are limited to a small number of options for those decisions that correspond only to a certain 2300 ENTREPRENEURSHIP AND SUSTAINABILITY ISSUES
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that is most often called optimal in economic literature. At the same time, under conditions of constant
uncertainty, it is not always possible to find the optimal and at the same time strictly formalized solution
(Golubkov, 2005). Famous scientists in the field of control theory M.K. Meskon, M. Albert and F. Hedouri believe that in the case
when managers “... are not able to assess what will happen if nothing is done, there is a danger not to resist the
demand for immediate action. Action for the sake of action increases the probability of responding to an external
symptom of a problem, rather than its main cause” (Meskon, 2002, p. 205). 1. Introduction Being in a similar situation, managers often initiate radical transformations based on reengineering, rebuilding
their business and IT processes and, what is most important, business strategies under new business conditions,
modern challenges, and threats, while destroying the established internal knowledge potential of their business
systems. Thus, in the process of such business transformations, managers make a huge mistake, replacing the
outdated (in their opinion) corporate memory with new knowledge at that moment and practically stop there. At
the same time, there is a desire to withdraw the most experienced knowledge carriers from business and replace
them with younger, but less competent employees. 2301 2301 ENTREPRENEURSHIP AND SUSTAINABILITY ISSUES
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http://doi.org/10.9770/jesi.2020.7.3(56) Researches of scientists and modern practice have proved that a fixed in time view of a problem situation is
typical for reengineering. The reason for this lies in the inability to take into account the characteristic features of
modern markets, continuously progressing, and very rapid changes occurring in a competitive environment
(Koulopoulos and Frappaolo, 1999, p. 114). Thus, the knowledge acquired by the business as a result of reengineering also very soon becomes obsolete. An
urgent need for a repeated and at the same time very costly transformation of the business model arises. According to the opinion of the famous scientists and practitioners Thomas M. Culopoulos and Karl Frappaolo
and with which one cannot disagree, the strength of modern business of the 21st century is not in the knowledge
that was used in the past and is outdated, “... but in the ability to constantly update the corporate wisdom
repository and use its contents for new purposes” (Koulopoulos and Frappaolo, 1999, p. 12). However, an
approach based on reengineering does not allow this process to be carried out continuously and requires more and
more costs for new transformations (Nagimzhanova et al., 2019; Magsumov, 2017; Yemelyanov et al., 2018). Unlike reengineering, knowledge management defines a constant readiness of a business for managerial
influences, contributes to continuous transformation and innovation at a speed that at least corresponds to the pace
of the modern development of a competitive environment. In modern conditions, changes are, of course, inevitable and continuous, therefore, innovative processes must be
constant and continuous. The information base for this is precisely the repository of corporate wisdom. In order to
remain successful and sustainable in today's constantly changing competitive environment, it is necessary to
continuously accumulate knowledge and expand it in accordance with the needs of the future market,
transforming corporate memory into corporate wisdom (Koulopoulos and Frappaolo, 1999, p. 116). Thus, a new, intangible, form of resources appears in business - intellectual capital. At the same time, scientists
and practitioners in the field of management have recently used the concept of “intellectual potential” of a
business (Novgorodov, 2017; Nigamaev et al., 2018; Kopteva et al., 2019; Prakash and Garg, 2019). Mentioned objects almost always existed in business, but they were identified relatively recently as a resource or
an asset. ENTREPRENEURSHIP AND SUSTAINABILITY ISSUES
ISSN 2345-0282 (online) http://jssidoi.org/jesi/ business and IT processes. Consumer (client) capital is an information base about the clients of a particular
business, including the evolution of relationships with them, as well as relationships (Stewart, 2001). In his turn, Karl-Eric Sveiby proposed his approach to the consideration of the elements of intellectual capital. In
his opinion, it is necessary to single out the competence of employees, internal and external structures (Sveiby,
2004). Moreover, under the competence of employees, a scientist means their abilities, including their education,
qualifications, experience, attitude to their functions and to the business in which they work. This element of
intellectual capital depends on specific employees, and if they leave the business, the competence leaves with
them. At the same time, the internal structure is nothing more than intangible assets belonging to a particular
business, enabling it to satisfy customer preferences of customers. It relates to the internal structure business
strategy, patents, know-how, information systems, information databases, organizational structure and
documented business and IT processes. And finally, the external structure, in his opinion, shows the relationship of among business and contractors. External structure includes brands, trademarks, image belonging to a particular business. At the same time, it should be mentioned that these scientists do not quite unambiguously correlate the objects of
intellectual capital to one or another of its elements. For example, Karl-Eric Sveiby defines the attitude to work to
the competence of employees, but at the same time, attitude to work is also considered as part of the corporate
culture of the business. Moreover, he relates the latter to the internal structure (Sveiby, 2004). Such approach
inherently complicates the practical use of these theories for the formation and application of knowledge
management systems. At the same time, these approaches to determining the elements of intellectual capital can
be applied to assess its state in a particular business and can be used as a starting point in the development of a
business strategy and management policy, as well as its development (Thalassinos, I.E. and Thalassinos, Y., 2018;
Hilkevics and Semakina, 2019). By choosing one of the above as a working model, it is possible in the context of
a single business to develop a classification of intellectual capital objects with a view to subsequently controlling
them and developing ways to develop intellectual capital management on its basis. 2301 Intellectual capital can be defined as the set of all the knowledge of employees of an individual business,
ensuring its resistance to permanent challenges from the competitive environment (Makarov, 2005), or as the set
of intangible assets of an individual business that can be used to create value for a consumer result (Nayanova,
2001). )
In its turn, the "intellectual potential" is a stock of knowledge, abilities, skills, culture and morality, health,
capable of capitalizing under certain conditions. Moreover, it does not have age limits and health restrictions
(Kotyrlo, 2011). The definition of knowledge as business assets and, accordingly, the existence of the need to manage this type of
asset are discussed in the works of scientists-economists as well as management practitioners (Kashirskaya et al.,
2019). Intellectual capital determines the competitiveness of the business in the information society and therefore
becomes one of the key resources. In its turn, the intellectual potential is not a capitalized element of the
development of society (Akhmadeev et al., 2019; Smolnikova et al., 2019). The well-known scientist in the specified subject field Thomas Stewart defines the intellectual capital as all
business knowledge that can be considered an asset and distinguishes three main elements in its structure -
human, structural and consumer capital. At the same time, human capital, in his opinion, represents the
knowledge, skills and creative potential of business employees. In addition, here he also relates the culture of
interpersonal relations. Structural (organizational) capital include patents, licenses, trademarks, brands belonging
to a particular business, as well as hardware and software, organizational structure and methods of organizing 2302 ENTREPRENEURSHIP AND SUSTAINABILITY ISSUES
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http://doi.org/10.9770/jesi.2020.7.3(56) 2. Methods y
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According to scientists and experts in the field of auditing, it is advisable to make an audit of this knowledge in
the following cases: According to scientists and experts in the field of auditing, it is advisable to make an audit of this knowledge in
the following cases: g
- during developing a business strategy in the field of knowledge management; - during developing a business strategy in the field of knowledge management; - during developing a business strategy in the field of knowledge management;
- if there are significant difficulties in finding the necessary information or a competent specialist expert in a
ti
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t - if there are significant difficulties in finding the necessary information or a competent specialist expert in a
particular subject area;
if th
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li it (
f
li
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l d particular subject area;
- if there is duplication of the collection of information and implicit (non-formalized) knowledge; e is duplication of the collection of information and implicit (non-formalized) knowledge; - in case of doubt about the value of an initiative related to the use of an information system, investment in certain
software products or business projects; - during relatively low efficiency of implementation of the results of scientific research and experimental
development (R&D); g the reorganization of a business, its merger or acquisition (Kashirskaya et al., 2019). ourse of the specified direction of the audit as part of a business audit the auditor may: - during the reorganization of a business, its merger or acquisition (Kashirskaya et al., 2019). 2. Methods In the opinion of well-known foreign scientists and practitioners, and with whom one cannot disagree, the most
acceptable tool for highly professionally implementing these functions in their entirety is a modern audit, but not
a traditional audit of financial statements, but an audit of the business as a whole. It is a business audit that should
include, as a separate area of audit, a knowledge audit (Kashirskaya et al., 2019). At the same time, in this context, an audit of knowledge should be understood as a systematic independent
scientific audit of corporate knowledge, both past and present, and especially future (corporate memory and
corporate wisdom) and the development of management recommendations for business management based on its
results. According to Anne Hilton, a knowledge audit specialist, only 15% of knowledge management developments are
completed with appropriate results (Andrusenko, 2007). At the same time, the main reason for failures when
introducing these systems into the business is the lack of a preliminary audit of corporate implicit (non-
formalized) knowledge (Vyatkina and Sitnov, 2018; Korchevenkov and Aleksandrova, 2018). In addition, the lack of a coherent methodology for auditing a business as a whole does not allow them to
determine appropriate approaches to identifying implicit (unformalized) knowledge necessary for a particular
business both in the current time and, which is especially important, in modern conditions of a rapidly changing
competitive environment, and especially in a strategic perspective. Thus, auditors are not able to prepare specific and appropriate management recommendations for the management
of this knowledge for any modern business. Thus, auditors are not able to prepare specific and appropriate management recommendations for the management
of this knowledge for any modern business. At the same time, it can be confidently sated that the audit of implicit (non-formalized) knowledge at the present
stage of development of a business audit as a whole is a very complex and time-consuming scientific audit study,
requiring high professionalism and competence from the auditors themselves. At the same time, it is necessary to
understand that the goals in each specific audit assignment may be different, and their achievement in the audit
study of implicit (non-formalized) business knowledge is ambiguous. ENTREPRENEURSHIP AND SUSTAINABILITY ISSUES
ISSN 2345-0282 (online) http://jssidoi.org/jesi/ In the economy of the 21st century, the list of business systems whose success depends on the proper attitude to
their knowledge is expanding rapidly. In large business, separate structural units are created and engaged in the
development of a new consumer result (product, good or service), its introduction to the market, market research
of its own consumer result, sale of high-tech products, and management of relations with contractors. The
knowledge of the employees of these structural units of the business is of great importance for the entire business. The creating of modern effective knowledge management systems allows the specified business to solve the
problems of distributing the knowledge system between the interested structural units of the business, as well as
among its regionally separated employees who perform their functions in its geographical segments (Kotyrlo E. S., 2011). In the context of the development of the information age, taking into account the needs of market participants,
preventive consistent and at the same time independent control of the entire set of knowledge is necessary,
focusing attention not only on the past, but also on the present and especially on the future of the controlled
business, while erasing the boundaries between directly controlling functions and management consulting. 2303 ENTREPRENEURSHIP AND SUSTAINABILITY ISSUES
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http://doi.org/10.9770/jesi.2020.7.3(56) - determine the structure of the intellectual capital of the business and establish the most effective methods and
procedures for its evaluation; - determine the structure of the intellectual capital of the business and establish the most effective methods and
procedures for its evaluation; sess the likely loss of knowledge due to the departure of their carriers from the business; - identify and assess the likely loss of knowledge due to the departure of their carriers from the business;
- establish the potential for creating in business groups of the most competent practicing employees who create
this or that knowledge; - establish the potential for creating in business groups of the most competent practicing employees who create
this or that knowledge; - systematize the identified knowledge assets and develop for management an appropriate methodology for their
assessment. - systematize the identified knowledge assets and develop for management an appropriate methodology for their
assessment. At the same time, it should be mentioned that conducting an audit of knowledge requires the implementation of a
significant amount of work to create the proper conditions for the preparation of an information base. At the same
time, the fundamental target setting is the formation among managers and knowledge carriers of the studied
business of an appropriate understanding of its importance for increasing the effectiveness of the knowledge
management system. It should be borne in mind that an important component of the audit of implicit (non-formalized) knowledge, as
noted earlier, is the individuality and atypicality of each business under study, its business strategies, as well as
the need for direct close cooperation with its knowledge holders and the involvement of various subject matter
experts areas. Thus, at the planning stage, it is necessary first of all to assess the status of the existing business knowledge
management system. For this purpose, according to a number of scientists and practitioners, as well as the studies
conducted, we can consider that the most aprroriate is the use of the Capability Maturity Model proposed by the
Software Engineering Institute by the US Department of Defense to classify and evaluate projects related to
software development and quality assurance during the implementation of these projects (Sitnov and Urintsov,
2014; Ibbs and Kwak, 1997). 2. Methods In the course of the specified direction of the audit as part of a business audit, the auditor may: In the course of the specified direction of the audit as part of a business audit, the auditor may:
d t
i
th
i
ti
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d th
di
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t diti
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f se of the specified direction of the audit as part of a business audit, the auditor may: determine the organizational aspects and the readiness of the business for non-traditional tr - develop management recommendations for the development of an adequate competitive environment
requirements, a business strategy in the field of knowledge management, creation or development of the existing
knowledge management system; - identify hidden reserves of implicit (unformalized) knowledge for their further effective use by the management
of the business under study; 2304 ENTREPRENEURSHIP AND SUSTAINABILITY ISSUES
ISSN 2345-0282 (online) http://jssidoi.org/jesi/ Based on the Maturity Models scale, which is based on determining the level of development of a business and IT
system as a whole and its knowledge management system in particular from nonexistent (first level) to optimized
(5th level), the auditor can determine: Based on the Maturity Models scale, which is based on determining the level of development of a business and IT
system as a whole and its knowledge management system in particular from nonexistent (first level) to optimized
(5th level), the auditor can determine: (5th level), the auditor can determine: (5th level), the auditor can determine: the current state of the business and its knowledge management system, that is, to assess w - the current state of the business and its knowledge management system, that is, to assess what stage the business
as a whole and its knowledge management system in particular are at the current time; - the current state of the business and its knowledge management system, that is, to asses
as a whole and its knowledge management system in particular are at the current time; s a whole and its knowledge management system in particular are at the current time; - the current status of best practice for knowledge management in the industry in which the business under study
operates, that is, compare the specific business system and its knowledge management system being studied by
the auditor with the best subject in the industry; - the current status of the business and its knowledge management system in accordance with international best
practices; - the status of the business and its knowledge management system after their proposed improvement, that is,
evaluate its business strategy for the results that it seeks to achieve in the field of knowledge management in the
long term. After identifying critical points and bottlenecks in the study area, Maturity Models allow the auditor to develop
preliminary corrective recommendations and provide them with the management of the business under study. Then the auditor develops a strategy and tactics for bringing the business as a whole and its knowledge
management system in particular to the desired level of knowledge management efficiency. After identifying critical points and bottlenecks in the study area, Maturity Models allow the auditor to develop
preliminary corrective recommendations and provide them with the management of the business under study. ENTREPRENEURSHIP AND SUSTAINABILITY ISSUES
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http://doi.org/10.9770/jesi.2020.7.3(56) level takes a systematic and structured approach to planning and control. The staff is trained to apply the
methodology and tools of knowledge management. The fourth maturity level (integration level) is characterized by full formalization with the official approval of all
knowledge management processes and documentation (mapping) of relevant information. A business that has
reached this level is able to effectively plan, manage and control the entire set of business processes they perform. Knowledge management processes are well defined, quantified, understood by staff and put into practice. Process
related data is standardized, collected and stored in a database to ensure effective and objective analysis and
quantification of processes, as well as to predict undesirable trends and prevent possible adverse situations. This
allows the business to create the foundation for making effective management decisions. And finally, at the highest, fifth level of maturity (level of improvement), knowledge management processes in
business are constantly being improved. Automatic collection of both explicit (formalized) and implicit
(unformalized) knowledge is provided. They are carefully analyzed and quantified to identify opportunities for
further improvements to management processes. This level assumes the availability and use of appropriate tools. Such tools may include, for example, organizational structures, procedures and information technologies that
provide audit, monitoring and examination of knowledge. ENTREPRENEURSHIP AND SUSTAINABILITY ISSUES
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http://doi.org/10.9770/jesi.2020.7.3(56) Assessment of implicit (non-formalized) knowledge by an auditor based on Maturity Models can serve as a
qualitative component in the development of management recommendations for the practical implementation of
the current and strategic management of the knowledge management system as a whole and its components in
particular (Sitnov and Urintsov, 2014). Moreover, compliance with one or another level of the Model allows to
determine the readiness of a business for modernization or updating. These Models allow the auditor to determine
audit procedures that allow giving answers to what needs should be done during the audit on the essence of the
audit engagement, as well as to establish the state in which the business knowledge management system is
located. In this study, the auditor must take into account that the first level of maturity corresponds to a situation when
there are no formally accepted procedures for knowledge management in general in business and, as a result,
which is especially important, implicit (unformalized) management plans, plans for its implementation are not
created, work is poorly defined in content, volume and cost. Knowledge management processes are completely
unpredictable and poorly controlled, and managers often do not understand key management issues. As a result,
the success of the knowledge management system depends more on the individual efforts of knowledge holders
than on the organization of management processes. A business at this level can be described as trying to
spontaneously master the basic processes of knowledge management. The second level of maturity (or as it is often called the “level of individual planning”) corresponds to the use of
separate informal and incomplete knowledge management procedures in an organization. Managers partially
apply and control management processes. However, in each case, planning and management depends on the
individual approach of a manager. The third level of maturity (management level) involves a partial formalization of knowledge management
processes and the use of a basic planning and management system in business. A business that has reached this 2305 ENTREPRENEURSHIP AND SUSTAINABILITY ISSUES
ISSN 2345-0282 (online) http://jssidoi.org/jesi/ - discuss the knowledge audit methodology; - answer all possible questions that have arisen from the staff of the studied business regarding the essential
aspects of the upcoming audit study. When planning an audit of the knowledge of a particular business, the auditor should take into account that almost
all of its subject areas should be subject to audit research. However, practice shows that the timing and scale of
the upcoming audit do not allow to fully cover the entire business under study. Therefore, to improve the quality
and effectiveness of the audit cycle, it is necessary to pre-select the most significant aspects for the business,
constantly supplementing and expanding the range of studies both in the audit process on the merits of the audit
engagement and in subsequent audit studies. It should be specially noted that the planning process must be carried out in three consecutive and interrelated
stages: preliminary planning (identifying areas of knowledge carriers that are significant for business), strategic
planning (developing a strategy for the upcoming audit) and ongoing planning (developing methods and preparing
tools for conducting substantive audits audit assignment. Despite the fact that each of these stages has its own characteristics and some resulting individuality, however,
their practical implementation is due to close interdependence, since any adjustments in one of them will
necessarily lead to changes in the others. Therefore, when planning audit of implicit (unformalized) knowledge, it
is necessary to be guided by the generally recognized principles of continuity, complexity and optimality. At the same time, continuity in this context should be understood as the installation of specific tasks
interconnected at all stages of the audit of knowledge for a group of auditors and specialists- experts. In its turn,
the complexity of planning an audit of knowledge refers to the interconnectedness and consistency of all the
previously mentioned planning stages. And, finally, the principle of optimal planning for this audit consists in the
choice by the auditor of the most important subject areas for the business being studied, which will allow to
obtain the greatest effect from the results of the entire audit cycle. Starting the stage of a detailed audit of the essence of the audit engagement, as noted earlier, to study implicit
(non-formalized) knowledge, the auditor should consider the possibility of using heuristic methods based on the
admissibility, rationality and even satisfactoriness of the prepared management recommendations. ENTREPRENEURSHIP AND SUSTAINABILITY ISSUES
ISSN 2345-0282 (online) http://jssidoi.org/jesi/ Then the auditor develops a strategy and tactics for bringing the business as a whole and its knowledge
management system in particular to the desired level of knowledge management efficiency. It should be especially noted that the nature of the relationship between the auditor and the managing and
managed business systems depends on the conditions in which the audit of implicit (informal) knowledge is
implemented regardless of its thematic focus (general audit of knowledge, assessment of corporate culture,
collection and systematization of informal knowledge , interaction with business knowledge holders leaving this
business, to identify their implicit (informal) knowledge, the existing innovative aspect, conducting on lying
training or retraining of the staff of the business, the effectiveness of the use of social networks, etc.). g
ore, even before the start of the audit, the essence of the audit engagement is necessary: g
g
Therefore, even before the start of the audit, the essence of the audit engagement is neces
i f
i
d b
i
l b
h
bj
i
d
f h
di
d g g
- inform interested business personnel about the objectives and terms of the audit study; m interested business personnel about the objectives and terms of the audit study; - to present to managers and knowledge holders the composition of the audit team and expert experts involved in
carrying out the audit assignment; - coordinate all the powers to access any, including confidential business information and its key knowledge
carriers; - agree on the duties and responsibilities of specific leading personnel of the business under study and key knowledge carriers; 2306 ENTREPRENEURSHIP AND SUSTAINABILITY ISSUES
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http://doi.org/10.9770/jesi.2020.7.3(56) 3. Results It should be mentioned that these approaches, implemented in an audit of knowledge, allow us to identify the likely
success factors of the business under study. For this, in the course of the audit study, it is necessary to focus not on a
separate group of specialists of knowledge carriers, but to try to cover the staff of the entire business. In this case, the
key aspect is precisely the assessment of factors that can serve either as barriers to the use of knowledge or to
facilitate an effective exchange of knowledge. Thus, it should be noted that a detailed audit of knowledge allows us to determine the patterns of knowledge flows in
business that form ideas about the approaches used to process information and, as a result, the efficiency of the use
and exchange of knowledge in this system. Practice shows that according to the results of the knowledge audit it is advisable not only to prepare a report and
management recommendations, but also to conduct knowledge mapping. Knowledge Maps are not only an appropriate way of fixing and exchanging explicit (formalized) knowledge, but
also a reflection of implicit (unformalized) knowledge with varying degrees of detail. These documents enable
managers of the business under study to understand what knowledge is needed for a particular business staff. In
addition, they allow you to separate explicit (formalized) knowledge, which is inherently accessible information,
from implicit (unformalized), requiring a special approach to them from the knowledge management system. According to I.V. Kozlova, and with which one cannot disagree, the goal of developing Knowledge Maps is the
formation of special documents in the form of a separate new intellectual product, which is essentially a source of
knowledge, as they reveal the links between sources of knowledge or indicate gaps in existing knowledge assets
(Kozlova, 2016). At the same time, Knowledge Maps should not be identified with knowledge stores, where the
entire body of business knowledge is directly stored. Knowledge cards are a kind of guide to the indicated
repositories, specialists, knowledge carriers, sources of knowledge, etc. The process of creating Knowledge Maps is
mainly focused on the definition and planning of the knowledge management system of any business (Kozlova,
2016). When creating Knowledge Maps to describe the subject area, it is most expedient to use thesaurus modeling of
knowledge. ENTREPRENEURSHIP AND SUSTAINABILITY ISSUES
ISSN 2345-0282 (online) http://jssidoi.org/jesi/ The specified
system of methods will allow us to assess the quality indicators characterizing the effectiveness of the functioning
of the knowledge management system, and as a result of management decisions. It should be borne in mind that the use of heuristic methods to assess implicit (non-formalized) knowledge and the
entire knowledge management system at a qualitative level when performing a knowledge audit is due to: g
g
y
q
- the qualitative nature of implicit (non-formalized) knowledge; - significant uncertainty of the probability of their formalization; chnologies that allow the construction and study of a formalized model of this knowledge. - the current lack of technologies that allow the construction and study of a formalized model of this knowledge. In this regard, the use by the auditor of expert assessment methods (questioning, interviewing, etc.) is noteworthy. However, these methods require a special approach to the formation of a group of specialists- experts with the
proper competence in the subject areas being studied. Moreover, a group of specialists - experts can be both
homogeneous and consist of specialists in various subject areas. g
,
y
p
(q
g,
g,
)
y
However, these methods require a special approach to the formation of a group of specialists- experts with the
proper competence in the subject areas being studied. Moreover, a group of specialists - experts can be both
homogeneous and consist of specialists in various subject areas. At the same time, it must be borne in mind that the practice of using these methods shows that their integrated
application is most effective for solving the same problem. In addition, each of the methods, as noted earlier,
involves some preparation for their implementation. In addition to the specified qualitative methods aimed at identifying implicit (non-formalized) knowledge, a
qualitative assessment of the knowledge management system and management decisions made on their basis, 2307 ENTREPRENEURSHIP AND SUSTAINABILITY ISSUES
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2020 Volume 7 Number 3 (March)
http://doi.org/10.9770/jesi.2020.7.3(56) during the audit of knowledge on the merits of the audit task, the auditor needs to conduct a study of knowledge
flows, that is, to identify the relationship of personnel, business and IT processes and technologies. These studies
will reveal the insufficiency or duplication of this or that knowledge, as well as the best practices and existing
barriers in the business system for using both explicit (formalized) and implicit (non-formalized) knowledge. 3. Results This approach allows you to effectively use thematic thesauruses, which forms the basis of a systematic
idea of the content of the concepts of the subject area and its structure, the development of logical, associative and
creative thinking, training of memory and imagination. At the same time, the thesaurus (from Greek language - treasure, treasury) is understood as structured and organized
knowledge containing the most complete amount of vocabulary on a certain topic with an indication of clearly
expressed semantic relations between concepts (Andrusenko, 2007). Among a significant number of Knowledge Maps used in practice, we may distinguish the most general types of
knowledge applicable to any business system (table 1). 2308 ENTREPRENEURSHIP AND SUSTAINABILITY ISSUES
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http://doi.org/10.9770/jesi.2020.7.3(56) 2020 Volume 7 Number 3 (Marc
http://doi.org/10.9770/jesi.2020.7.3(5
Table 1. Knowledge Maps and their characteristics
Types of Knowledge Maps
Characteristic
Process Oriented Knowledge Maps
Reflect knowledge and its sources supporting the main
business and IT processes of the business system. These maps
are m\formed according to the results of a study of business
operations and the external environment of the business. Conceptual Knowledge Maps
Reflect the hierarchical classification in the form of concepts
and semantic relations between them. Used when comparing
similar business projects, turning knowledge into related and
explicit (Kozlova, 2016). Competency Maps
Reflect the skills of one or another specialist, his advancement
on the hierarchical ladder and professionalism. Allow the
management system to search for experts in various subject
areas within the business system
Social Net Maps
Reflect knowledge graph and communication models of
business systems among various groups of practitioners,
business partners and other social environments. Allow to
analyze ways of sharing knowledge in the process of
collaboration. Strategic Knowledge Maps
Reflect the share of business initiative in the development of a
product, product or service
Advanced Development Distribution Maps
Show the experience and data of the use of various business
processes by the business system. Directs a business system
specialist who is interested in advanced developments in a
specific subject area to business resources and groups that
actively use advanced ideas
Source: own research 4. Discussion As it was mentioned earlier, a report based on the results of a knowledge audit, as well as generated Knowledge
Maps, are not a stage in completing the entire cycle of a specified audit. Research and practice of the
implementation of the audit of knowledge shows that the greatest effect of the implementation of management
recommendations developed on the basis of the audit is possible only with continuous monitoring of the process
of their implementation and use. This approach allows you to timely carry out the necessary additional research
and adjustments to management decisions made on the basis of audit recommendations. Conclusions Thus, it should be admitted that the knowledge audit cycle does not end with the stage of preparation and
presentation of the audit report, but continues throughout the support of management decisions based on its
results. An audit of knowledge accompanies these decisions until they are fully implemented, that is, until the
final effect is obtained. Knowledge audit, similar to other types and methods of audit, aims to assess the current
state of the business, but the main attention here is aimed at determining the availability of knowledge, further
needs for them, establishing knowledge flows and their use in business processes to add value to the organization. Knowledge audit is an important tool for assessing the readiness of a business to implement a knowledge
management system and further monitor its functioning. 2309 ENTREPRENEURSHIP AND SUSTAINABILITY ISSUES
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ISSN 2345-0282 (online) http://jssidoi.org/jesi/ Liudmila KASHIRSKAYA Liudmila KASHIRSKAYA Liudmila KASHIRSKAYA
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Professor, Doctor of the Economic Sciences, Financial University under the Government of the Russian Federation, Russia. ORCID ID: https://orcid.org/0000-0003-2221-4037 Dilmurod DAVLATZODA Candidate of Economic Sciences, Associate Professor, Russian-Tajik (Slavonic) University (RTSU), Tajikistan. ORCID ID: https://orcid.org/0000-0003-0115-7731 ENTREPRENEURSHIP AND SUSTAINABILITY ISSUES
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This work is licensed under the Creative Commons Attribution International License (CC BY).
http://creativecommons org/licenses/by/4 0/ Tatiana VOROZHEYKINA Doctor of Economics, Associated Professor, Department of Production Organization, Russian State Agrarian University - Moscow
Agricultural Academy named after K.A. Timiryazev, Russia. ORCID ID: http://orcid.org/0000-0001-7295-1372 Register for an ORCID ID:
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Predicting return of lung function after a pulmonary exacerbation using the cystic fibrosis respiratory symptom diary-chronic respiratory infection symptom scale
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Predicting return of lung function after a pulmonary
exacerbation using the cystic fibrosis respiratory
symptom diary-chronic respiratory infection
symptom scale Eliana R. Gill University of Washington
Christopher H. Goss
University of Washington
Scott D. Sagel
University of Colorado Anschutz Medical Campus
Michelle L. Wright
The University of Texas at Austin
Sharon D. Horner
The University of Texas at Austin
Julie A. Zuñiga
The University of Texas at Austin Keywords: Posted Date: September 13th, 2023
DOI: https://doi.org/10.21203/rs.3.rs-3232522/v1 Posted Date: September 13th, 2023 DOI: https://doi.org/10.21203/rs.3.rs-3232522/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Additional Declarations: No competing interests reported. Version of Record: A version of this preprint was published at BMC Pulmonary Medicine on July 24th,
2024. See the published version at https://doi.org/10.1186/s12890-024-03148-w. Version of Record: A version of this preprint was published at BMC Pulmonary Medicine on July 24th,
2024. See the published version at https://doi.org/10.1186/s12890-024-03148-w. Page 1/16 Results A regression model predicted that recovery of lung function decreased by 0.2 points for every increase in
CRISS points, indicating that participants with a CRISS score greater than 48.3 were at 14% greater risk
of not recovering to baseline lung function by Visit 2, than people with lower scores. Conclusion Monitoring CRISS scores in PwCF is an efficient, reliable, non-invasive way to determine a person’s status
at the beginning of a PEx. The results presented in this paper support the usefulness of studying
symptoms in the context of PEx in PwCF. Page 1/16 Page 2/16 Methods A secondary analysis of a longitudinal, observational study (N = 56) was conducted. Data was collected
at four time points: year-prior-to-enrollment annual appointment, termed “baseline”, day 1 of PEx
diagnosis, termed “Visit 1”, day 10–21 of PEx diagnosis, termed “Visit 2” and two-weeks post-
hospitalization, termed “Visit 3”. A linear regression model was performed to analyze the research
question. Background Pulmonary exacerbations (PExs) in people with cystic fibrosis (PwCF) are associated with increased
healthcare costs, decreased quality of life and the risk for permanent decline in lung function. Symptom
burden, the continuous physiological and emotional symptoms on an individual related to their disease,
may be a useful tool for monitoring PwCF during a PEx, and identifying individuals at high risk for
permanent decline in lung function. The purpose of this study was to investigate if the degree of
symptom burden severity, measured by the Cystic Fibrosis Respiratory Symptom Diary (CFRSD)- Chronic
Respiratory Infection Symptom Scale (CRISS), at the onset of a PEx can predict failure to return to
baseline lung function by the end of treatment. Introduction However, symptom burden score at onset of PEx
therapy has not been analyzed as a predictor to lung function loss in PwCF, and may help us to identify
people at increased risk for poor recovery from PExs. We hypothesize that higher symptom burden, as measured by the CFRSD-CRISS, at the beginning of a
PEx may predict people who will not recover their lung function by the end of treatment, and identify a
group of individuals at risk for permanent decline in lung function. Therefore, the purpose of this study
was to investigate if the degree of CRISS score severity at the onset of a PEx can predict failure to return
to baseline lung function by the end of treatment. Introduction Cystic fibrosis (CF) is one of the most common, lethal genetic disorders in the United States, and affects
almost 40,000 people (Cystic Fibrosis Foundation, 2022). The major health risk for people with CF
(PwCF) is acute pulmonary exacerbation (PExs), which are characterized by worsening respiratory
symptoms, and associated with decreased quality of life, morbidity and mortality (Goss, 2019; Liou et al.,
2001; Britto et al., 2002). PExs are also a major driver of healthcare costs, with the cost of a single Page 3/16 episode ranging from $60,800-$74,830 (Gold et al., 2022). PExs increase the risk for decline in lung
function: Sanders et al. (2010) has shown that 25% of people who are treated for a PEx fail to recover to
their baseline lung function by 3 months after antibiotic treatment. Further, PExs are associated with 50%
of permanent decline in lung function, and the more frequently PwCF experience PExs the more rapid the
decline and risk for respiratory failure (de Boer et al., 2011; Waters et al., 2012; Sanders et al., 2011). Therefore, it is critical to understand what characteristics can predict failure to recover baseline lung
function. When PwCF are diagnosed with a PEx they experience an increase in physiological symptoms, or
symptom burden (Bell et al., 2020; Rosenfeld et al., 2001). Symptom burden results from the continuous
physiological and emotional symptoms related to the disease and/or treatment for a PEx (Schmid-
Mohler et al., 2019). Higher symptom burden in chronic illnesses is associated with an increase in
healthcare utilization (Deng et al., 2022; Zhang et a., 2020). In chronic obstructive pulmonary disease
people with high symptom burden had significantly increased healthcare utilization and were more likely
to have died by the 5-year follow-up than people with moderate or low symptom burden (Park & Larson,
2014). Symptom burden is often measured in PwCF to evaluate efficacy of treatment when PExs occurs. Symptom burden, as measured by the Cystic Fibrosis Respiratory Symptom Diary (CFRSD)-Chronic
Respiratory Infection Scale (CRISS) in PwCF has been associated with c-reactive protein (CRP), a marker
of systemic inflammation (VanDevanter et al., 2022), suggesting that symptom burden increases in
response to inflammation or infection, and may be a useful measure for predicting treatment outcomes. The change in symptom burden CRISS score from a person’s baseline during a PEx is a viable efficacy
endpoint in clinical trials (VanDevanter et al., 2021). Timing of Assessments and Measures For the present study we used data from four time points: 1) annual visit data from the year prior to
enrollment into the study, termed annual visit; 2) the first day of PEx diagnosis, termed Visit 1; 3) Day 10–
21 of the PEx, termed Visit 2; 4) Two-weeks post-hospitalization, termed Visit 3. We measured seven
demographic variables: age, sex, race, ethnicity, health insurance, smoking history and exposure to
secondhand smoke; and six CF related variables: ppFEV1, homozygous delF508 mutation, positive
Pseudomonas aeruginosa infection, nutritional status, nights spent in the hospital, and nights spent on
home IV antibiotics. Symptoms and burden were collected and measured using the CFRSD-CRISS, which is composed of
eight items. The response for each item is scored on a 5-point likert-scale, ranging 0 for no symptoms to
4 for extremely severe symptoms, which address: difficulty breathing, feeling feverish, having
chills/sweats, increased cough, increased mucus production, fatigue, chest tightness, and wheezing
(Goss et al., 2009). Participants missing more than 1 response to the CFRSD were removed from
analysis for incomplete data. Each item response has a corresponding score that is totaled for a raw
summated score, ranging from 0–24 (Goss et al., 2009). The raw summated score is then converted to a
continuous Rasch-logit score, ranging from 0-100, termed the CRISS score. The higher the CRISS score
the worse the symptom burden. Spirometry measures were gathered at the annual visit, Visit 1 and Visit 2, and the ppFEV1 was
calculated using reference equations (Wang et al., 1993; Hankinson et al., 1999). Failure to recover
baseline lung function by Visit 2 was calculated as the ppFEV1 at Visit 2 minus the ppFEV1 at the annual
visit. A result of 0 indicated return to baseline lung function, positive result indicated improved recovery
of ppFEV1 and a negative result indicated failure to recover baseline ppFEV1 by Visit Study Population Study Population Page 4/16
This study is a secondary analysis of a longitudinal, observational study that explored changes in clinical
outcomes and systemic measurements of inflammation in response to antibiotic therapy for PExs in
PwCF (Sagel et al. 2015). The original study enrolled 123 participants from 6 CF Foundation (CFF)
Centers in the United States who were diagnosed with an acute PEx; defined as presenting at least 3 of
11 criteria for PEx and requiring at least 2 IV antibiotics. Data access were acquired from the CFF Page 4/16 Therapeutics Development Network Coordinating Center (TDNCC), in Seattle, Washington after review
and approval. Data from the original study and matched CFF registry data were provided. Inclusion
criteria for the study included PwCF 10 years of age and older. We further restricted our analysis to those
with symptom data gathered on Day 1 of PEx diagnosis, and percent-predicted forced expiratory volume
in 1 second (ppFEV1) data gathered at the previous year annual visit and as well as Day 10–21 of PEx
diagnosis. No data received from the TDNCC contained personally identifiable information or qualifying
HIPAA identifiers, and thus The University of Texas at Austin’s Institutional Review Board (IRB)
determined that this study met the criteria for exemption from IRB review under 45 CFR 46.104 (4)
secondary research data or specimens (no consent required) (IRB ID: STUDY00000967). Timing of Assessments and Measures Statistical Analysis Data were assessed for normality and missingness. Descriptive statistics were used to examine the
mean and standard deviation of continuous variables, and frequencies for categorical variables. We used
linear regression to test the relationship between CRISS score and lung function recovery. We controlled Page 5/16 Page 5/16 for age, gender, and P. aeruginosa infection due to research showing that PwCF who are female, older
and having chronic P. aeruginosa infection are more likely to respond to a lesser extent to PEx treatment
(Sanders et al., 2010; Szczesniak et al., 2017). We visualized the distribution of residuals; measured the
covariance ratio levels for levels outside 1 plus three-times the leverage and the residuals for values
greater than 3; we tested for independence of variable, outliers, the homogeneity of variance and
multicollinearity. R version 4.2.1 was used for all statistical analysis (The R Foundation for Statistical
Computing, 2023). Results A total of 56 participants were included in this analysis; attrition was related to missing ppFEV1 data (n =
67). The demographic and clinical characteristics of the included cohort can be found in Table 1, and
were compared to participants not included. This subset was representative of the overall study cohort. The majority of participants were Non-Hispanic White (n = 52, 92.9%), female (n = 37, 66%) and mean age
was 25.1 (Standard Deviation [SD] = 9.81) years. The majority of participants were positive for P. aeruginosa (n = 44, 78.6%). The median length of hospitalization was 16 nights, and median time spent
on home IV antibiotics was 12 nights. The majority of our participants did not use oxygen throughout the
year (n = 38, 67.9%) or at the beginning of PEx treatment (n = 49, 91.5%), and no participants smoked (n
= 0, 0%). We assessed body mass index (BMI) as a measure of nutritional status for participants 21
years of age and older, and the Center for Disease Control clinical growth charts weight-for-age for
participants 12–20. The mean BMI for participants 21 years of age and older was 20.71 kg/m2 (SD =
2.63), and the majority of participants less than 21 years of age were normal weight-for-age percentile (n
= 13, 61.9%). A total of 56 participants were included in this analysis; attrition was related to missing ppFEV1 data (n =
67). The demographic and clinical characteristics of the included cohort can be found in Table 1, and
were compared to participants not included. This subset was representative of the overall study cohort. The majority of participants were Non-Hispanic White (n = 52, 92.9%), female (n = 37, 66%) and mean age
was 25.1 (Standard Deviation [SD] = 9.81) years. The majority of participants were positive for P. aeruginosa (n = 44, 78.6%). Results Symptom
burden significantly improved with IV antibiotic treatment (p < 0.001), and while CRISS scores were still
significantly better (p < 0.01) at the follow-up appointment 2 weeks post-hospitalization, scores
increased again after systemic antibiotic treatment was completed (Fig. 1). Table 2
Symptom Prevalence, Visit 1
N = 56
Frequency (%)
Mean Severity (SD)
Min, Max
Difficulty Breathing
32 (57.1%)
1.23 (1.27)
0, 4
Feverish
11 (19.6%)
0.39 (0.82)
0, 3
Chills/Sweats
10 (17.9%)
0.34 (0.82)
0, 4
Fatigue
45 (80.4%)
2.09 (1.3)
0, 4
Chest Tightness
23 (41.1%)
0.93 (1.26)
0, 4
Cough
55 (98.2%)
2.29 (0.76)
0, 4
Mucus production
49 (87.5%)
1.95 (1.05)
0, 4
Wheezing
24 (42.9%)
0.64 (0.84)
0, 3
CRISS Score
–
44.75 (10.67)
0, 61
ongitudinal analysis of CRISS scores over 3 visits. Visits: 1 = Visit 1; 2 = Visit 2; 3 = Visit 3. e higher numbers indicate more severe symptom burden. Figure 1. Longitudinal analysis of CRISS scores over 3 visits. Visits: 1 = Visit 1; 2 = Visit 2; 3 = Visit 3. CRISS score higher numbers indicate more severe symptom burden. Figure 1. Longitudinal analysis of CRISS scores over 3 visits. Visits: 1 = Visit 1; 2 = Visit 2; 3 = Visit 3. CRISS score higher numbers indicate more severe symptom burden. Results The majority of patients recovered to baseline lung function (n = 34, 60.7%),
and the average amount of lung function recovered by Visit 2 from annual visit was 1.52% (SD = 4.35,
range: -6.28-12.8). However, 39.3% of participants still failed to recover to baseline lung function, and
10.4% failed to recover within 10% of their baseline lung function by Visit 2. The most prevalent symptom
experienced by participants was increased cough (n = 55, 98.2%), followed by increased mucus (n = 49,
87.5%) and fatigue (n = 45, 80.4%). The most severe symptom experienced was also increased cough,
2.29 (SD = 0.76), followed by fatigue, 2.09 (SD = 1.03) then increased mucus, 1.95 (SD = 1.05) based on a
raw score. A summary of frequency and severity of measured symptoms can be found in Table 2. The
mean CRISS score at Visit 1 was 44.75 (SD = 10.67) and at Visit 2 was 23.69 (SD = 14.83). Symptom
burden significantly improved with IV antibiotic treatment (p < 0.001), and while CRISS scores were still
significantly better (p < 0.01) at the follow-up appointment 2 weeks post-hospitalization, scores
increased again after systemic antibiotic treatment was completed (Fig. 1). The mean ppFEV1 at the annual visit was 58.4% (SD = 21.6), Visit 1 was 52.28% (SD = 20.24) and Visit 2
was 60.59% (SD = 23.65). The majority of patients recovered to baseline lung function (n = 34, 60.7%),
and the average amount of lung function recovered by Visit 2 from annual visit was 1.52% (SD = 4.35,
range: -6.28-12.8). However, 39.3% of participants still failed to recover to baseline lung function, and
10.4% failed to recover within 10% of their baseline lung function by Visit 2. The most prevalent symptom
experienced by participants was increased cough (n = 55, 98.2%), followed by increased mucus (n = 49,
87.5%) and fatigue (n = 45, 80.4%). The most severe symptom experienced was also increased cough,
2.29 (SD = 0.76), followed by fatigue, 2.09 (SD = 1.03) then increased mucus, 1.95 (SD = 1.05) based on a
raw score. A summary of frequency and severity of measured symptoms can be found in Table 2. The
mean CRISS score at Visit 1 was 44.75 (SD = 10.67) and at Visit 2 was 23.69 (SD = 14.83). Results The median length of hospitalization was 16 nights, and median time spent Page 6/16 Table 1 ab e
Demographic Characteristics
Secondary analysis (N = 56)
Parent Study (N = 123)
Demographic Variables
N (%)
N (%)
Age
10–20
21 (37.5%)
61 (50.8%)
20+
35 (62.5%)
59 (49.2%)
Gender, Female
37 (66.7%)
72 (60%)
Race/Ethnicity
White
52 (92.95%)
116 (94.3%)
Non-Hispanic
51 (91.1%)
113 (91.8%)
Hispanic
1 (1.8%)
3 (2.4%)
Black
2 (3.6%)
2 (1.6%)
Bi-Racial
2 (3.6%)
2 (1.6%)
Insurance Type
Medicaid
31 (53.6%)
37 (30.8%)
Medicare
5 (8.9%)
9 (7.5%)
Private
21 (37.5%)
30 (35%)
Private & Medicaid
8 (14.3%)
10 (8.3%)
Other
3
3 (2.5%)
No Insurance
0
1 (0.8%)
Smokes
0 (0%)
0 (0%)
Second-hand smoke
-
-
Daily
2 (3.6%)
2 (1.7%)
Several Time per Week
2 (3.6%)
3 (2.5%)
Several Times per Month
4 (7.14%)
6 (5%)
Never
19 (33.9%)
23 ( 19.2%)
Oxygen Use
-
-
Throughout the Year
18 (32.1%)
12 (10%) Demographic Characteristics Secondary analysis (N = 56)
Parent Study (N = 123)
Demographic Variables
N (%)
N (%)
During of PEx
7 (8.5%)
12 (10%)
BMI, < 21 years old
Underweight (< 5%)
6 (28.6%)
7 (18.4%)
Normal weight (5%-85%)
13 (61.9%)
29 (76.3%)
Overweight (85%-99%)
2 (9.5%)
2 (5.3%)
Homozygous DelF508
35 (62.5)
66 (53.7%)
Pseudomonas aeruginosa +
culture
44 (78.6%)
55 (44.7%)
Mean
(SD)
Median
Min &
Max
Mean
(SD)
Median
Min &
Max
Age, all
25.3
(9.81)
23
12, 50
22.38
(9.83)
21
10, 58
10–20
16.1
(2.5)
16
12, 20
15.06
(3.33)
16
10, 20
20+
30.8
(8.3)
29
21, 50
29.9
(8.51)
27
21, 58
BMI, 21 + years of age
20.1
(2.6)
22
15.5,
23.8
21.2
20.9
16.1,
33.5
ppFEV1
52.3
(20.2)
55.4
17.5,
93.5
54.7
(21.3)
51.9
17.5,
95.5
Hospital Nights
23.9
(23.1)
15.5
0, 87
25 (29.2)
15
0, 194
Adult
22.3
(23.6)
16
0, 87
26.8
(35.8)
13.5
0, 194
Child
26.8
(22.6)
15
3, 72
23.1
(20.2)
17.5
0, 86
Home Intravenous
Antibiotics
18.6
(24.8)
11.5
0, 110
16 (25.9)
6
0, 180
Adult
23.6
(27.3)
19
0, 110
22.4
(30.6)
14
0, 180
Child
10.1
(17.5)
0
0, 71
9.2 (17.4)
0
0, 90 The mean ppFEV1 at the annual visit was 58.4% (SD = 21.6), Visit 1 was 52.28% (SD = 20.24) and Visit 2
was 60.59% (SD = 23.65). Discussion In this study of 56 people living with CF, we found that CRISS scores at the onset of PEx significantly
predicted failure to recover ppFEV1 by end of systemic antibiotic treatment, even when controlling for
age, sex, and P. aeruginosa infection. Interestingly, these confounding variables chosen a priori were not
significant. It is possible that while these variables have a significant relationship to incomplete PEx
treatment response (Sanders et al., 2010), they do not influence the relationship between symptom
burden and failure to recover baseline ppFEV1 between Day 10–21. Future research should investigate
other variables that may influence this relationship such as use of highly effective CFTR modulator
therapy and nutrition status. The most common symptoms were increased cough, mucus and fatigue; increased cough and fatigue
were the two most severe symptoms experienced, and are similar to previous research (Gold et al.,
2019). Our results support previous findings that, while CRISS scores are sensitive to IV antibiotics, the
majority of CRISS scores increased again after treatment was stopped (VanDevanter et al., 2017). As
VanDevanter et al. (2017) implored, PEx research should not only focus on the improvement in clinical
end-points seen at the cessation of antibiotic therapy, but they must prioritize the optimization of
longitudinal response to PEx treatment, such as symptom burden and ppFEV1. The present study
supports that symptoms must be monitored even after the cessation of antibiotic therapy, since
symptom burden increases again after treatment and higher overall symptom burden may predict a
lower likelihood of returning to baseline after treatment. Previously, studies have focused on the change in CRISS score during PEx treatment to be used as a
reference for end to clinical treatment, but none have used symptom burden at PEx onset to predict
PwCF at high risk for incomplete treatment response. Our results suggest that the higher the symptom
burden at the onset of treatment for a PEx, the lower the chances of returning to baseline ppFEV1 at the
completion of antibiotic therapy. Based on this model, a participant with a CRISS score at Visit 1 of 48.3
or greater is less likely to regain baseline lung function by Visit 2. The mean CRISS score of these
participants is 44.8 and our model suggests that less than one standard deviation (10.8) above the mean
has a 14% higher chance of not recovering their baseline lung function by Day 10–21 of their PEx
treatment. CRISS Score and Lung Function We found that CRISS score at Visit 1 significantly predicts failure to recover ppFEV1 at Visit 2, even when
controlling for age, sex, and P. aeruginosa infection. The model was statistically significant, Adjusted R-
squared: 0.14 p = 0.02. The coefficients were intercept = 9.66, CRISS score = -0.2, age = 0.12, P. aeruginosa = -1.97, and gender = -1.1. While the model was overall statistically significant, the CRISS
score was the only significant predictor within the model (p < 0.01) while the three variables we
controlled for were not significant (p > 0.05). The model results predict that recovery of lung function Page 9/16 decreased by 0.2 points for every increase in CRISS points, indicating that participants with a CRISS
score greater than 48.3 were at 14% greater risk of not recovering to baseline lung function by Visit 2,
than people with lower scores, when all else is held constant. decreased by 0.2 points for every increase in CRISS points, indicating that participants with a CRISS
score greater than 48.3 were at 14% greater risk of not recovering to baseline lung function by Visit 2,
than people with lower scores, when all else is held constant. Discussion Three recent studies (combined N = 1,146) measured the CRISS score at onset of a PEx in
PwCF, and their mean or median CRISS scores ranged from 49-56.6 (Roberts et al., 2018; VanDevanter et
al., 2021; VanDevanter et al., 2022). This conveys that, on average, PwCF at the beginning of a PEx
treated with IV antibiotics consistently have CRISS scores above 48.3. Our results suggest that
measuring symptom burden at PEx onset may be a useful tool for identifying PwCF at risk for incomplete
treatment response before the end of antibiotic therapy. Page 10/16 There is a need for improved monitoring of lung function following PEx treatment. Heltshe et al. (2023)
found that 49.6% of participants who received antibiotic treatment returned to baseline lung function
when compared to participants not undergoing PEx therapy. The most improvement in lung function
following IV antibiotic therapy has been shown to occur within the first 7–10 days of treatment (Goss et
al., 2021), and any recovered lung function may begin to decline again anywhere between 1–16 weeks
after therapy has ended (Cunningham et al., 2003; Béghin et al., 2009). However, as Heltshe et al. (2023)
has identified, previous research on recovery of lung function has been obscured by inherent progression
of the CF disease, natural variability in ppFEV1 and inconsistent observation times (p. 2), which may have
mischaracterized that PExs treatment are inadequate. A weakness of the presented secondary analysis
is the lack of long term follow-up after treatment has ended and no inclusion of comparators. However,
PwCF who do not recover their baseline ppFEV1 by Day 10–21 of a PEx, a period when they have been
receiving IV antibiotics, may be at even higher risk of not recovering to their baseline lung function by 3
months. Utilizing CRISS score we have still identified a group at high risk for failure to recover baseline
lung function. Our results support the inclusion of CRISS score in clinical practice to provide an
additional measure of individuals response to PEx as well as monitoring CF disease progression, and
further research should seek to incorporate consistent measurement times and comparative groups. CRISS score and CRP have been shown to be significantly associated at the beginning of a PEx
(VanDevanter et al., 2022), and CRP has been shown to significantly predict failure to recover baseline
ppFEV1 when admission levels are greater than 75mg/L, (Sharma et al., 2017). Conclusion Measuring symptom burden via the CRISS score is an efficient, reliable, non-invasive way to determine a
patient’s status at the beginning of a PEx. Administering the CFRSD-CRISS at the beginning of a PEx may
allow clinicians to identify individuals at high-risk for not responding to treatment and permanent decline
in lung function. By using a patient's unique symptom experience clinicians may be able to tailor their
treatment to the individual to improve lung function recovery between Day 10–21 of a PEx in PwCF. The
results presented in this paper support the usefulness of studying symptoms in the context of PEx in
PwCF, and future research is needed to validate these findings. Discussion The significant
relationship between CRP and CRISS score, as well as their abilities to independently predict failure to
recover baseline lung function, conveys the clinical relevance of symptom burden as an objective, non-
invasive predictor and further research is needed to validate these findings for potential incorporation
into clinical practice. Declarations Ethics approval and consent to participate Acknowledgements We would like to acknowledge and thank the people with cystic fibrosis who participated in this study
and provided valuable information. We would like to acknowledge and thank the people with cystic fibrosis who participated in this study
and provided valuable information. under 45 CFR 46.104 (4) secondary research data or specimens (no consent required) (IRB ID:
STUDY00000967). under 45 CFR 46.104 (4) secondary research data or specimens (no consent required) (IRB ID:
STUDY00000967). Consent for publication Not applicable. Availability of data and materials Datasets generated and/or analyzed during the current study are available from the Cystic Fibrosis
Foundation’s Therapeutics Development Network (https://www.cff.org/researchers/therapeutics-
development-network). Data are available at no-cost upon application and peer-review. Competing Interests The authors declare that they have no competing interests. The authors declare that they have no competing interests. This work was supported by the Cystic Fibrosis Foundation, award numbers: GILL21H0 and SAGEL07B0,
and the NIH, award number: 1T32NR019035. NIH/NCATS Colorado CTSA Grant Number UL1 TR002535. Authors contributions Authors contributions ERG wrote the main manuscript and performed the analyses. CHG and JAZ provided major contributions
to study creation and assisted with writing of the manuscript. SDS collected primary data used in the
study and contributed to the editing and writing of the manuscript. MLW and SHD contributed to study
creation and contributed to writing of the study, as well as the figures and tables presented. All authors
reviewed the manuscript prior to submission. Acknowledgements Ethics approval and consent to participate Institutional Review Board (IRB) at The University of Texas at Austin reviewed this study for ethics
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345–352. https://doi.org/10.1093/aje/153.4.345 16. Park, S. K., & Larson, J. L. (2014). Symptom cluster, healthcare use and mortality in patients with
severe chronic obstructive pulmonary disease. Journal of Clinical Nursing, 23(17-18), 2658–2671. https://doi.org/10.1111/jocn.12526 17. R Core Team (2021). R: A language and environment for statistical computing. R Foundation for
Statistical Computing, Vienna, Austria. Retrieved from: https://www.R-project.org/. 18. Roberts, J. M., Dai, D. L. Y., Hollander, Z., Ng, R. T., Tebbutt, S. J., Wilcox, P. G., Sin, D. D., & Quon, B. S. (2018). Multiple reaction monitoring mass spectrometry to identify novel plasma protein biomarkers
of treatment response in cystic fibrosis pulmonary exacerbations. Journal of Cystic Fibrosis, 17(3),
333–340. https://doi.org/10.1016/j.jcf.2017.10.013 19. References Rosenfeld, M., Emerson, J., Williams-Warren, J., Pepe, M., Smith, A., Montgomery, A. B., & Ramsey, B. (2001). Defining a pulmonary exacerbation in cystic fibrosis. Journal of Pediatrics, 139(3), 359–365. https://doi.org/10.1067/mpd.2001.117288 20. Sagel, S. D., Thompson, V., Chmiel, J. F., Montgomery, G. S., Nasr, S. Z., Perkett, E., Saavedra, M. T.,
Slovis, B., Anthony, M. M., Emmett, P., & Heltshe, S. L. (2015). Effect of treatment of cystic fibrosis
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Respir Crit Care Med, 182(5), 627–632. https://doi.org/10.1164/rccm.200909-1421OC 22. Sanders, D. B., Bittner, R. C. L., Rosenfeld, M., Redding, G. J., & Goss, C. H. (2011). Pulmonary
exacerbations are associated with subsequent FEV1 decline in both adults and children with cystic
fibrosis. Pediatric Pulmonology, 46(4), 393–400. https://doi.org/10.1002/ppul.21374 23. Schmid-Mohler, G., Yorke, J., Spirig, R., Benden, C., & Caress, A. L. (2019). Adult patients’ experiences
of symptom management during pulmonary exacerbations in cystic fibrosis: A thematic synthesis
of qualitative research. Chronic Illness, 15(4), 245–263. https://doi.org/10.1177/1742395318772647 harma, A., Kirkpatrick, G., Chen, V., Skolnik, K., Hollander, Z., Wilcox, P., & Quon, B. S. (2017). Clinical
tility of C-reactive protein to predict treatment response during cystic fibrosis pulmonary
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zczesniak, R. D., Li, D., Su, W., Brokamp, C., Pestian, J., Seid, M., & Clancy, J. P. (2017). Phenotypes
f rapid cystic fibrosis lung disease progression during adolescence and young adulthood. Am J
Respir Crit Care Med, 196(4), 471–478. https://doi.org/10.1164/rccm.201612-2574OC
VanDevanter, D. R., Heltshe, S. L., Spahr, J., Beckett, V. V., Daines, C. L., Dasenbrook, E. C., Gibson, R. ., Raksha, J., Sanders, D. B., Goss, C. H., Flume, P. A., & STOP Study Group (2017). Rationalizing 24. Sharma, A., Kirkpatrick, G., Chen, V., Skolnik, K., Hollander, Z., Wilcox, P., & Quon, B. S. (2017). Clinical
utility of C-reactive protein to predict treatment response during cystic fibrosis pulmonary
exacerbations. PloS One, 12(2), e0171229. https://doi.org/10.1371/journal.pone.0171229 25. Szczesniak, R. D., Li, D., Su, W., Brokamp, C., Pestian, J., Seid, M., & Clancy, J. P. (2017). Phenotypes
of rapid cystic fibrosis lung disease progression during adolescence and young adulthood. Am J
Respir Crit Care Med, 196(4), 471–478. https://doi.org/10.1164/rccm.201612-2574OC 26. VanDevanter, D. R., Heltshe, S. References L., Spahr, J., Beckett, V. V., Daines, C. L., Dasenbrook, E. C., Gibson, R. L., Raksha, J., Sanders, D. B., Goss, C. H., Flume, P. A., & STOP Study Group (2017). Rationalizing Page 14/16 Page 14/16 endpoints for prospective studies of pulmonary exacerbation treatment response in cystic fibrosis. Journal of Cystic Fibrosis, 16(5), 607–615. https://doi.org/10.1016/j.jcf.2017.04.004 endpoints for prospective studies of pulmonary exacerbation treatment response in cystic fibrosis. Journal of Cystic Fibrosis, 16(5), 607–615. https://doi.org/10.1016/j.jcf.2017.04.004 27. VanDevanter, D. R., Heltshe, S. L., Sanders, D. B., West, N. E., Skalland, M., Flume, P. A., Goss, C. H., &
STOP-OB Study (2021). Changes in symptom scores as a potential clinical endpoint for studies of
cystic fibrosis pulmonary exacerbation treatment. Journal of Cystic Fibrosis 20(1), 36–38. https://doi.org/10.1016/j.jcf.2020.08.006 28. VanDevanter, D. R., Heltshe, S. L., Skalland, M., West, N. E., Sanders, D. B., Goss, C. H., & Flume, P. A. (2022). C-reactive protein (CRP) as a biomarker of pulmonary exacerbation presentation and
treatment response. Journal of Cystic Fibrosis, 21(4), 588–593. https://doi.org/10.1016/j.jcf.2021.12.003 29. Wang, X., Dockery, D.W., Wypij, D., Fay, M.E. & Ferris, B.G. (1993). Pulmonary function between 6 and
18 years of age. Pediatric Pulmonology 15, 75–88. 29. Wang, X., Dockery, D.W., Wypij, D., Fay, M.E. & Ferris, B.G. (1993). Pulmonary function between 6 and
18 years of age. Pediatric Pulmonology 15, 75–88. 30. Waters, V., Stanojevic, S., Atenafu, E. G., Lu, A., Yau, Y., Tullis, E., & Ratjen, F. (2012). Effect of
pulmonary exacerbations on long-term lung function decline in cystic fibrosis. European Respiratory
Journal, 40(1), 61–66. https://doi.org/10.1183/09031936.00159111 30. Waters, V., Stanojevic, S., Atenafu, E. G., Lu, A., Yau, Y., Tullis, E., & Ratjen, F. (2012). Effect of
pulmonary exacerbations on long-term lung function decline in cystic fibrosis. European Respiratory
Journal, 40(1), 61–66. https://doi.org/10.1183/09031936.00159111 31. Zhang, J. C., El-Majzoub, S., Li, M., Ahmed, T., Wu, J., Lipman, M. L., Moussaoui, G., Looper, K. J.,
Novak, M., Rej, S., & Mucsi, I. (2020). Could symptom burden predict subsequent healthcare use in
patients with end stage kidney disease on hemodialysis care? A prospective, preliminary study. Renal Failure, 42(1), 294–301. https://doi.org/10.1080/0886022X.2020.1744449 Figures Page 15/16 Figure 1
Longitudinal analysis of CRISS scores over 3 visits. Visits: 1= Visit 1; 2= Visit 2; 3= Visit 3. CRISS score
higher numbers indicate more severe symptom burden. Figure 1 Figure 1 Longitudinal analysis of CRISS scores over 3 visits. Visits: 1= Visit 1; 2= Visit 2; 3= Visit 3. CRISS score
higher numbers indicate more severe symptom burden. Page 16/16
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MODERN COST MANAGEMENT TECHNICS AND AREVIEW ABOUT GRADUATE THESIS’ STUDYING SUCH TECHNICS AT 2000-2014 PERIOD
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ÖZET Modern maliyet belirleme yaklaşımları maliyetlerin belirlenmesinde ortaya çıkan yanlışlıkların ve hataların
giderilmesinde, işletmelere klavuzluk yapmaktadır. Bu yaklaşımlar; tam zamanlı üretim, kalite maliyetleri, değer
mühendisliği, kaizen maliyetleme, hedef maliyetleme, faaliyet tabanlı maliyetleme, stratejik maliyetleme,
stratejik yönetim muhasebesi, mamul yaşam dönemi boyunca maliyetleme olarak geçmektedir. Bu araştırmanın
amacı da, Yükseköğretim Kurulu Ulusal Tez Merkezi’ne yüklenmiş ve erişime açık olduğu tespit edilen lisansüstü
tezlerden hareketle içerik analizi yapılmasıdır. Literatürün bir parçasını oluşturan bu çalışmalar kendisinden
sonraki çalışmalara referans teşkil ettiğinden bu şekilde yapılacak bir içerik analizi ile bazı anlamlı sonuçların
ortaya konulması hedeflenmiştir. Anahtar Sözcükler: Muhasebe, Modern Maliyetleme, İçerik Analizi. YRD.DOÇ.DR. OSMAN KÜRġAT ONAT
Mehmet Akif Ersoy Üniversitesi, İktisadi ve İdari Bilimler Fakültesi, Sağlık Yönetimi Bölümü
osmankursatonat@mehmetakif.edu.tr Mehmet Akif Ersoy Üniversitesi, İktisadi ve İdari Bilimler Fakültesi, Sağlık Yönetimi Bölümü
osmankursatonat@mehmetakif.edu.tr MODERN MALĠYET YÖNETĠMĠ TEKNĠKLERĠ VE 2000-
2014 DÖNEMĠNDE BU TEKNĠKLERĠ ELE ALAN
LĠSANSÜSTÜ TEZLERE DAĠR BĠR DEĞERLENDĠRME
MODERN COST MANAGEMENT TECHNICS AND AREVIEW ABOUT GRADUATE THESIS’
STUDYING SUCH TECHNICS AT 2000-2014 PERIOD YRD.DOÇ.DR. OSMAN AKIN
Mehmet Akif Ersoy Üniversitesi, Bucak İşletme Fakültesi, İşletme Bölümü
osmanakin@mehmetakif.edu.tr YRD.DOÇ.DR. OSMAN KÜRġAT ONAT YRD.DOÇ.DR. OSMAN KÜRġAT ONAT
Mehmet Akif Ersoy Üniversitesi, İktisadi ve İdari Bilimler Fakültesi, Sağlık Yönetimi Bölümü
osmankursatonat@mehmetakif.edu.tr 1. GĠRĠġ Maliyet kavramı, ele alındığı, kullanıldığı yere göre birçok anlam taşımaktadır. Maliyet kavramı bir şeyi
elde etmek için katlanılan her türlü fedakarlıklar toplamıdır. Buradan çıkarılacak anlam ise, kişi ya da
işletmelerin yaptıkları her faaliyetin bir maliyeti olduğudur. Bir işletmenin masa üretmesi, bir marketin
satış yapması gibi daha birçok faaliyetin maliyeti vardır (Bekçi ve Özal, 2010: 79). İşletmelerde maliyet kavramı ve bunun doğru bir şekilde hesaplanması işletmenin birim maliyetlerinin
bilinmesi ve bunu takiben satış fiyatlarının doğru tespit edilmesinde önemli bir rol oynamaktadır. Bu
nedenle geleneksel olarak nitelendirilen ve genel üretim maliyetlerinin hesaplanmasında bazı eksiklikler
oluşturan maliyet muhasebesi, işletmeleri yanlış kararlar vermesine neden olabilmektedir. İşletmelerin
bu eksikliği gidermek amacıyla arayışlarının sonucu olarak modern maliyet muhasebesi yaklaşımları
işletmeler açısından büyük önem taşımakta ve stratejik üstünlük sağlamaktadır. ABSTRACT Modern cost determination methods guide companies at correcting mistakes and error soccuring during cost
calculation. These approaches are just-in-time production, quality costs, value engineering, kaizen costing,
target costing, activitity based costing and life cycle costing. Aim of this study is content analysis of grduate
thesis’ uploaded and open Access data Council of Higher Education National Thesis Center. Because of those
studies that compose some part of literatüre are being reference to future studies some significant results are
aimed to be obtained. Keywords: Accounting, Modern Cost Accounting, Content Analysis. 6 AKIN & ONAT 2. KAVRAMSAL ÇERÇEVE Modern maliyet yönetim teknikleri literatürde faaliyet tabanlı maliyetleme, hedef maliyetleme, tam
zamanında maliyetleme, kaizen maliyetleme, stratejik maliyet yönetimi, mamül yaşam dönemi
maliyetlemesi olarak sayılmaktadır. Aşağıda bu maliyet yönetim teknikleri açıklanmıştır. Modern maliyet yönetim teknikleri literatürde faaliyet tabanlı maliyetleme, hedef maliyetleme, tam
zamanında maliyetleme, kaizen maliyetleme, stratejik maliyet yönetimi, mamül yaşam dönemi
maliyetlemesi olarak sayılmaktadır. Aşağıda bu maliyet yönetim teknikleri açıklanmıştır. Japon menşeili olan Hedef Maliyetleme Yöntemi (Target Costing), Pazara Dayalı Hedef Maliyet
Yönetimi (Target Cost Management) olarak da adlandırılmaktadır. Stratejik maliyet yönetiminin bir
enstrümanı olarak performansa dayalı maliyet planlama ve kontrolü olarak da tanımlanan Hedef
Maliyet Yöntemi işletmelerin rekabet gücünü artırmak için, ürettikleri ürünle ilgili faaliyetlerinin pazara
dayalı ve maliyet odaklı olarak yönetilmesini amaçlamaktadır. Hedef Maliyet Yöntemi’nin hareket
noktasını, sürekli değişen ve şiddetlenen rekabet koşullarında tüm işletme faaliyetlerine tutarlı bir
biçimde stratejik yönetim oluşturmaktadır. Bu nedenle stratejik bir kar ve maliyet yönetim süreci
olarak da ifade edilen Hedef Maliyet Yöntemi, tekniği özetle, “bir mamule ait beklenen kar oranını
kazandıracak kabul edilebilir maliyet düzeyi” biçiminde tanımlanan hedef maliyet kavramına
dayanmaktadır (Alagöz ve Ceran, 2006: 62-63) Tam zamanında üretim, işletmelerin müşterilerinden aldıkları siparişleri zamanında ve kaliteli bir
şekilde teslim etmelerine yöneliktir (Savaş, 2003: 204). Tam zamanında kavramı, gerekli görülen veya
talep edilen faaliyetlerin hemen devreye sokulmasını baz alan bir felsefedir. Dolayısıyla tam zamanında
üretim, bir üretim hattında üretilen bir parçanın izleyen safha tarafından ihtiyaç duyulduğu kadar ve
hemen üretildiği bir sistem olarak tanımlanabilir. Tam zamanında üretim felsefesinin temelini sıfır stok,
kısa üretim süreleri, küçük-seri ikmal miktarları ile yüksek kalite ve sıfır hata olmak üzere dört unsur
oluşturmaktadır. Tam zamanında felsefesi, işletmelerin işletme fonksiyonları ve yönetim anlayışında
önemli düzeyde değişikliklere neden olmuştur. Bunun sonucunda da maliyet ve yönetim
muhasebesinin bu yapıya adapte olması zorunluluğu doğmuştur. Bu değişiklikler; direkt maliyet 7 Mehmet Akif Ersoy ÜNiversitesi
İktisadi ve İdari Bilimler Fakültesi Dergisi
3(5), Bahar, 2016, ss. 6-15. Mehmet Akif Ersoy University
Journal of Economics and Administrative Sciences
3(5), Spring 2016, pp. 6-15. unsurlarında artış meydana gelmesi, maliyet merkezlerinin otomasyona dayalı olarak saptanması,
dağıtım anahtarlarının değişmesi, satın alma fiyat farkının azalması, raporlama detay ve aralıklarında
azalışların meydana gelmesi olarak sayılabilir (Atmaca ve Terzi, 2007: 295-296). Faaliyet Tabanlı Maliyetleme (FTM) stratejik karar almada etkinliği güçlendirmek için geleneksel
muhasebe sisteminin kısıtlarını aşmak için 1980’li yıllarda geliştirilen bir bilgi sistemidir. FTM’nin
dayandığı temel fikir; bir maliyet objesinin üretiminin (ürünler, müşteriler vb.) kaynakları (maaşlar,
ekipmanlar) tüketen faaliyetleri gerçekleştirdiğidir. 2. KAVRAMSAL ÇERÇEVE Her işletmenin stratejisi diğerlerinden farklı olduğu için, her işletme almış olduğu özel
stratejilerini destekleyecek farklı maliyet yönetim sistemine sahip olmalıdır (Bekçi ve Özal, 2010: 83). pozisyonlarını sürekli geliştirmek ve maliyetlerini düşürmek için maliyet yönetim tekniklerini
uygulamasıdır. Stratejik maliyet yönetimi, hem kısa dönemde hem de uzun dönemde finansal
amaçlarını maliyet avantajı için yönetmektir. Bir firmanın maliyet yapısının açıkça anlaşılması,
sürdürülebilir rekabet avantajı aramada yararlı olur. Stratejik maliyet yönetimi, maliyet unsurlarının
daha ön planda tutulduğu, kesin, net ve biçimsel olarak, maliyet çözümlemelerine dayanan bir maliyet
yönetimidir. Buna göre maliyet verileri, sürekli ve güçlü rekabete dayalı avantajlar kazanmak için
kullanılır. Her işletmenin stratejisi diğerlerinden farklı olduğu için, her işletme almış olduğu özel
stratejilerini destekleyecek farklı maliyet yönetim sistemine sahip olmalıdır (Bekçi ve Özal, 2010: 83). Araştırma, bir süreç olarak nitelendirildiğinde amaçlara ulaşmayı ve problem çözmeyi hedefleyen,
sonuçları değerlendirmeye yönelik gayrettir. Bilimsel araştırma olarak bu konuda içerik analizinden
yararlanılmıştır. İçerik analizleri akademik kariyerlerine devam eden uzmanlara ve öğrencilere kaynak sağlayan
araçlardır. İçerik analizi ile ilgili araştırmalar aynı zamanda literatürde bibliyometrik araştırma olarak da
anılmakta ve bunlar bilim dallarının kendi alanlarındaki yayın yeterliliğini; yayın sayısı, yayın niteliği,
yayın yapılan dergilerin ait olduğu endekslerin seçimi gibi kriterler ışığında değerlendirerek geleceğe
yönelik bilim politikalarının oluşturulmasına yardımcı olur. 3. ARAġTIRMANIN AMACI VE ÖNEMĠ Yapılan bu çalışmanın amacı, 2000-2014 yılları arasında modern maliyet yönetim yöntemleri ile ilgili
yapılmış olan yüksek lisans ve doktora tezlerinin içerik analizinin yapılmasıdır. Bu çalışma, anılan
dönemde ileri maliyet muhasebesi yaklaşımlarına yönelik hazırlanış tezlerin içeriğini ortaya koyması ve
gelecekte bu doğrultuda yapılacak olan başka çalışmalara bilgi ve literatüre katkı sağlaması yönüyle
önem arz etmektedir. 2. KAVRAMSAL ÇERÇEVE FTM’nin temel amacı, endirekt maliyetlerin ürünlere
(hizmetlere) ve müşterilere doğru bir şekilde dağıtımının sağlanması ve daha iyi operasyonel, stratejik
kararlar almak için tam maliyetlemeye göre daha doğru bir ürün (servis) birim maliyetinin
hesaplanmasıdır. Genel üretim giderlerinin FTM yoluyla dağıtılması iki aşamada gerçekleşmektedir. İlk
aşamada; FTM modeli farklı maliyet havuzlarındaki maliyetleri farklı faaliyetlere dağıtmak için “kaynak
maliyet etkenlerine” dayanmaktadır. İkinci olarak, faaliyetlerde biriken bu maliyetler “faaliyet maliyet
etkenleri” yardımıyla söz konusu faaliyeti kullandığı ölçüde maliyet objelerine dağıtılmaktadır (Cengiz,
2011: 35). Mamül yaşam dönemi maliyetleme, fiziksel varlıkların ekonomik ömürleri boyunca ortaya çıkan tüm
maliyetleri tanımlayıp, ölçerek bugünkü değer yöntemiyle elde etme ve bunlara sahip olma maliyetinin
optimizasyonuna çalışır. Mamül yaşam dönemi maliyetleme yöntemi, yöneticilere bir ürünün tüm
yaşamı boyunca maruz kaldığı maliyetleri yönetme ve anlama konusunda bilgi sağlayan bir sistemdir
(Güneş ve Aksu, 2003: 44). Kaizen maliyetleme, yeni ürünlerin ve var olan ürünlerin odak noktasını karlılığa ve üretim aşamasına
yönlendirmiştir (Williamson, 1997: 22). Kaizen maliyetleme, bir mamulün hayatının üretim safhasında
maliyet azaltımı için uygulanan sürekli iyileştirme olarak tanımlanabilir. Kaizen maliyetleme, mevcut
mamülleri üretebilmek için kullanılan üretim süreçlerinin etkinliğini arttıracak alternatif yollar arayarak
mevcut mamullerin üretim maliyetini düşürmektedir. Çok kısa ömürlü mamullere sahip çoğu işletmede,
üretim süreçlerinin ömrü mamullerin ömründen uzundur. Bundan dolayı, mamulün kendisinden ziyade
üretim safhasındaki üretim süreçlerine odaklanmak suretiyle büyük tasarruflar başarılabilir. Hedef
maliyetleme gibi kaizen maliyetleme de maliyet azaltımı amaçlar seti belli olduğunda çok etkilidir. Hedef maliyetlemeden farklı olarak kaizen maliyetleme, mamul tasarımıyla çok ilgili olmayıp daha çok
verilen mamulün maruz kaldığı üretim sürecine odaklanmaktadır. Kaizen maliyetlemenin temel amacı,
etkin olmayan her türlü unsurun üretim süreçlerinden kaldırılmasıdır (Altınbay, 2006: 104). Değer mühendisliği, varolan ürünleri geliştirmek, yeni ürünler ortaya koymak, karmaşık çevresel
projelerin planlarını yapmak, faaliyet analizi yapmak, kalite ve güvenirlik sistemlerini iyileştirmek,
gereksiz maliyetlerden kaçınmak, riski azaltmak ve başarıyı arttırmak amaçlarıyla uygulanan bir
tekniktir (Örnek, 2003: 215). Stratejik yönetimin önemli fonksiyonlarından olan firmanın rekabet avantajında sürdürülebilir rekabetçi
pozisyon geliştirmek, işletme için devamlı başarı sağlar. Stratejik maliyet yönetimi, işletmelerin stratejik 8 AKIN & ONAT pozisyonlarını sürekli geliştirmek ve maliyetlerini düşürmek için maliyet yönetim tekniklerini
uygulamasıdır. Stratejik maliyet yönetimi, hem kısa dönemde hem de uzun dönemde finansal
amaçlarını maliyet avantajı için yönetmektir. Bir firmanın maliyet yapısının açıkça anlaşılması,
sürdürülebilir rekabet avantajı aramada yararlı olur. Stratejik maliyet yönetimi, maliyet unsurlarının
daha ön planda tutulduğu, kesin, net ve biçimsel olarak, maliyet çözümlemelerine dayanan bir maliyet
yönetimidir. Buna göre maliyet verileri, sürekli ve güçlü rekabete dayalı avantajlar kazanmak için
kullanılır. 5. ARAġTIRMANIN KAPSAMI VE SINIRLILIKLARI Araştırmanın varsayımları ve kısıtları şu şekilde sıralanmaktadır: Araştırmaya konu olan tüm tezler; tez konuları, başlıkları, anahtar sözcükleri, kullandıkları yöntem gibi
faktörler esas alınarak araştırma yapılmış ve tezlerin bilimsel esaslara göre hazırlandığı kabul edilmiştir. YÖK Ulusal Tez Merkezi veri tabanında kayıtlı tezlerin ileri maliyet muhasebesi alanındaki bütün tezleri
kapsadığı, elektronik ortamda doğru kaydedildiği ve tezlerin bilimsel yöntemler kullanılarak hazırlandığı
araştırmanın temel varsayımlarındandır. Araştırmada yer alan tezler; 2000-2014 yılları arasında üniversitelerin sosyal bilimler enstitüleri
bünyesinde hazırlanan tezlerdir. Tez taramamızda ana çerçeveyi oluşturan tez sayısı 187’dir. Araştırmanın son 15 yılı kapsaması çalışmanın bir diğer kısıtıdır. 4. ARAġTIRMANIN YÖNTEMĠ Sosyal bilimler alanında sıkça başvurulan analiz yöntemlerinden biri olan içerik analizi, çalışmaların
incelenmesine olanak sağlamaktadır. İçerik analizinin temel amacı elde edilen verileri açıklamak, veriler
arasında ilişki kurmak ve yorumlamaktır. Bu amaçla veriler düzenlenerek kodlanır, başlıklar belirlenir ve
düzenlenir elde edilen bulgular değerlendirilir (Akın ve Topçuoğlu, 2013: 3). İçerik analizi, metinlere ve kullanıldıkları faaliyetlere yönelik anlamlı, geçerli ve tutarlı çıkarımlar
yapabilmek için kullanılan bir bilimsel araştırma yöntemidir. Çeşitli konulara uygulanan birtakım
metodolojik araç ve teknikler bütünü olan içerik analizi, kontrollü olarak yorumlanan ve genelde
tümdengelime dayanan bir okuma aracı olarak değerlendirilebilmektedir (Gürel ve Alem, 2010: 336). 9 Mehmet Akif Ersoy ÜNiversitesi
İktisadi ve İdari Bilimler Fakültesi Dergisi
3(5), Bahar, 2016, ss. 6-15. Mehmet Akif Ersoy University
Journal of Economics and Administrative Sciences
3(5), Spring 2016, pp. 6-15. Mehmet Akif Ersoy University
Journal of Economics and Administrative Sciences
3(5), Spring 2016, pp. 6-15. Mehmet Akif Ersoy ÜNiversitesi
İktisadi ve İdari Bilimler Fakültesi Dergisi
3(5), Bahar, 2016, ss. 6-15. Çalışmada lisansüstü tezlerin yazım yılı, yapıldığı üniversite, içeriğinde kullanılan araştırma yöntemi ve
araştırma yapılan sektör ile ilgili veriler toplanmıştır. Toplamda 187 adet teze ulaşılmış, ulaşılan tezler
yüksek lisans ve doktora ayırımı, yıllara göre, üniversitelere göre ve kullanılan yönteme göre ayırımı
yapılarak elde edilen veriler Excel programında düzenlenmiş ve bu veriler incelenerek yorumlanmıştır. 6. ARAġTIRMANIN BULGULARI Araştırma kapsamında öncelikli olarak elde edilen bulgular değerlendirilecek olunursa tezlerin dağılımı
Tablo 1’deki gibidir: Tablo 1. Tezlerin Yıllara Göre Dağılımı
Yıllar
Yüksek Lisans
Yüzde
Doktora
Yüzde
2000-2005
63
43,75
13
30,23
2006-2011
62
43,05
22
51,16
2012-2014
19
13,20
8
18,61
Toplam
144
100
43
100 Tablo 1. Tezlerin Yıllara Göre Dağılımı Tablo 1 incelendiğinde; 2000-2005 yılları arasında yazılan tezlerin 63'ü (%43,75) yüksek lisans, 13'ü
(%30,23) doktora, 2006-2011 yılları arasındaki tezlerin 62'si (%43,05) yüksek lisans, 22'si (%51,16)
doktora ve 2012-2014 yılları arasında yazılan tezlerin ise 19'u (%13,20) yüksek lisans, 8'i (%18,61)
doktoradır. 10 AKIN & ONAT Tablo 2. Tezlerin Konulara Göre Dağılımı
Konu
Yüksek Lisans
Yüzde
Doktora
Yüzde
Tam Zamanlı Üretim
2
1,39
0
0
Kalite maliyetleri
44
30.56
5
11,62
Değer Mühendisliği
5
3,47
2
4,65
Kaizen Maliyetleme
2
1,39
0
0
Hedef Maliyetleme
22
15,27
9
20,93
Faaliyet Tabanlı Maliyetleme
35
24,31
11
25,59
Stratejik Maliyetleme
12
8,33
7
16,28
Stratejik Yönetim Muhasebesi/Yönetim Muhasebesi
20
13,89
9
20,93
Mamul Yaşam Dönemi
2
1,39
0
0
TOPLAM
144
100
43
100 Tablo 2. Tezlerin Konulara Göre Dağılımı Tezlerin konulara göre dağılımını gösteren Tablo 2’ ye baktığımızda en fazla 44 adet (%30,56) tezin
kalite maliyetleri konusunda yazıldığı görülmekte ve bunları da yüksek lisans tezleri oluşturmaktadır. Bunu takiben de 35 adet (%24,31) faaliyet tabanlı maliyetleme ve 22 adet (%15,27) hedef
maliyetleme konuları yüksek lisans tezi olarak yazılmıştır. Doktora çalışmalarını incelediğimizde ise 11
adet (%25,59 ,) tezin faaliyet tabanlı maliyet konusu üzerine yazıldığı ve 9 adet (%20,93) tezin ise stratejik yönetim
muhasebesi ve hedef maliyetleme konularında yazıldığı görülmektedir. Çalışılan konuların tümü
incelendiğinde de ağırlıklı olarak turizm, tekstil, sağlık, gıda, mermer, otomotiv sanayi sektörlerinin
çalışmalarda kullanıldığı görülmektedir. 11 11 Mehmet Akif Ersoy University
Journal of Economics and Administrative Sciences
3(5), Spring 2016, pp. 6-15. Mehmet Akif Ersoy ÜNiversitesi
İktisadi ve İdari Bilimler Fakültesi Dergisi
3(5), Bahar, 2016, ss. 6-15. Tablo 3.Tezlerin Üniversitelere Göre Dağılımı
Konu
Yüksek Lisans
Yüzde
Doktora
Yüzde
Ankara Ünv. 2
3
5
2,67
Anadolu Ünv. 2
1
3
1,60
Atatürk Ünv. 2
3
5
2,67
Adnan Menderes Ünv. 1
3
4
2,13
Akdeniz Ünv. 1
5
6
3,20
Adıyaman Ünv. 1
0
1
0,53
Afyon Kocatepe Ünv. 1
1
2
1,06
Beykent Ünv. 1
0
1
0,53
Celal Bayar Ünv. 3
0
3
1,60
Cumhuriyet Ünv. 1
0
1
0,53
Çankaya Ünv. 1
0
1
0,53
Çanakkale On Sekiz Mart Ünv. 6
0
6
3,20
Çukurova Ünv. 6. ARAġTIRMANIN BULGULARI Tezlerin Yöntemlere Göre Dağılımı Çalışma kapsamındaki yüksek lisans tezlerinin 23 adedi (%27,72) anket, 17 adedi (%20,48) mülakat,
3 adedi (%3,61) vaka analizi, 13 adedi (%15,67) model kurgusu, 7 adedi(%8,43) literatür taraması
şeklinde olup, 20 adedi (%24,09) ise firma verilerinden yararlanılmıştır. Doktora tezlerinin ise; 13 adedi (%34,22) anket, 7 adedi (%18,42) mülakat ve vaka analizi, 3 adedi
(%7,89) model kurgusu ve literatür taraması şeklinde olup, 5 adedi de (%13,16) firma verilerinden
yararlanılmıştır. Yapılan araştırmalarda genel olarak anket yöntemi uygulanmıştır. Doktora tezlerinin ise; 13 adedi (%34,22) anket, 7 adedi (%18,42) mülakat ve vaka analizi, 3 adedi
(%7,89) model kurgusu ve literatür taraması şeklinde olup, 5 adedi de (%13,16) firma verilerinden
yararlanılmıştır. Yapılan araştırmalarda genel olarak anket yöntemi uygulanmıştır. 6. ARAġTIRMANIN BULGULARI 4
0
4
2,13
Dumlupınar Ünv. 5
2
7
3,74
Dokuz Eylül Ünv. 7
2
9
4,81
Ege Ünv. 2
2
1,06
Eskişehir Osman Gazi Ünv. 2
1
3
1,60
Fatih Ünv. 2
0
2
1,06
Gazi Ünv. 4
0
4
2,13
Gaziantep Ünv. 1
0
1
0,53
Gebze İleri Teknoloji Enstitüsü
1
0
1
0,53
Gümüşhane Ünv. 1
0
1
0,53
Hacettepe Ünv. 1
1
2
1,06
Harran Ünv. 2
0
2
1,06
Marmara Ünv. 22
8
30
16,04
Maltepe Ünv. 1
0
1
0,53
Mersin Ünv. 2
0
2
1,06
Mimar Sinan Güzel Sanatlar Ünv. 2
0
2
1,06
Muğla Ünv. 1
1
2
1,06
Mustafa Kemal Ünv. 1
0
1
0,53
Niğde Ünv. 3
0
3
1,60
Kadir Has Ünv. 1
0
1
0,53
Karabük Ünv. 1
0
1
0,53
Karamanoğlu Mehmet BeyÜnv. 2
0
2
1,06
Kara Harp Okulu Ünv. 1
0
1
0,53
Kocaeli Ünv. 9
1
10
5,34
Sakarya Ünv. 12
2
14
7,48
Selçuk Ünv. 5
0
5
2,67
Süleyman Demirel Ünv. 5
2
7
3,74
İstanbul Ünv. 11
4
15
8,02
İstanbul Teknik Ünv. 2
0
2
1,06
İnönü Ünv. 3
0
3
1,60
Uludağ Ünv. 4
1
5
2,67
Trakya Ünv. 1
0
1
0,53
Yıldız Teknik Ünv. 2
0
2
1,06
Zonguldak Karaelmas Ünv. 1
0
1
0,53
TOPLAM
144
43
187
100 Tablo 3.Tezlerin Üniversitelere Göre Dağılımı 12 12 AKIN & ONAT Muhasebe alanında hazırlanan lisansüstü tezler açısından ilk 5 üniversite% 16,04 ile Marmara
Üniversitesi, %8,02 ile İstanbul Üniversitesi, %7,48 ile Sakarya Üniversitesi, %5,34 ile Kocaeli
Üniversitesi, %4,81 ile Dokuz Eylül Üniversitesi ve %3,74 ile Süleyman Demirel Üniversitesidir. Muhasebe alanında hazırlanan lisansüstü tezler açısından ilk 5 üniversite% 16,04 ile Marmara
Üniversitesi, %8,02 ile İstanbul Üniversitesi, %7,48 ile Sakarya Üniversitesi, %5,34 ile Kocaeli
Üniversitesi, %4,81 ile Dokuz Eylül Üniversitesi ve %3,74 ile Süleyman Demirel Üniversitesidir. Tablo 4. Tezlerin Yöntemlere Göre Dağılımı
Yöntem
Yüksek Lisans
Yüzde
Doktora
Yüzde
Toplam
Anket
23
27,72
13
34,22
36
Mülakat
17
20,48
7
18,42
24
Vaka Analizi
3
3,61
7
18,42
10
Model Kurgusu
13
15,67
3
7,89
16
Literatür
7
8,43
3
7,89
10
Firma Verileri
20
24,09
5
13,16
25
Toplam
83
100
38
100
121 Tablo 4. 7. SONUÇ Küreselleşen dünyada işletmelerin diğer işletmelerle rekabetini sürdürebilmeleri, kaliteli ve müşteri
odaklı hizmet verebilmeleri gerekmektedir. Günümüzde işletmeler sürekli bir rekabet içerisinde olup
fark yaratmak için farklı yollara başvurmaktadırlar. Bu noktada devreye çağdaş maliyet yaklaşımları
girmektedir. Çünkü işletmelerin kârını maksimize etmeye çalışır ve geleceğe yönelik planlarla disiplinli
ilerlemesini sağlamaktadırlar. Bu bağlamda, gerçekleştirmiş olduğumuz araştırmada adı geçen tam zamanlı üretim, kalite maliyetleri,
değer mühendisliği, kaizen maliyetleme, hedef maliyetleme, faaliyet tabanlı maliyetleme, stratejik
maliyetleme, stratejik yönetim muhasebesi, mamul yaşam dönemi boyunca maliyetleme, yönetim
muhasebesi konularına yönelik bir değerlendirmeyi kapsamaktadır. Söz konusu konular ile ilgili 15 yılda birçok çalışma yapılmıştır. En çok çalışmanın 2000-2005 yılları
arasında aralığında, 63 adet yüksek lisansve 13 adet doktora tezi yazıldığı görülmektedir. Bu tezler
genel olarak anket ve firma verilerinden yararlanarak oluşturulmuştur. 13 13 Mehmet Akif Ersoy ÜNiversitesi
İktisadi ve İdari Bilimler Fakültesi Dergisi
3(5), Bahar, 2016, ss. 6-15. Mehmet Akif Ersoy ÜNiversitesi
İktisadi ve İdari Bilimler Fakültesi Dergisi
3(5), Bahar, 2016, ss. 6-15. Mehmet Akif Ersoy University
Journal of Economics and Administrative Sciences
3(5), Spring 2016, pp. 6-15. Araştırmada elde edilen sonuçlar Modern Maliyet yaklaşımları konularında yazılmış olan yüksek lisans
ve doktora tezlerini kapsamaktadır. Araştırma sonucunda elde edilen bulgular ise;2000 ve 2014 yılları
arasında ile ilgili hazırlanan lisansüstü tezler incelenmektedir. Bu alanda yapılan çalışmaların büyük bir
çoğunluğunu yüksek lisans tezlerinin (144 adet) oluşturduğu görülmektedir. Bu sonucun oluşmasının
nedenleri arasında öğrenci popülasyonu açısından yüksek lisans yapan öğrenci sayısının daha fazla
olması gösterilebilir. Üniversitelere göre dağılımı bazında incelediğimizde ise en modern maliyet yönetimi yaklaşımları ile
ilgili olarak gerek yüksek lisans (22 adet), gerekse doktora (8 adet) tezin Marmara Üniversitesinde
yazıldığı görülmektedir. Söz konusu araştırma modern maliyet yönetimi ile ilişkili lisansüstü çalışmalar dikkate alınarak
halihazırda lisansüstü tezlerin hangi konularda ve hangi yöntem aralığında yoğunlaştığı araştırılmıştır. Araştırmacılar gelecekte yapılması planlanan çalışmalarını yöneltebilecekleri alanlar konusunda
belirlemelerde bulunurken çalışmanın analizlerinden faydalanarak daha az araştırılmış konular ve sık
kullanılmamış olan yöntemlere odaklanabileceklerdir. Savaş, O. (2003). ''Tam Zamanında Üretim Sisteminin Gerektirdiği Maliyet Muhasebesinin Temel
Nitelikleri'', Erciyes Üniversitesi İktisadi ve İdari Bilimler Dergisi, Sayı: 20, .203-218, Ocak-
Haziran. İraz, R.,Adem Ö., Zerenler M., (2007). ''Değer Mühendisliği Uygulamalarının Fonksiyonel Etkinlik
Açısından İşletmelerin Somut ve Soyut Varlıklarına Yönelik Olası Etkileri'', Selçuk Üniversitesi
İktisadi ve İdari Bilimler Fakültesi Sosyal ve Ekonomik Araştırmalar Dergisi,51-68. Örnek, Ş. A. (2003). ''Bir Yönetim Tekniği Olarak Değer Mühendisliği'', Dokuz Eylül Üniversitesi Sosyal
Bilimler Enstitüsü Dergisi, Cilt: 5, Sayı: 2., 213-230. Williamson, A. (1997). ''Targetand Kaizen Costing Manufacturing Engineer'', Sayı: 76, 22-24. KAYNAKÇA Acar, D. (2005). “Küresel Rekabette Maliyet Yönetimi ve Yaklaşımları: Tekstil Sektörü ile İlgili Bir
Araştırma”, Ankara: Asil Yayın Dağıtım. Akın, O. ve Topçuoğlu, F. (2013). ''2000-2013 Yılları Arasında Çalışma Sermayesi Yönetimi Alanında
Yapılan Lisansüstü Tezlere İlişkin Bibliyometrik Analiz Çalışması” ISAF Kongre Bildiri Kitabı,
Gaziantep. Alagöz, A. ve Ceran, Y. (2006). “Stratejik Maliyet Ve Kar Planlama Aracı Olarak Hedef Maliyet Yönetimi
(Target Cost Management)”. www.alialagoz.com.tr adresinden alınmıştır Altınbay, A. (2006). “Kaizen Maliyetleme Sistemi: Dinamik Bir Maliyet Yönetim Sistemi”. Afyon
Kocatepe Üniversitesi İktisadi ve İdari Bilimler Fakültesi Dergisi, 8(1), 103-121. Atmaca, M. ve Terzi, S. (2007). “Stratejik Maliyet Yönetimi Açısından Tam Zamanında Üretim Felsefesi
İle Kısıtlar Teorisinin Karşılaştırmalı Olarak İncelenmesi”. Marmara Üniversitesi İktisadi ve
İdari Bilimler Fakültesi Dergisi, 12(1), 293-309. Bekçi, İ. ve Özal, H. (2010). “Stratejik Maliyet Yönetiminin Sağlık Sektöründe Uygulanabilirliğine
Yönelik Bir Araştırma”. Akademik Araştırmalar ve Çalışmalar Dergisi, 2(3), 78-97. Cengiz, E. (2011). “Faaliyet Tabanlı Maliyetleme ve Sürece Dayalı Faaliyet Tabanlı Maliyetleme
Arasındaki Farklar- Bir Mobilya Üreticisi Firmada Vaka Çalışması”. Muhasebe ve Finansman
Dergisi(Nisan), 33-58. Feil, P.,Yook, H.K. ve Kim, W. (2004).''Japaneesetargetcosting: a historicalpersfective'', International
Journal Of Strategic Cost Management, 10-19. Güneş, R. ve Aksu, İ. (2003). “Mamul Yaşam Dönemi Maliyetlemesi”. Süleyman Demirel Üniversitesi
İktisadi ve İdari Bilimler Fakültesi Dergisi, 8(2), 43-61. Gürel, E.,ve Alem, J. (2010). ''Postmodern Bir Durum Komedisi Üzerine İçerik Analizi: Simpsonlar'',
Uluslararası Sosyal Araştırmalar Dergisi, Sayı:3/10, .336. 14 AKIN & ONAT İraz, R.,Adem Ö., Zerenler M., (2007). ''Değer Mühendisliği Uygulamalarının Fonksiyonel Etkinlik
Açısından İşletmelerin Somut ve Soyut Varlıklarına Yönelik Olası Etkileri'', Selçuk Üniversitesi
İktisadi ve İdari Bilimler Fakültesi Sosyal ve Ekonomik Araştırmalar Dergisi,51-68. Örnek, Ş. A. (2003). ''Bir Yönetim Tekniği Olarak Değer Mühendisliği'', Dokuz Eylül Üniversitesi Sosyal
Bilimler Enstitüsü Dergisi, Cilt: 5, Sayı: 2., 213-230. Savaş, O. (2003). ''Tam Zamanında Üretim Sisteminin Gerektirdiği Maliyet Muhasebesinin Temel
Nitelikleri'', Erciyes Üniversitesi İktisadi ve İdari Bilimler Dergisi, Sayı: 20, .203-218, Ocak-
Haziran. 15 15
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https://openalex.org/W3171732767
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Does the medical insurance system really achieved the effect of poverty alleviation for the elderly in China? Characteristics of vulnerable groups and failure links
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Ma et al. BMC Public Health (2020) 20:435
https://doi.org/10.1186/s12889-020-08554-3 Ma et al. BMC Public Health (2020) 20:435
https://doi.org/10.1186/s12889-020-08554-3 Open Access Does the medical insurance system really
achieved the effect of poverty alleviation
for the middle-aged and elderly people in
China? Characteristics of vulnerable groups
and failure links Meiyan Ma1, Ye Li1*, Nianshi Wang1†, Qunhong Wu1*, Linghan Shan1†, Mingli Jiao1†, Xuelian Fu2, Heng Li3,
Tao Sun4, Bin Yi5, Wanxin Tian1, Qi Xia1, Baoguo Shi6, Yanhua Hao1, Hui Yin1, Ning Ning1, Lijun Gao1,
Libo Liang1 and Jiahui Wang1 * Correspondence: liye8459@163.com †Nianshi Wang, Linghan Shan and Mingli Jiao contributed equally to this
work. 1Department of Social Medicine, School of Health Management, Harbin
Medical University, Harbin 150086, Heilongjiang, China
Full list of author information is available at the end of the article 1Department of Social Medicine, School of Health Management, Harbin
Medical University, Harbin 150086, Heilongjiang, China
Full list of author information is available at the end of the article Abstract Background: We examined the physiological, household, and spatial agglomeration characteristics of the health
poverty population in China. We identified weak links that affect the implementation of the medical insurance and
further improve its effectiveness for health poverty alleviation. Methods: A national representative sample from the China Health and Retirement Longitudinal Study (CHARLS)
was analyzed. The WHO recommended method was adopted to calculate catastrophic health expenditure (CHE)
and impoverishment by medical expenses (IME). We created a binary indicator for IME as the outcome variable and
applied the treatment-effect model to analyze the determinants of IME. Results: The incidence of IME was 7.2% of the overall population, compared to 20.3% of the sample households
trapped in CHE. The incidence of IME enrolled in insurance schemes was 7.4% higher than that of uninsured
families (4.8%). Economic level, living area, family size, age of household head, having hospitalized members, and
participating in insurance were statistically significant for the occurrence of IME. (Continued on next page) * Correspondence: liye8459@163.com Background health services,which all contribute to more economic
risks and health poverty. Poverty continues to be a worldwide problem, as of
2015, there are still more than 10% of the world’s popu-
lation lived on less than US$1.90 a day [1, 2]. Measure-
ment of poverty has gradually changed from a single
economic dimension to multiple dimensions integrating
economic, cultural, health, and dignity indicators and
some scholars define poverty as a lack of basic needs,
health, and social capital [3, 4]. People who fall into
healthy poverty often cannot access health services,
which affects their health level, and ultimately their abil-
ity to obtain income. The poverty then worsens and they
may easily become trapped in a cycle of “poverty-health
deterioration-more
poverty”
[5].In
India,
health
deprivation is second only to economic income in terms
of factors leading to high poverty rates [6]. True health
poverty means both the loss of health ability and the
deprivation of social health rights. To reduce the disease burden, medical insurance has
played a certain role in economic protection, China’s
basic medical insurance system consists of three types of
medical insurance scheme: the Medical Insurance for
Urban Employees (MIUE) is designed for cost sharing
between employers and employees, with risk polling
managed at the municipal level. The Medical Insurance
for Urban Residents (MIUR) is for urban residents who
are not covered by MIUE and is co-financed by enrollees
and local government. The New Cooperative Medical
Scheme (NCMS) is designed for rural residents covering
mostly inpatient services and a few outpatient services
[13]. The coverage of the basic medical insurance
schemes has achieved the breath of universal coverage
with participation rate of 97%. However, studies have demonstrated that the actual ef-
fect of this coverage has been offset by the rapid escal-
ation of medical expenses [14]. In 2012, the proportion
of Chinese residents’ Out-of-Pocket (OOP) expenses to
total health expenditure was 35.69%, 2.3 times higher
than the international average (15.28%) [15]. The WHO
(World Health Organization)2017 Global UHC Report
demonstrates that 17.7% of Chinese people spend more
than 10% of their household budget on OOP medical
payments, far higher than OECD countries [16]. The
WHO proposes a reasonable OOP proportion of 15%~
20%, but as of 2017, the ratio in Chinese residents is as
high as 28.77% [17]. (Continued from previous page) Conclusions: The original poverty-promoting policies has not reached the maximum point of convergence with
China’s current demand for health. The overlapped health vulnerabilities exacerbated the risk of poverty among the
elderly and households with high health needs and utilization. In addition, the medical insurance schemes have
proven to be insufficient for protection against economic burden of poor households. So, special health needs, age,
and household capacity to pay should be comprehensively considered while strengthening the connection
between the disease insurance scheme with supplementary insurance. Keywords: Medical insurance, Poverty alleviation, Healthy poverty, Catastrophic health expenditure,
Impoverishment by medical expenses © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,
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data made available in this article, unless otherwise stated in a credit line to the data. Ma et al. BMC Public Health (2020) 20:435 Page 2 of 15 Background Although previous studies have
shown that participating in medical insurance is condu-
cive to reducing the risk of catastrophic health expendi-
tures (CHE) in the family, and it can reduce the basic
economic burden of the family. However, under the
background of the reform of the medical insurance sys-
tem, there are still many people in China who have a
heavy OOP burden. In China,there are still more than
34 million poor people, of which 33% are due to lack of
labor caused by disease, and 12% are due to CHE and
high medical expenses [10].Kumar K found that, the
OOP’s share in total expenditure in China was 15.1%,
much
higher
than
India
(11.5%)
[18].In
addition, Since the beginning of reform and opening up in
1978, China has significantly alleviated its poverty. Be-
tween 1978 and 2018, the poor population decreased
from 770 million to 16.6 million [7].Moreover, the
global population of extreme poverty fell from 1.9 bil-
lion in 1990 to 836 million in 2015,and in this
process, China’s contribution to poverty reduction
exceeded 70% [8]. However, with changes to China’s
population structure and chronic disease-based dis-
ease spectrum, traditional poverty alleviation methods
cannot accurately identify the characteristics of the
poor, causing bottlenecks in poverty alleviation. From
1978 to 1999, China reduced poverty at a rate of
1.5% per year. However, in the following 8 years, this
rate was only approximately 0.26% [9]. In 2017,ap-
proximately 45% of poor people are impoverished due
to
major
illness
and
disabilities
and
260
million
people in China have chronic diseases, causing ap-
proximately 70% of the disease burden [10, 11]. Add-
itionally, the prevalence of chronic disease in the
elderly
is
2.9
times
that
of
the
total
population
[12].The inherent weakness of elderly who are vulner-
able to suffer from chronic diseases, not only lead to
the shortage of labor, but also increase demand for Ma et al. BMC Public Health (2020) 20:435 Page 3 of 15 population having access to health services. Background A study
from
China
has
shown
that,
the
annual
medical
spending of depression has reached to nearly $42.67
per person [27],but the welfare package of the med-
ical insurance only covers 33 kinds of drugs for men-
tal disorders [28].Moreover, in 2018, the actual rate
of reimbursement for NCMS hospitalization was only
50%, far lower than the 75% required by the policy
[29].And the OOP of Chinese accounted for 30%,
which is much higher than the national average of
the WHO member countries (18.6%) [30]. The depth
(welfare package coverage) and height (compensation
level)
of
medical
insurance
are
still
need
to
be
strengthened, so as to effectively play to the economic
protection
role
for
poverty
alleviation
system. In
short,
although
China
has
gradually
implemented
some healthy poverty alleviation plans, there are still
many weak links, such as, lacking precise and quanti-
tative identification mechanism of marginal groups
that are prone to fall into poverty due to illness and
lacking assistance programs for vulnerable groups (the
elderly, severely disabled people, people suffering sud-
den catastrophic medical expense) [31]. How to give
full play to the basic role of the medical insurance
system in precise poverty alleviation policies? It is the
key to achieving health poverty reduction. numerous studies have shown that,high medical ex-
penses increase the risk of poverty. E.g,Ravi et al. esti-
mated that, OOP health expenses increased hidden
poverty’s rate by 7.5 points in India [13, 19, 20]. Especially in China where OOP accounting for a rela-
tively high proportion, we found that,the OOP by resi-
dents are the key factors affecting the occurrence of
impoverishment by medical expense (IME). It can be
seen that, previous poverty alleviation policies concen-
trated on accelerating China’s poverty reduction through
economic construction, education, and human capital
investment to reduce material poverty. The traditional
poverty alleviation policy is no longer suitable for
China’s current poverty-stricken conditions, nor can it
precisely target the most vulnerable people who are poor
due to disease. Improvement in the health of the popula-
tion can reduce the occurrence of poverty [21]. Background Both
China and international organizations have emphasized
that, the health poverty alleviation is of great significance
to guaranteeing the poor population access to basic
medical and health services, and preventing “disease
from poverty” and “return to poverty from disease”, and
has become a promotion key nodes in poverty reduction
[22, 23].So, China must change the way of poverty allevi-
ation only through economic means and make health
poverty the focus. p
y
The introduction of the “Healthy China Project” in
2002, firstly shifted people’s poverty reduction focus
to a healthy perspective, but only gave a framework
for action [24]. With the promulgation of the targeted
poverty alleviation policy in 2016, healthy poverty al-
leviation has been taken as an absolute area of prior-
ity intervention. The accurate identification of health
poverty population contains two approaches: firstly, it
identifies the poor people and household by the na-
tional poverty alleviation standards, and selects “im-
poverishment households by medical expense” and
“health poverty population” from all the poor people
and poor households who set up files according to
the household surveys. Secondly, it identifies health
poverty population by a certain comprehensive stan-
dards, that is, classify “the people who are likely to be
poor due to illness” and “vulnerable groups” into the
health poverty alleviation targets. The main object of
health poverty alleviation is rural poor population. The key scope of health poverty alleviation is poor
areas [25, 26].With the implementation of health pov-
erty
alleviation
policies,
the
government
conducts
series of actions to achieve convergence of basic med-
ical insurance, major disease insurance, medical assist-
ance and emergency medical assistance. However, as
a basic institutional means of health poverty allevi-
ation, the medical insurance system has only achieved
population width coverage with almost 95% of the Based on the perspective of healthy poverty, we adopt
the WHO recommended method to verify the health-
reducing effect of the health care system, that is, whether
the medical insurance system reduces the risk of IME
among the middle-aged and elderly people. In addition,
we also analyze the physiological, household, and spatial
agglomeration
characteristics
of
the
health
poverty
population and scientifically identify groups at high risk
of health poverty. It is important to understand whether
medical insurance system increases the accessibility and
affordability of health care, which provides good evi-
dence for further ways to alleviate health poverty. Calculation method for CHE and impoverishment by
medical expense (IME) We adopted the WHO recommended method to calcu-
late CHE and IME. CHE was defined as an OOP pay-
ment for health care equaling or exceeding 40% of a
household’s capacity to pay [32]. IME was defined as
consumption expenditure equal to or higher than house-
hold subsistence expenditure but lower than the subsist-
ence expenditure (SE) net of OOP health payments. The
key expenditure indicators involved in the calculation
process are as follows: In
order
to
identify
good
instruments,
we
first
reviewed the existing literatures. We identified two po-
tential instruments: the self-assessment health status and
the average community participation rate of medical
health
insurance schemes. Generally
speaking,
self-
reported groups with poor health (whether poor or rich)
are more likely to choose to participate in medical insur-
ance to protect their health [36].In additional, the com-
munity participation rate of basic medical insurance for
residents has an important impact on the willingness of
households to participate in the insurance. The people
living in the same community has certain common char-
acteristics such as the household economic level and
health management awareness, which all contribute to
their participation to medical insurance. Moreover, some
of the basic medical insurance schemes in China are
community-based units. For example, NCMS and MIUR
use the community as a promotion unit, and the village Data collection and quality control Data were collected through questionnaires comprising
the following eight parts: A. Household registration
form, B. Demographic background, C. Family informa-
tion, D. Health status and function, E. Health care and
insurance, F. Work, retirement, and pension, G/H. In-
come, expenses, and assets. Face-to-face household in-
terviews
were
conducted
by
qualified
investigators. Quality control was implemented by supervisors and in-
cluded GPS comparison, data verification, recording
verification, and telephone verification. Based on the re-
search needs of IME, we largely focused on demographic
information, health status, medical insurance, income,
and expenditure. Treatment effect model and instrumental variables The relationship between participation in medical insur-
ance system and IME is characterized by a joint causal-
ity. Participation in medical insurance system exists
hidden selection bias, unobserved characteristics such as
self-selection of participation, self-assessment health sta-
tus are correlated with initial treatment choice. To ad-
dress the bias resulting from hidden selection bias and
joint causality, we applied an instrumental variable (IV)
approach known as the treatment-effect model [33, 34]. Instrument indicators We applied the treatment-effect model to instrument
the indicators of participation in medical insurance sys-
tem, that is to say, participation in medical insurance
system is endogenous. We need to find instrument vari-
ables which are related with endogenous predictors (par-
ticipation in medical insurance system) but not related
to the error term of outcome variables (IME) [35].For
the further identify whether endogenous variables exist-
ing in the regression equation, we use DWH (Durbin-
Wu-Hausman) test to examines whether the endogenous
predictor is truly endogenous (The heterogeneity test re-
sult shows that, P = 0.0224 < 0.05, so we use the DWH
test). And the DWH test,shows, P = 0.0450 < 0.05,The re-
sults confirm that there is indeed endogeneity in our
model. Data source and sampling method Data source and sampling method
The data used to calculate the rates of IME and CHE
were obtained from the 2015 China Health and Retire-
ment Longitudinal Study (CHARLS) database, which is
designed to collect microscopic information from middle
aged and elderly people over the age of 45. The filter
questionnaire was used to determine whether the candi-
date has 45 or older respondents. If there were several
households living in the sample address and more than
one household had age-appropriate respondents, the sys-
tem would randomly select one household to conduct
the survey, and then list all the household members and
their age. If there was only one age-appropriate respond-
ent, he/she directly became the "main respondent". If Page 4 of 15 Page 4 of 15 Ma et al. BMC Public Health (2020) 20:435 Ma et al. BMC Public Health (2020) 20:435 there were multiple age-appropriate respondents, one
was randomly selected as the "main respondent". The
survey used four-stage stratified cluster sampling to se-
lect eligible individuals. First, all the counties in 28 prov-
inces countrywide were sorted according to per capita
GDP, and 150 counties (cities) were then randomly se-
lected by PPS (probabilities proportionate to size as
measured by population). Second, three villages or com-
munities in each county were randomly selected and a
data-set of 450 samples was generated. Then,80 house-
holds in each village or community were randomly se-
lected. 21,101 samples were included in the survey. Finally, A total of 19,144 samples (9167 households)
were finally obtained after cleaning the incomplete data
and missing values. expenditure as a share of household consumption ex-
penditure fell between the 45th and 55th percentiles of
the entire sample, was treated as the poverty line. The
SE of each household was calculated as the poverty line
multiplied by the standard household size. A household’s
capacity to pay (CTP) was defined as non-subsistence
spending of a household as a share of total household
consumption expenditure. Outcome variable and covariates We created a binary indicator for IME as the outcome
variable (1 = occurrence, 0 = no occurrence). Based on a
large literature review, variables on social-demographic
characteristic (age, gender, occupation, marital status,
education level of household head, family size, presence
of over 65-year-olds) and health service needs and
utilization (having hospitalization members, chronic pa-
tients, hospitalization rate, and non-admission rate (de-
fined
as
the
percentage
of
patients
requiring
hospitalization but who were not hospitalized) were used
as control variables. According to the WHO classifica-
tion criteria, 14 types of chronic disease in the question-
naire were reclassified into six categories: cardiovascular,
respiratory, mental, cancer, diabetes, and other chronic
diseases. [2] High health utilization but low reimbursement. Households
with
mental
illness
had
a
higher
hospitalization rate (17.7%) but a hospitalization reim-
bursement ratio (54.9%) that was lower by nearly 8%
than cancer households (62.9%). Patients who partici-
pated in MIUR demonstrated a similar pattern. Out-of-pocket health expenditure (OOP) The payments made by households for their health ser-
vices without third-party compensation; Household con-
sumption expenditure (exp) comprises both monetary
and in-kind payment on all goods and services, and the
money value of the consumption of home-made prod-
ucts. Household subsistence expenditure (SE) was calcu-
lated
using
food
expenditure
as
a
share
of
total
household
consumption
expenditure. The
weighted
average food expenditure of a household, whose food Ma et al. BMC Public Health (2020) 20:435 Page 5 of 15 (61.1%) of elderly members had chronic diseases. Finally,
92.6% had medical insurance (Table 2). cadre mobilize the villagers to participate in the medical
insurance schemes, which makes the high correlation
between the community participate rate and the house-
hold participation willingness [37, 38]. Region level: poverty, catastrophic health expenditure,
impoverishment by medical expense in different
provinces p
The poverty rate ranges from 4~45%, with the highest
rate in Guizhou (43.75%), which is approximately 10
times that in Tianjin (4.47%). As shown in the figure,
poverty rates are generally higher in central (21–
33%)and western provinces (22–29%)than in eastern
provinces (18–25%).To verify the disease burden of
middle-aged and older over 45, we also measured the in-
cidence of IME and CHE in different provinces. Among
them, Neimenggu (10.41%), Anhui (10.38%), Shandong
(9.63%), Sichuan (8.48%), and Chongqing (8.09%) were
the provinces most burdened by IME; In terms of CHE
rate,
the
top
five
incidence
rates
are:
Neimenggu
(28.81%), Chongqing (25.73%), Anhui (24.36%), Shan-
dong (24.26%), Qinghai (23.68%) (Fig. 1). The treatment-effect model consists of a two-stage re-
gression. In the first stage, we regress the outcome vari-
able on the covariates. Based on the results of the first
phase, we add instrument variables and perform a quad-
ratic regression on the outcome variable. Health-care needs and service utilization Good instrument should satisfy two main criteria
known as relevance and validity criteria, that is to say,
good instruments would be correlated with the endogen-
ous variable (relevance criteria) but not to be correlated
with the error terms in the model of the outcome vari-
able (validity criteria). The overall monthly prevalence of the surveyed sample
was 11.8%, chronic disease was 60.7%, hospitalization
rate 13.5%, and hospitalization reimbursement ratio
49.7%. These indicators demonstrate different trends for
different populations as follows (Table 3): p p
(
)
[1] The population with high level of health service de-
mand but low utilization of health services and low re-
imbursement
rate. For
example,
respiratory
disease
members had a high demand for health services (preva-
lence
rate
17.4%)
but
low
level
of
health
service
utilization (no-admission rate 7.6%) and an adequate re-
imbursement level (53.3%). Additionally, the prevalence
of families participating in integrated insurance (16.8%)
and the prevalence of chronic diseases (57.7%) were sig-
nificantly higher than for the MIUE (11.8 and 57%), but
the hospitalization reimbursement ratio (42.2%) was
23.6% lower than for the MIUE (65.8%). People from dif-
ferent
economic
groups
demonstrated
a
similar
phenomenon. We use the Over-identification Test and F value in
GMM regression to check the validity and relevance
[39, 40]. In addition, in order to further investigate
the weak tool variable problem, we also perform re-
dundancy testing. The results show that community
participation rate becomes our ultimate effective in-
strumental variable. The results regarding the validity
and relevance are presented in Table 1. Basic information The total sample comprised 9167 households and 19,144
individuals, of which 52.5% of household heads were
male, and up to 65.8% of the middle-aged and elderly
people were < 64 years. Most had primary and junior
high school education level (56.6%), and more than half Table 1 Validity test and correlation test presented of people
Instrumental variable
DWH Test
Over-identification Test
F Test
Redundancy Test
Community participation rate
chi2 [1]=4.0201
(p = 0.0450 < 0.05)*
chi2 [1]=0.1053
(p = 0.7455 > 0.05)*
F = 35.9921 > 10
Chi-sq [1] =0.0000*
Self-assessment health
Chi-sq [1] =0.6096 Table 1 Validity test and correlation test presented of people
Instrumental variable
DWH Test
Over-identification Test
F Test
Redundancy Test
Community participation rate
chi2 [1]=4.0201
(p = 0.0450 < 0.05)*
chi2 [1]=0.1053
(p = 0.7455 > 0.05)*
F = 35.9921 > 10
Chi-sq [1] =0.0000*
Self-assessment health
Chi-sq [1] =0.6096 Ma et al. Basic information BMC Public Health (2020) 20:435 Page 7 of 15 Page 7 of 15 Table 3 Health-care needs and service utilization
Prevalence
(%)
Chronic disease
prevalence (%)
Hospitalization
rate (%)
Non-admission
rate (%)
Hospitalization
reimbursement ratio (%)
Family size
1
15.9
63.2
13.4
5.9
39.3
2–3
14.0
61.8
13.8
5.9
49.9
> 3
14.2
58.6
12.9
5.5
55.1
Hospitalization
members
Yes
22.5
65.7
100.0
/
49.7
No
13.1
60.0
0.0
4.8
/
Disabled members
Yes
20.1
63.7
15.6
9.0
56
No
13.9
59.8
13.4
5.5
50.9
Chronic disease
Respiratory diseases
17.4
/
16.7
7.6
53.3
Cardiovascular
diseases
13.9
/
14.9
6.4
50.2
Mental illness
14.8
/
17.7
6.3
54.9
Diabetes
11.5
/
15.2
5.9
62.9
Cancer
18.5
/
9.7
6.2
57.4
Others
15.9
/
14.8
6.8
47.5
Medical insurance
schemes
MIUE
11.8
57%
16.4
4.3
65.8
MIUR
14.6
60.1
16
6.5
58.7
NCMS
14.6
61.8
13
6.1
42.2
Integrated insurance
16.8
57.7
12.5
4.4
34.1
Others and no have
insurance
12.6
58.1
15.7
5.7
48.5
Economic quintile
Lowest
14.5
61.0
13.4
5.9
49.9
2
14.4
65.0
12.6
6.2
42
3
14.1
61.0
12.5
5.5
59.6
4
14.6
59.9
13.4
5.8
47.1
Highest
13.4
56.7
15.8
5.5
50.5
Overall
11.8
60.7
13.5
5.8
49.7 (10.1%) was 2.2 times that of the 45–54 age group. Moreover, MIUE had the least risk of economic bur-
den, and the incidence of IME was 3.5%, only half
the total sample population (7.2%). The proportion of
OOP to household payment capacity was 13.9%, sig-
nificantly
lower
than
MIUR
(16.4%)
and
NCMS
(19.0%). The incidence of IME in NCMS (7.9%) was
equivalent to that of the integrated medical insurance
family, but the burden of health expenditure (19.0%)
was 2.1 times more. Meanwhile, except for the sub-
poverty group, the incidence of IME decreased with
the improvement of the economic quintile. The poor-
est group shared the highest IME rate (approx. 8.0%),
which was 2.7 percentage points higher than the rich-
est group (5.3%). was higher than that in Guangxi and Sichuan. Moreover,
the poverty rate in Guizhou (43.75%) was much higher
than in Neimenggu (15.25%). However, after paying for
health services, the risk of IME (10.41%) was twice as
high as in Guizhou (4.17%). Basic information BMC Public Health (2020) 20:435 Page 6 of 15 It is worth noting that, in the case of a similar level of
poverty, residents’ utilization of health services caused
significant differences in the incidence of CHE and IME:
the levels of poverty in Chongqing, Guangxi, and Si-
chuan were originally equal, but after paying medical ex-
penses, the incidence of CHE in Chongqing (25.74%)
Table 2 Sample characteristics
Variables
Variable value
Percentage(%)
Mean
SD
Outcome variable
0 = No
92.8
0.07
0.259
1 = Yes
7.2
Participate in medical insurance scheme
0 = No
7.4
0.971
0.001
1 = Yes
92.6
Gender of household head
0 = Female
47.5
1.475
0.499
1 = Male
52.5
Marital status of household head
0 = Others
14.2
0.858
0.348
1 = Married
85.7
Education level of household head
1 = Illiteracy
18.9
1.923
0.590
2 = Elementary to junior high
56.6
3 = High school and above
12.2
Family size
1 = 1
61.3
1.447
0.607
2 = 2–3
32.6
3 = More than 3 people
6.1
Age of household head
1 = 45–54
33.3
2.080
0.968
2 = 55–64
32.5
3 = 65–74
23.2
4= > 75
9.0
Having members over 65 years old
0 = No
65.8
1.671
0.469
1 = Yes
32.3
Occupation of household head
1 = Agriculture
27.4
1.551
0.807
2 = Employed
10.2
3 = Self employed
4.7
4 = Free
1.2
5 = Others
4.6
6 = Retirement
9.5
7 = Unemployed
7.3
Having hospitalization members
0 = No
86.2
0.131
0.337
1 = Yes
13.0
Having NCD members
0 = No
38.9
0.611
0.487
1 = Yes
61.1
Household consumption per capita quintile
Lowest
20.6
2.963
1.410
2
20.4
3
20.1
4
19.8
Highest
19.1
Region
1 = Eastern
34.9
1.874
0.746
2 = Middle
42.7
3 = Western
22.3
Urban and rural
0 = Rural
79.0
0.209
0.406
1 = Urban
20.8 1.671
0.469 It is worth noting that, in the case of a similar level of
poverty, residents’ utilization of health services caused
significant differences in the incidence of CHE and IME: the levels of poverty in Chongqing, Guangxi, and Si-
chuan were originally equal, but after paying medical ex-
penses, the incidence of CHE in Chongqing (25.74%) the levels of poverty in Chongqing, Guangxi, and Si-
chuan were originally equal, but after paying medical ex-
penses, the incidence of CHE in Chongqing (25.74%) Ma et al. Family level: poverty, catastrophic health expenditures
and related indicators The muti-dimensional analysis on the incidence of
CHE and IME in elder households demonstrated that
the IME occurred in 7.2% of the overall population
compared to 20.3% of sample households trapped in
CHE. OOP accounted for 18.72% of family payment
capacity
and
households
enrolled
in
insurance
schemes were more likely to suffer from IME (7.4%)
and CHE (20.5%) than uninsured households (4.8 and
17.9%) (Table 4). Treatment-effect model analysis As the age of household head increased, the risk of
households being trapped in IME also increased. The
incidence of IME among the elderly > 75 years old Treatment-effect model analysis
The model demonstrated that economic levels, living
area,
family
size,
age
of
household
head,
having Ma et al. BMC Public Health (2020) 20:435 Page 8 of 15 Fig. 1 Poverty, catastrophic health expenditure, impoverishment by medical expense Fig. 1 Poverty, catastrophic health expenditure, impoverishment by medical expense Medical insurance level:factors affecting IME by different
medical insurance systems hospitalization members, and participating in insurance
were statistically significant for IME (P < 0.05) (Table 5). All the results indicated that people with medical insur-
ance were more likely to suffer from IME. To identify
the key bottlenecks, we conducted a series of calcula-
tions (seen in Fig. 2): Economic level and family size with the middle-
aged and elderly people demonstrated a negative
correlation
with
incidence
of
IME:
the
family’s
economic income rises by higher level, the risk of
IME
was
0.6%age
points
reduction
(a
similar
phenomenon
occurred
family
size);
age
was
positively correlated with rate of IME, that is, as the
age of the head of household increased, the risk of
family impoverishment by medical expense increased
by
1.8
percentage
points. Households
with
hospitalization members increased the risk of IME
and the risk of poverty was 2.3 percentage points,
higher
than
for
families
without
hospitalization
members. The model results demonstrated that liv-
ing in urban areas reduced the probability of IME
more than living in rural areas. However, participa-
tion in medical insurance was more likely to cause
IME, and increased the risk of being trapped in
IME by 16.5 percentage points. Households
with
mental
illness,
cancer,
diabetes,
hospitalization, and > 65-year-old members as well as
those living in rural areas and of a lower economic level
were at high risk of IME. For households enrolled in
MIUR, NCMS, and households without medical insur-
ance, having members with mental illness was the pri-
mary factor in impoverishment, with IME at 25.0, 11.89,
and 11.11% respectively. The top five influencing factors
of integrated medical insurance families demonstrated
the highest rate of IME, generally above 10.64%. Having
a household head > 75 years old was the first influencing
factor (IME 22.22%), at 15 percentage points higher than
the overall population (7.23%). Discussion Since the 1990s, health poverty has been officially
adopted as an important agenda worldwide [41].The
Sustainable Development Goals (SDGs) clearly demon-
strate that health protection policies should be devel-
oped for the most vulnerable populations [42].In 2016,
“Guiding Opinions on Implementing a Healthy Poverty
Alleviation Project” clearly stated that the Health Pov-
erty Alleviation Project was key to preventing IME [43]. The rate of CHE for middle-aged elderly people in
China (20.3%) was higher than in other low and middle
income countries. Amaya-Lara found that,9.6% of Co-
lombian
households
had
catastrophic
expenditure
[44].In India, the incidence of catastrophic health ex-
penditure for older people over 65 was only 7%, much
lower than in China [45].And in Iran,the rate of cata-
strophic health expenditure headcount ratio varied from
0.5 to 14.3% and from 0.48 to 13.27% for rural and
urban households, respectively [46]. The incidences of Since the 1990s, health poverty has been officially
adopted as an important agenda worldwide [41].The
Sustainable Development Goals (SDGs) clearly demon-
strate that health protection policies should be devel-
oped for the most vulnerable populations [42].In 2016,
“Guiding Opinions on Implementing a Healthy Poverty
Alleviation Project” clearly stated that the Health Pov-
erty Alleviation Project was key to preventing IME [43]. Against the background of population aging, the
prevalence of chronic diseases, hospitalization rate, and
disability due to illness increase the health needs of the
elderly. However, the elderly is disadvantaged in the
process of obtaining resources. Therefore, it is crucial to
determine the risk characteristics of this important
group with physiological, social, and health vulnerability
and to maximize the effectiveness of the dividend effect
of welfare policies. Through our comprehensive analysis,
we find that the vulnerable middle-aged and elderly
people population in China is characterized by the fol-
lowing characteristics: The rate of CHE for middle-aged elderly people in
China (20.3%) was higher than in other low and middle
income countries. Amaya-Lara found that,9.6% of Co-
lombian
households
had
catastrophic
expenditure
[44].In India, the incidence of catastrophic health ex-
penditure for older people over 65 was only 7%, much
lower than in China [45].And in Iran,the rate of cata-
strophic health expenditure headcount ratio varied from
0.5 to 14.3% and from 0.48 to 13.27% for rural and
urban households, respectively [46]. The incidences of Treatment-effect model analysis For people enrolled in
NCMS, the rate of the top five influencing factors
ranged between 10 and 12%, second only to integrated Ma et al. BMC Public Health (2020) 20:435 Page 9 of 15 Page 9 of 15 Table 4 Poverty, CHE, and IME in different households Table 4 Poverty, CHE, and IME in different households
Incidence of
IME (%)
Incidence of
CHE (%)
OOP/CTP
(%)
Out-of-pocket
(yuan)
Capacity to pay
(yuan)
Family size
1
8.1
23.0
21.1
336.7
1590.6
2–3
5.7
16.1
15.3
397.3
2588.1
> 3
6.2
15.8
14.8
348.1
2345
Age of household head
45–54
4.4
12.4
12.6
310.1
2460.5
55–64
7.4
20.6
19.4
372.2
1910.6
3 = 65–74
9.7
27.0
25.6
401.2
1564.1
> 75
10.1
32.0
26.8
375.8
1399.2
Hospitalization members
Yes
9.9
29.3
31.5
640.2
2030.0
No
6.8
19.0
16.2
315.6
1948.0
Medical insurance
MIUE
3.5
15.7
13.9
438.4
3133.2
MIUR
5.6
15.8
16.4
377.1
2291.0
NCMS
7.9
21.4
19.0
320.0
1676.6
Integrated insurance
8.5
18.2
7.8
210.7
2674.5
Others and no have
insurance
4.8
17.9
22.7
601.0
2638.7
Household consumption per
capita quintile
Lowest
8.0
19.4
19.0
315.6
1660.7
2
8.3
22.6
21.5
382.5
1773.7
3
7.6
20.9
19.4
347.1
1785.5
4
6.6
19.2
17.3
331.1
1913.7
Highest
5.3
19.4
15.1
412.3
2722.3
Participate in insurance
Yes
7.4
20.5
17.7
331.9
1873.2
No
4.8
17.9
22.7
601.0
2638.7
Overall
/
7.2
20.3
18.2
357.1
1961.7 IME (7.4%) and CHE (20.5%) participating in medical in-
surance were 2.6% higher than those of uninsured
households (4.8 and 17.9%). Our research also found
that families with mental illness, elderly people > 75, in-
patients, disabled, and chronic patients have a higher
risk of falling into poverty due to medical expenses. medical insurance. Cancer was the only important factor
affecting the IME of NCMS, with the incidence as high
as 11.77%. Regional level: inter-regional macroeconomics is not the
main driver for reducing the risk of health poverty When the original poverty rate is at the same level, there
is a tendency for IME to occur in the central and Ma et al. BMC Public Health (2020) 20:435 Page 10 of 15 Table 5 Results of the Treatment-effect model
Coef. Std. Err. z
P > |z|
[95% Conf Interval]
IME
Gender of household head
0.0147
0.0078
1.87
0.061
−0.0006
0.0300
Education level of household head
0.0118
0.0070
1.69
0.091
−0.0018
0.0256
Marital status of household head
−0.0039
0.0108
−0.37
0.714
−0.0251
0.0172
Household consumption per capita quintile
−0.0060
0.0028
−2.13
0.033
−0.0116
−0.0004
Region
0.0044
0.0052
0.85
0.395
−0.0057
0.0146
Urban and rural
−0.0280
0.0102
−2.73
0.006
−.0481
−0.0079
Type of chronic disease
0.0528
0.0330
1.60
0.110
−.0119
0.1175
Occupation of household head
0.0002
0.0018
0.14
0.887
−0.0034
0.0039
Family size
−0.0159
0.0064
−2.48
0.013
−0.0284
−0.0033
Age of household head
0.0183
0.0039
4.65
0.000
0.0106
0.0261
Having hospitalization members
0.0230
0.0113
2.04
0.041
0.0009
0.0452
Cardiovascular diseases
−0.0453
0.0337
−1.34
0.180
−0.1115
0.0209
Chronic respiratory disease
−0.0482
0.0321
−1.50
0.134
−0.1113
0.0147
Other chronic diseases
−0.0589
0.0334
−1.76
0.078
−0.1245
0.0066
Cancer
−0.0375
0.0496
−0.76
0.450
−0.1348
0.0597
Diabetes
−0.0583
0.0315
−1.85
0.064
−0.1201
0.0034
Mental illness
0.0099
0.0366
0.27
0.787
−0.0619
0.0817
Having disabled members
0.0184
0.0215
0.85
0.393
−0.0238
0.0607
Participate in insurance
0.1655
0.0831
1.99
0.047
0.0025
0.3285 regions, but there are still differences in poverty caused
by the use of health services. western regions due to the purchase of health services. The incidence of IME is significantly higher in the cen-
tral and western regions than in the east, which is con-
sistent
with
the
distribution
of
poverty-stricken
populations in China. Guo also demonstrated significant
regional differences in the rural poverty-stricken groups
and gradually gather in the central and western regions
over time in China between 1978 and 2014 [47]. Therefore, while paying attention to economic devel-
opment and driving poverty alleviation, we should accur-
ately target the characteristics of the poor, capture the
poverty-reducing characteristics at the individual, family,
and institutional levels, prioritize vulnerability, and im-
prove the targeting accuracy of poverty alleviation pol-
icies for the poor [49]. Inter-regional macroeconomics is not the main driver
for reducing the risk of health poverty. For example,
Shaanxi and Jiangsu belong to different regions but have
the same poverty rate. Regional level: inter-regional macroeconomics is not the
main driver for reducing the risk of health poverty In 2015, Shaanxi’s GDP was
180.218 billion-yuan, accounting for only a quarter of
Jiangsu (70,116.38 billion yuan), but the incidence of
IME (5.26%) was lower than in Jiangsu (6.13%). Individual level: the superposition of multiple health
vulnerabilities exacerbated the risk of poverty among
middle-aged and elderly people High health service needs and utilization households
with chronic disease and disability members, but with
lower reimbursement ratio increase the difficulty for res-
idents to obtain health rights. For example, the preva-
lence and hospitalization rates of respiratory disease
patients were 17.4 and 16.7%, respectively, but the OOP
proportion was as high as 46.7%, nearly 10% higher than
for diabetic patients (37.1%). Callander calculated that
Australian households with chronic disease (COPD) pay
109% more for care than those without health problems
[50].Middle-aged and elderly patients with chronic dis-
eases will increase the demand and utilization of health
services as the outpatient visit rate and hospitalization
rate increase. If there is a low actual reimbursement Thus, the economic development-centered approach
to regional poverty alleviation has not reached the max-
imum point of convergence with China’s current de-
mand for health. Basu found that, in India, when poverty
develops to a certain stage, the poverty reduction effect
incurred by the increase in economic income would re-
duce under the influence of other specific factors (1951–
1991) [48]. Our data can better explain that residents’
physiological characteristics and medical insurance im-
pact a family’s IME and CHE more than the overall
macroeconomic level. The indiscriminate poverty allevi-
ation policy has reduced poverty in provinces and Page 11 of 15 Ma et al. BMC Public Health (2020) 20:435 Fig. 2 Factors affecting IME by different medical insurance system ratio and a high proportion of OOP, it will inevitably
lead to a catastrophic increase in health expenditure risk. This shows that, people with chronic diseases or low so-
cial status have a heavy burden of disease due to low co-
payments
or
no
compensation
[51–53]. Therefore,
higher cost sharing is important for reducing the burden
on residents. The age of the head of household affects the incidence
of IME. As the head of the household grows,the risk of
IME increases by 1.83 percentage point. Thus, aging is
one of the key factors hindering poverty reduction. In-
come inequality and reduced autonomy have increased
the sensitivity of older groups to health rights and health
equity [54]. According to the Global Burden of Disease
Study, 23% of the global burden of disease occurs in
older people, and chronic non-communicable diseases
have a major impact on this burden [55]. Individual level: the superposition of multiple health
vulnerabilities exacerbated the risk of poverty among
middle-aged and elderly people Although the health vulnerability of the middle-aged
and elderly people is often regarded as an intrinsic prop-
erty, individuals can reduce their exposure to health
rights by providing corresponding social support. More
importantly, such support reduces barriers to entry for
health services for the elderly, including raising the gov-
ernment’s
high
co-payments
and
deductibles,
and
strengthening the policy inclination of the elderly [56]. Family level: families with low risk of mutual aid Family level: families with low risk of mutual aid
Family size is negatively correlated with incidence of
IME,as the member of family grows, the risk of IME is
reduced by 1.5 percentage points. Connie noted that
family size is related to the number of individuals enter-
ing the labor market, and the two are negatively corre-
lated. Once the family faces unemployment, it prolongs
the poverty time and poverty rate [57]. Medical insurance is less effective for low-income
families. The proportion of OOP in poor households
accounted for 21.5% of the household’s ability, which
is 6% higher than the wealthiest households (15.1%). Furthermore, for every 1% increase in household in-
come, the incidence of IME is reduced by 0.6%. Sujin
Kim also found that after the implementation of med-
ical insurance, the coefficient of change of the CHE
rate in high-income groups (1.424) exceeded that in
low-income groups (0.544); that is, the policy protec-
tion of low-income groups was less than that of high-
income groups [58]. The rural poor remain the key target group for poverty
alleviation. The incidence of IME in rural areas (7.72%)
is higher than in urban areas (5.39%). Living in the city
reduces the rate of IME by 2.8%. The hospitalized reim-
bursement ratio of MIUE is 68.07%, while that of the
NCMS is only 37.1%, accounting for only half of the
MIUR (25.0%). Urban households with higher income
levels are better able to withstand the burden of health Fig. 2 Factors affecting IME by different medical insurance systems Page 12 of 15 Ma et al. BMC Public Health (2020) 20:435 expenditures. Ye Li used China’s fourth health service
survey data to calculate that the CHE of MIUE and
MIUR were 9.4 and 8.5% respectively, far lower than the
NCMS (14.8%) [59]. transportation expenses, nursing expenses, lost time, and
preventive health care expenses are not within the scope
of reimbursement, which greatly increases the house-
hold’s disease burden. The difference between the type and internal design of the
medical insurance leads to a certain gap between different
income groups. There is a lack of policy inclination for low-
income groups, while high income groups have a more
advantageous compensation level by medical insurance
First, the difference between the medical insurances
leads to different risks of IME. Medical insurance level:the design of medical insurance
lacks policy inclination for special populations Participating in medical insurance increased the risk of
IME by 16.5 percentage points. The protective effect of
the medical insurance system has been offset by the
rapid rise in medical expenses,health-care needs and ser-
vice utilization. It can be seen that the medical insurance
system has not played a full protective role and is likely
to increase the medical burden,other scholars have
reached similar conclusions. Some scholars have found
that under the 30% threshold for catastrophic medical
expenditures, rural residents participating in basic med-
ical insurance will increase the risk of catastrophic med-
ical expenditures for families by 31.8% [60]. Wagstaff
et al. found that,insurance significantly increases the
risks of catastrophic expenditures (at the 10% threshold-
s)by 42.2% [61].To explore the failure of medical insur-
ance, we analyzed the top five factors affecting different
medical insurances and found that chronic patients, in-
patients, disabled people, and people over 65 are the key
populations in need of poverty alleviation. The specific
reasons for failure are as follows: Additionally, the internal system design of medical in-
surance lacks economic support for low-income families
and protects the rich more. In our study, the sub-poor
group had a hospitalization reimbursement ratio of only
42%,
lower
than
the
highest
at
50.5%. A
similar
phenomenon occurred in India. With only government
expenditure, the poorest households receive only 10% of
medical care, while the wealthiest families receive up to
33% of social subsidies [65]. The medical insurance does not impose policy inclinations
on the middle-aged and elderly people groups with high
health service demand and utilization, but only reduces the
access standards of this group According to our data, the overall hospitalization reim-
bursement ratio for the elderly over 45 years old is
49.7%, which is lower than the. European Commission
(EU)‘s standard for the reimbursement of major illnesses
of not less than 60% [62]. In the case of cancer patients,
the hospitalization reimbursement ratio for cancer pa-
tients is only 57.4%. Goss calculated the cancer costs in
different countries and found that the proportion of
household expenses for patients in the US was only
20.9%, while in China it was as high as 78.8% [63]. Family level: families with low risk of mutual aid According to our data,
the NCMS has a high incidence of IME, reaching 7.9%,
approximately 2.42 times that of MIUE. Although med-
ical insurance integration has been implemented in some
places to alleviate the huge differences between groups,
the incidence of IME is the highest (8.5%), at 0.6 percent
higher than the NCMS. Therefore, it has not been pos-
sible for residents to achieve equal access to healthcare
services. Su, Min reached the same conclusion [64]. The
fundamental reason for this lack of equality is the low fi-
nancing level, combined with a reimbursement package
that does not consider the traffic, economic level, and
family-mutual aid capabilities. Medical insurance level:the design of medical insurance
lacks policy inclination for special populations Be-
cause it is difficult to design access mechanisms for
major diseases that cover all chronically ill patients with
large medical expenses, MIUE and MIUR with higher in-
come levels are also suffering from IME, and poverty is
as high as 25%. Simultaneously, the difference in reim-
bursement ratio for chronic diseases caused by medical
insurance introduces risks within the system, which
makes the reimbursement of mental illness patients
(53.9%) of MIUE nearly 28.4% lower than those with
low-grade diabetes (81.5%). Some chronic drugs are not
included in the reimbursement range, which increases
patients’
OOP
expenses. Meanwhile,
the
patient’s Funding This work was supported by the National Natural Science Foundation
(71874045,71403073,71333003,71804036),China Postdoctoral Science
Foundation (2016 M590296),Heilongjiang Postdoctoral Foundation (LBH-
Z14166,LBH-Z17156) and Heilongjiang Provincial Health and Family Planning
Commission Foundation (2014–427). The funding body had no role in the
design of the study and collection, analysis,and interpretation of data and in
writing the manuscript. Competing interests
h
h
d
l
h The authors declare that they have no competing interests. References
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China Hospital Development institute of Shanghai Jiao Tong University,
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Purushotham A, Sullivan R, Badwe R, Banavali SD, Nair R, Kumar L, Parikh P,
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published maps and institutional affiliations.
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DENGUE UMA DOENÇA EMERGENTE NO ESTADO DO AMAZONAS
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DENGUE UMA DOENÇA EMERGENTE NO ESTADO
DO AMAZONAS DENGUE UMA DOENÇA EMERGENTE NO ESTADO DO AMAZONAS Cínthia Maria Teixeira Sampaio
Mestre do Programa de Pós-graduação em Geografia - UFAM
Universidade Federal do Amazonas
cinthia-sampaio2011@hotmail.com Adoréa Rebello da Cunha Albuquerque
Professora Doutora do Programa de Pós-graduação em Geografia - UFAM
Universidade Federal do Amazonas
adorea27@yahoo.com.br Adoréa Rebello da Cunha Albuquerque
Professora Doutora do Programa de Pós-graduação em Geografia - UFAM
Universidade Federal do Amazonas
adorea27@yahoo.com.br RESUMO: A dengue é classificada como uma doença emergente já havia sido controlada e ressurge,
o Município de São Gabriel da Cachoeira apresentou maior prevalência de doença (700,16), Manaus
(657,15), Humaitá (539,18). Compreender o processo do aumento da dengue no contexto das relações
socioambientais. A metodologia sistematização de dados disponível no DATASUS, na FVS – AM e no
Sistema de Informações de Agravos de Notificação – SINAN, utilizou-se a ferramenta QGIS 2.18. O
estudo foi realizado através do número de internações hospitalares por dengue no período de 2007 a
2012, para o cálculo da prevalência utilizou-se o número de casos, divididos pela população de
determinada área geográfica por cada 100.000 habitantes. Os resultados do aumento da dengue em
decorrência do saneamento básico eficaz que não atende a demanda dos domicílios. Palavras-chave: emergente, saneamento, ambiente. Palavras-chave: emergente, saneamento, ambiente. INTRODUÇÃO Descreve Luna (2002, p.231), postula-se que o conceito – doenças emergentes e
reemergentes – emergem frente às limitações do paradigma hegemônico da teoria da
transição Epidemiológica para explicar um novo quadro de morbimortalidade,
especialmente à emergência da epidemia de HIV/AIDS nos países centrais. A
Epidemiologia segue em paralelo à teoria da transição Demográfica, enfatiza-se que
as doenças degenerativas substituiriam gradativamente às doenças emergentes e
com o ressurgimento das epidemias, os países desenvolvidos e os países em
desenvolvimento se inserem no processo saúde-doença. Segundo a FVS – AM (Fundação de Vigilância Sanitária do Amazonas, 2016, p.1), a
presença do Aedes aegypti na cidade de Manaus foi detectada, a partir de Novembro
de 1996 e a do Aedes albopictus em Setembro de 1997. Portanto houve a migração
do mosquito transmissor para outros Municípios do Estado do Amazonas. Em
Fevereiro de 1998 foram registrados os primeiros casos autóctones de DENV-1 na
Capital do Estado, dando início à epidemia. No entanto, a dengue se manifestou de
forma endêmica com o registro de, pelo menos, quatro epidemias nos anos de 1998,
2001, 2011 e 2013. (ISSN 2237 - 1419) 192 DENGUE UMA DOENÇA EMERGENTE NO ESTADO
DO AMAZONAS MATERIAIS E MÉTODOS Compreender o processo do aumento das doenças no contexto das relações
socioambientais. Como objetivos específicos: Apontar os dados demográficos do
Instituto Brasileiro de Geografia e Estatística (IBGE), sobre a população urbana do
Estado do Amazonas; Mencionar os dados da Secretaria de Estado de Meio Ambiente
(SEMA), sobre os fatores ambientais. Para os cálculos foi aplicada a taxa de prevalência de acordo com os estudos
desenvolvidos pelo Wagner (1998), a taxa de prevalência é a soma dos números de
casos da Dengue, divididos pela soma do total de indivíduos estudados. RESULTADOS E DISCUSSÕES De acordo com Dallari (1988, p.58), muito já se escreveu a respeito da conceituação
da saúde durante a história da humanidade. Hipócrates, filósofo grego que viveu no
século IV. a.C refere à influência da cidade e do tipo de vida de seus habitantes sobre
a saúde e afirma que o médico não cometerá erros ao tratar as doenças, pois, envolve
o ambiente de determinada localidade, precisa ser compreendido, e inserido no
processo saúde-doença. Como mostra a (Fig. 1), a seguir. EVISTA GEONORTE, V.9, N.33, p.192-196, 2018. (ISSN 2237 - 1419)
OI: 10.21170/geonorte.2018.V.9.N.33.192.196
Figura 1 - Mapa dos Municípios do Estado do Amazonas com maiores incidência de
Dengue. Fonte: FVS – AM, (2017). Figura 1 - Mapa dos Municípios do Estado do Amazonas com maiores incidência de
Dengue. Fonte: FVS – AM, (2017). Figura 1 - Mapa dos Municípios do Estado do Amazonas com maiores incidência de
Dengue. Fonte: FVS – AM, (2017). REVISTA GEONORTE, V.9, N.33, p.192-196, 2018. DOI: 10.21170/geonorte.2018.V.9.N.33.192.196 (ISSN 2237 - 1419) (ISSN 2237 - 1419) 193 DENGUE UMA DOENÇA EMERGENTE NO ESTADO
DO AMAZONAS DENGUE UMA DOENÇA EMERGENTE NO ESTADO
DO AMAZONAS Segundo o (IBGE, 2017), a população do Estado do Amazonas corresponde a
3.483.985 milhões de habitantes, o equivalente a população total. Moram nas cidades
2.755.490, milhões de habitantes, o equivalente a 79%, enquanto a rural o contingente
populacional é de 728.495 milhões de habitantes, correspondendo a 21% do total. A
grande maioria concentrada na capital do Estado, o que amplia ainda mais o
desequilíbrio da distribuição da população (Censo, 2010). Conforme o (IBGE, 2017), em relação ao saneamento básico no Estado do
Amazonas, aproximadamente 24% da população urbana dispunha de abastecimento
de água1 via rede geral, enquanto a população rural 19,3% (Censo, 2010). Segundo
o (DATASUS, 2018), a distribuição dos serviços de saneamento básico em 2010 no
Estado do Amazonas na zona urbana, os serviços de coleta de lixo e limpeza foi de
84,19%, a coleta por caçamba 9,75%, a queima de lixo na propriedade 26,58%,
enterrado na propriedade 0,97%, jogado em terreno baldio ou logradouro 2,79%,
jogado no rio 0,76% e outro destino 0,74. Os Municípios com prevalência como a
(Tabela 1), de dengue no Amazonas a maioria insere-se no espaço urbano, com
saneamento básico eficaz. Tabela 1 – Casos confirmados e taxas de prevalência de dengue no Estado do Amazonas,
2007 a 2012. 1 Indicadores de saneamento básico (PNAD). A cobertura ou proporção da população servida por rede de abastecimento de água: População
residente em domicílios particulares permanentes servidos por rede geral de abastecimento de água, com ou sem canalização interna/ População total
residente em domicílios particulares permanentes X 100. REVISTA GEONORTE, V.9, N.33, p.192-196, 2018.
(ISSN 2237 - 1419)
DOI: 10.21170/geonorte.2018.V.9.N.33.192.196 RESULTADOS E DISCUSSÕES Municípios
Casos
Confirmados
Taxa
de
prevalência
(em 100 mil
habitantes)
Borba
189
90,41
Coari
1.699
373,76
Humaitá
1.315
539,18
Itacoatiara
1.462
27,88
Manacapuru
1.494
278,81
Manaus
70.157
657,15
Nova Olinda do Norte
419
223,24
Novo Aripuanã
565
465,81
Parintins
508
79,63
Presidente Figueiredo
108
66,90
Rio Preto da Eva
60
37,83
São Gabriel da Cachoeira
1.637
700,26
Tabatinga
593
196,83
Tefé
2.060
533,43
Fonte: DATASUS (2007 a 2012) - IBGE (Pop. Residente AM). Acesso 10/01/2018. Org.: Cinthia
Sampaio, 2018. a 1 – Casos confirmados e taxas de prevalência de dengue no Estado do Amazonas,
2007 a 2012. 1 Indicadores de saneamento básico (PNAD). A cobertura ou proporção da população servida por rede de abastecimento de água: População
residente em domicílios particulares permanentes servidos por rede geral de abastecimento de água, com ou sem canalização interna/ População total
residente em domicílios particulares permanentes X 100. (ISSN 2237 - 1419) 194 DENGUE UMA DOENÇA EMERGENTE NO ESTADO
DO AMAZONAS
Descrevem Souza (2010, p.19), é no ambiente urbano que o mosquito encontra o local
propício para depositar seus ovos. Esse local é denominado de criadouro e, trata-se
de qualquer recipiente que armazene ou possa a vir a armazenar água. DENGUE UMA DOENÇA EMERGENTE NO ESTADO
DO AMAZONAS DENGUE UMA DOENÇA EMERGENTE NO ESTADO
DO AMAZONAS DENGUE UMA DOENÇA EMERGENTE NO ESTADO
DO AMAZONAS Descrevem Souza (2010, p.19), é no ambiente urbano que o mosquito encontra o local
propício para depositar seus ovos. Esse local é denominado de criadouro e, trata-se
de qualquer recipiente que armazene ou possa a vir a armazenar água. CONCLUSÕES Os fatores sociais, o Estado do Amazonas, a ocupação urbana ultrapassa os limites
da cidade é fragmentado e segregado, nos lugares privilegiados da cidade de média
e alta renda, a estrutura urbana é mais adequada, as chuvas provocam problemas
que afetam a mobilidade do trânsito, gerando algumas perdas econômicas, enquanto
que nos lugares de baixa renda, os serviços de saneamento básico é eficaz, como:
abastecimento de água, coleta de lixo e rede de esgoto, não atende a demanda de
todos os domicílios. REFERÊNCIAS DALLARI, S. G. "The Right To Health." Revista de Saúde Pública, v.22, n.1, p. 57 -
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63, 1988. IBGE, Instituto Brasileiro De Geografia e Estatística. Estimativas da População
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195 (ISSN 2237 - 1419) REVISTA GEONORTE, V.9, N.33, p.192-196, 2018. DOI: 10.21170/geonorte.2018.V.9.N.33.192.196 195 DENGUE UMA DOENÇA EMERGENTE NO ESTADO
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Brasil, Ministério da Saúde. 2018. DATASUS. Indicadores de Morbidade e Fatores de
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Aceito em 30/05/2018 DENGUE UMA DOENÇA EMERGENTE NO ESTADO
DO AMAZONAS
Brasil, Ministério da Saúde. 2018. DATASUS. Indicadores de Morbidade e Fatores de
Risco – Taxa de Incidência da Dengue. Disponível em: <http://tabnet.datasus. gov.br/cgi/tabcgi.exeidb2018/d0203> Acesso em: fev. de 2018. Recebido em 13/03/2018
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Aceito em 30/05/2018 (ISSN 2237 - 1419) 196
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https://research-information.bris.ac.uk/ws/files/339244813/Sim_to_Real_Transfer_for_Fixed_Wing_Uncrewed_Aerial_Vehicle_Pitch_Control_by_High_Fidelity_Modelling_and_Domain_Randomization.pdf
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Sim-to-Real Transfer for Fixed-Wing Uncrewed Aerial Vehicle: Pitch Control by High-Fidelity Modelling and Domain Randomization
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IEEE robotics & automation letters
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Wada, D., Araujo-Estrada, S., & Windsor, S. P. (2022). Sim-to-real
transfer for fixed-wing uncrewed aerial vehicle: Pitch Control by High-
Fidelity Modelling and Domain Randomization. IEEE Robotics and
Automation Letters, 7(4), 11735-11742. https://doi.org/10.1109/LRA.2022.3205442 Wada, D., Araujo-Estrada, S., & Windsor, S. P. (2022). Sim-to-real
transfer for fixed-wing uncrewed aerial vehicle: Pitch Control by High-
Fidelity Modelling and Domain Randomization. IEEE Robotics and
Automation Letters, 7(4), 11735-11742. https://doi.org/10.1109/LRA.2022.3205442 Publisher's PDF, also known as Version of record
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applicable terms of use of the publisher. Wada, D., Araujo-Estrada, S., & Windsor, S. P. (2022). Sim-to-real
transfer for fixed-wing uncrewed aerial vehicle: Pitch Control by High-
Fidelity Modelling and Domain Randomization. IEEE Robotics and
Automation Letters, 7(4), 11735-11742.
https://doi.org/10.1109/LRA.2022.3205442 Wada, D., Araujo-Estrada, S., & Windsor, S. P. (2022). Sim-to-real
transfer for fixed-wing uncrewed aerial vehicle: Pitch Control by High-
Fidelity Modelling and Domain Randomization. IEEE Robotics and
Automation Letters, 7(4), 11735-11742. https://doi.org/10.1109/LRA.2022.3205442 Sim-to-Real Transfer for Fixed-Wing Uncrewed
Aerial Vehicle: Pitch Control by High-Fidelity
Modelling and Domain Randomization Daichi Wada
, Sergio Araujo-Estrada, and Shane Windsor Abstract—Deep reinforcement learning has great potential to
automatically generate flight controllers for uncrewed aerial ve-
hicles (UAVs), however these controllers often fail to perform as
expected in real world environments due to differences between the
simulation environment and reality. This letter experimentally in-
vestigated how this reality gap effect could be mitigated, focusing on
fixed-wing UAV pitch control in wind tunnel tests. Three different
training approaches were conducted: a baseline approach that used
simple linear dynamics, a high-fidelity modeling approach, and a
domain randomization approach. It was found that the base line
controller was susceptible to the reality gap, while the other two ap-
proaches successfully transferred to real tests. To further examine
the controllers’ capabilities to generalize, a variety of configuration
changes were experimentally implemented on the UAV, such as
increased inertia, extended elevator area, and aileron offset. While
the high-fidelity controller failed to cope with these changes, the
controllerwithdomainrandomizationmaintaineditsperformance. These results highlight the importance of selecting appropriate
sim-to-real transfer approaches and how domain randomization
is applicable to fixed-wing UAV control with uncertainty in real
environments. robotics and aviation. Supervised learning has potential benefits
for efficiency and smooth interpolation in applications where a
baseline controller already exists [1]. However, when a teaching
controller does not exist, or the goal is to create a new controller
with improved performance beyond that of existing controllers,
deep reinforcement learning is a more suitable approach. By
designing reward functions that elicit desired behaviors, the
controller is automatically trained to behave in an optimized
way, potentially offering time savings and performance benefits
over manual design processes [2], [3], [4], [5], [6], [7], [8]. Although deep reinforcement learning has produced convinc-
ing results in simulation, experimental demonstration is often
challenging due to discrepancies between simulated and real
environments; this is known as the “reality gap” [9], [10]. Deep
reinforcement learning usually optimizes a controller for a single
theoretical scenario and the controller’s performance can be poor
if the target domain is too different from the simulation [11]. To close the reality gap, sim-to-real transfer approaches have
been studied. One approach has been to improve the accuracy
of the simulation by using higher fidelity modeling, for example
by more accurate system identification [12], [13] or by utiliz-
ing another neural network to better approximate the systems
dynamics [14], [15]. 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/ 11735 IEEE ROBOTICS AND AUTOMATION LETTERS, VOL. 7, NO. 4, OCTOBER 2022 Manuscript received 24 February 2022; accepted 26 July 2022. Date of
publication 9 September 2022; date of current version 16 September 2022.
This letter was recommended for publication by Associate Editor I. Sa and
Editor P. Pounds Liu upon evaluation of the reviewers’ comments. This work
was supported in part by JSPS KAKENHI under Grant JP19K04850, in part
by European Research Council (ERC) through European Union’s Horizon
2020 Research and Innovation Programme under Grant 679355 and in part by
UKRI Trustworthy Autonomous Systems Node in Functionality under Grant
EP/V026518/1. (Corresponding author: Daichi Wada.) Sim-to-Real Transfer for Fixed-Wing Uncrewed
Aerial Vehicle: Pitch Control by High-Fidelity
Modelling and Domain Randomization Orthogonal to the high-fidelity modeling,
another solution is to generate controllers with robustness. This
is a viable approach especially when it is not feasible to fully re-
produce the rich dynamics of the real environment. An example
is the domain randomization approach, where parameters in the
source domain, such as the visual appearance of objects [16],
[17], [18], or dynamic parameters of the system [11], [12], [19],
[20], are randomized during training so that the controllers learn
robustness to environmental or system uncertainty. Index Terms—Aerial systems: mechanics and control, motion
control, reinforcement learning. Sergio Araujo-Estrada is with the Department of Aerospace Engineering, Uni-
versity of Bristol, Bristol BS8 1TR, U.K., and with the Department of Aeronau-
tical and Astronautical Engineering, University of Southampton, Southampton
SO16 7PX, U.K. (e-mail: s.araujo-estrada@soton.ac.uk). Daichi Wada is with the Aeronautical Technology Directorate, Japan
Aerospace Exploration Agency, Mitaka, Tokyo 181-0015, Japan (e-mail:
wada.daichi@jaxa.jp). Shane Windsor is with the Department of Aerospace Engineering, University
of Bristol, Bristol BS8 1TR, U.K. (e-mail: shane.windsor@bristol.ac.uk). This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons This
letter
has
supplementary
downloadable
material
available
at
https://doi.org/10.1109/LRA.2022.3205442, provided by the authors.
Digital Object Identifier 10.1109/LRA.2022.3205442 Manuscript received 24 February 2022; accepted 26 July 2022. Date of
publication 9 September 2022; date of current version 16 September 2022.
This letter was recommended for publication by Associate Editor I. Sa and
Editor P. Pounds Liu upon evaluation of the reviewers’ comments. This work
was supported in part by JSPS KAKENHI under Grant JP19K04850, in part
by European Research Council (ERC) through European Union’s Horizon
2020 Research and Innovation Programme under Grant 679355 and in part by
UKRI Trustworthy Autonomous Systems Node in Functionality under Grant
EP/V026518/1. (Corresponding author: Daichi Wada.)
Daichi Wada is with the Aeronautical Technology Directorate, Japan
Aerospace Exploration Agency, Mitaka, Tokyo 181-0015, Japan (e-mail:
wada.daichi@jaxa.jp).
Sergio Araujo-Estrada is with the Department of Aerospace Engineering, Uni-
versity of Bristol, Bristol BS8 1TR, U.K., and with the Department of Aeronau-
tical and Astronautical Engineering, University of Southampton, Southampton
SO16 7PX, U.K. (e-mail: s.araujo-estrada@soton.ac.uk).
Shane Windsor is with the Department of Aerospace Engineering, University
of Bristol, Bristol BS8 1TR, U.K. (e-mail: shane.windsor@bristol.ac.uk).
This
letter
has
supplementary
downloadable
material
available
at
https://doi.org/10.1109/LRA.2022.3205442, provided by the authors.
Digital Object Identifier 10.1109/LRA.2022.3205442 B. Sim-to-Real Transfer and Domain Randomization between the type of reality gaps and their consequence in control
performance [24], [25]. The effect of model errors, namely
friction and delay, were investigated experimentally using wind
tunnel testing. This study extends this line of previous research
on closing reality gaps by introducing a comparative perspective
of different sim-to-real transfer approaches. With an integrated
understanding of the cause and effect of reality gaps and how
they can be overcome, we aim to provide examples of how
different sim-to-real approaches influence the performance of
reinforcement-learning-based controllers for fixed-wing UAVs. This idea has been experimentally tested in various appli-
cations, such as for control of robot arms [19], quadruped
robots [12], and dexterous robot hands [20]. As an extended al-
ternative, more adaptive approaches have been proposed, where
controllers identify, implicitly or explicitly, environmental pa-
rameters using a limited number of on-site trials, and then tune
the outputs correspondingly. For example, latent space adapta-
tion methods identify a latent representation of the dynamics
on-site and modulate the systems actions [33], [34], [35]. This study investigates deep reinforcement-learning-based
pitch control for a fixed-wing UAV, with a focus on sim-to-real
transfer. The fixed-wing UAV is tested in a one degree-of-
freedom configuration in a wind tunnel, in order to allow as-
sessment with a simple and clear basis (Fig. 1). Two sim-to-real
transfer approaches, namely high-fidelity modeling and domain
randomization, are compared. Furthermore, a variety of exper-
imental modifications were implemented on the model config-
uration, such as additional inertia, altered control effectiveness
and changes to aerodynamic performance. By examining the
controllers’ behavior, the difference between the two sim-to-real
transfer approaches is highlighted in terms of their capabilities
under unpredicted dynamic variations. Domain randomization approaches do not require on-site
tuning when moving from sim-to-real. This is an advantage in
applications such as aviation, where flight trials are expensive
in terms of both time and cost and safety issues mean lose
of control has to be avoided during any trial-and-error tuning. In this context, domain randomization has been successfully
applied to quadrotors, such as flight through a narrow gap [22]
and flight control of hybrid UAVs with a variety of flight
modes [21]. Investigation of trajectory tracking performance has
been also carried out from the viewpoint of how to design control
outputs (action spaces) [23]. However, studies of sim-to-real for
fixed-wing UAVs specifically remain rare. II. RELATED WORKS A. Neural-Network-Based Control in Aviation
(Simulation Studies) I. INTRODUCTION F
OR nonlinear control, neural-network-based controllers
trained using machine learning techniques have the poten-
tial to be an alternative approach to custom designed controllers,
with many potential applications in a variety of fields such as F Manuscript received 24 February 2022; accepted 26 July 2022. Date of
publication 9 September 2022; date of current version 16 September 2022. This letter was recommended for publication by Associate Editor I. Sa and
Editor P. Pounds Liu upon evaluation of the reviewers’ comments. This work
was supported in part by JSPS KAKENHI under Grant JP19K04850, in part
by European Research Council (ERC) through European Union’s Horizon
2020 Research and Innovation Programme under Grant 679355 and in part by
UKRI Trustworthy Autonomous Systems Node in Functionality under Grant
EP/V026518/1. (Corresponding author: Daichi Wada.) In the aviation field specifically, sim-to-real transfers have
been demonstrated for quadrotors [21], [22], [23], but demon-
strations for fixed-wing UAVs remain rare. Fixed-wing UAVs
differ from quadrotors in that rather than directly controlling
the balance of thrust between rotors to control flight trajectory,
control surfaces are deflected to modify the aerodynamic forces
produced by the wing and tail. Fixed wing aerodynamics have
quite different characteristics from rotor aerodynamics, such as
fixed wings stalling at high angles of attack and having differ-
ent time scales for their dynamic effects. These fundamental
differences suggest the need for specific investigation of the
sim-to-real transfer considerations for fixed-wing UAV control. A series of previous studies investigated typical relationships B. Sim-to-Real Transfer and Domain Randomization Therefore, inspired
by the previous success in quadrotors, this study applies domain
randomization to a fixed-wing UAV to explore the reality gap
effect and how it can be mitigated. IEEE ROBOTICS AND AUTOMATION LETTERS, VOL. 7, NO. 4, OCTOBER 2022 11736 IEEE ROBOTICS AND AUTOMATION LETTERS, VOL. 7, NO. 4, OCTOBER 2022 Fig. 1. A fixed-wing UAV in the wind tunnel. The pitch angle is controlled by
deflecting the elevator. All above aviation-related applications have been explored
in simulation and the challenge remains to transfer these ap-
proaches to real environments. B. Sim-to-Real Transfer and Domain Randomization B. Sim-to-Real Transfer and Domain Randomization The basic concept of sim-to-real transfer is to use a policy
learned in simulation in a real environment while keeping the
theoretical performance, even though differences exist between
the simulated and real environments (known as the reality gap). To close the reality gap, previous works have proposed various
methods to mitigate the gap effect including Bayesian opti-
mization [30], deep inverse dynamics models [31], progressive
networks [32], and domain randomization. In domain random-
ization, a policy is trained to work across all of the simulated
environments with randomized parameters, thus becoming ro-
bust to real-world variability. Fig. 1. A fixed-wing UAV in the wind tunnel. The pitch angle is controlled by
deflecting the elevator. between the type of reality gaps and their consequence in control
performance [24], [25]. The effect of model errors, namely
friction and delay, were investigated experimentally using wind
tunnel testing. This study extends this line of previous research
on closing reality gaps by introducing a comparative perspective
of different sim-to-real transfer approaches. With an integrated
understanding of the cause and effect of reality gaps and how
they can be overcome, we aim to provide examples of how
different sim-to-real approaches influence the performance of
reinforcement-learning-based controllers for fixed-wing UAVs. between the type of reality gaps and their consequence in control
performance [24], [25]. The effect of model errors, namely
friction and delay, were investigated experimentally using wind
tunnel testing. This study extends this line of previous research
on closing reality gaps by introducing a comparative perspective
of different sim-to-real transfer approaches. With an integrated
understanding of the cause and effect of reality gaps and how
they can be overcome, we aim to provide examples of how
different sim-to-real approaches influence the performance of
reinforcement-learning-based controllers for fixed-wing UAVs. This study investigates deep reinforcement-learning-based
pitch control for a fixed-wing UAV, with a focus on sim-to-real
transfer. The fixed-wing UAV is tested in a one degree-of-
freedom configuration in a wind tunnel, in order to allow as-
sessment with a simple and clear basis (Fig. 1). Two sim-to-real
transfer approaches, namely high-fidelity modeling and domain
randomization, are compared. Furthermore, a variety of exper-
imental modifications were implemented on the model config-
uration, such as additional inertia, altered control effectiveness
and changes to aerodynamic performance. By examining the
controllers’ behavior, the difference between the two sim-to-real
transfer approaches is highlighted in terms of their capabilities
under unpredicted dynamic variations. A. Aircraft Model As described later in this section, actor-critic algorithms are
advantageous for domain randomization in that the critic func-
tion can utilize the randomized parameters in training, which
are generally not explicitly known to the controller. Although
there are other promising actor-critic algorithms including Twin
Delayed Deep Deterministic policy gradient (TD3) [40] and
Soft Actor-Critic (SAC) [22], [41], this study was inspired by
the success of previous studies employing PPO [8], [20], [23]. Table I summarizes the hyper-parameter settings for training
with PPO. The neural networks and training process were built
in a Python environment using the PyTorch framework (Ver. 1.7.1). To assess the generalization performance of the controllers
presented here, four experimental model variants were tested. Fig. 2(b) depicts the variant with added masses to increase
inertia. Two sets of 200 g masses were mounted to the pitching
shaft at a 15 cm radial distance from the center of the shaft. Fig. 2(c) depicts the variant with the extended elevator, which
doubled the area of the elevator. This modification changed the
equilibrium point and the effectiveness of the elevator deflection. Fig. 2(d) depicts the variant with the deflected aileron. The
aileron deflection was set to 40 ◦, which changed the equilibrium
pitch angle. We designed a neural network controller which output the
elevator angle rate based on the state input. The elevator angle
rate rather than the elevator angle itself was chosen as an output
so that the output was directly used in a penalty function as
described later. The state space consisted of the error between
the target and observed pitch angle, observed pitch angle, pitch
rate, elevator angle and wind speed. The neural network archi-
tecture is illustrated in Fig. 3. The policy function observed
what was observable in the real application, that is, the dynamic A. Aircraft Model An off-the-shelf radio control aircraft (WOT4 Foam-E Mk2+,
Ripmax) was modified into a span-wise half model, mounted
to the side wall of a wind tunnel. The model was free to
pitch around the shaft of the wing at its root, as depicted in
Fig. 2(a). The model was equipped with an air speed sensor
(custom-built based on SDP31, Sensirion), an elevator servo and
an inertial measurement unit (IMU) (Pixhawk 1, 3DR). See [25]
for full details. The signal processing and elevator control was
conducted at a rate of 20 Hz. The wind speed was kept constant
for each test case. Two theoretical models were developed; an approximated lin-
ear model and a high-fidelity model including frictional effects. The dynamics of these models were expressed as: Iyy ¨α = 1
2ρV 2S c CM −g4 tanh(g5q),
(1)
CM = CM0 + CMαα + CMq
c
2 V q + CMδe δe,
(2) (1) (2) where Iyy is moment of inertia in pitch, ρ is air density, V is air
speed, S is wing area, c is chord length, α is pitch angle, q is pitch
rate, δe is elevator angle and g4 and g5 are the coefficients for
the Coulomb friction effect. Each of the CMx terms represents
individual aerodynamic coefficients. These parameter values
are given in Table I. For the linear model, the friction effect
was set to g4 = 0. Detailed information about the parameter
identification and the model fidelity have been discussed in
previous research [25]. learning stability by limiting the divergence between the old and
updated policies using the so-called clipped surrogate loss func-
tion. As described later in this section, actor-critic algorithms are
advantageous for domain randomization in that the critic func-
tion can utilize the randomized parameters in training, which
are generally not explicitly known to the controller. Although
there are other promising actor-critic algorithms including Twin
Delayed Deep Deterministic policy gradient (TD3) [40] and
Soft Actor-Critic (SAC) [22], [41], this study was inspired by
the success of previous studies employing PPO [8], [20], [23]. Table I summarizes the hyper-parameter settings for training
with PPO. The neural networks and training process were built
in a Python environment using the PyTorch framework (Ver. 1.7.1). learning stability by limiting the divergence between the old and
updated policies using the so-called clipped surrogate loss func-
tion. WADA et al.: SIM-TO-REAL TRANSFER FOR FIXED-WING UNCREWED AERIAL VEHICLE WADA et al.: SIM-TO-REAL TRANSFER FOR FIXED-WING UNCREWED AERIAL VEHICLE 11737 Fig. 2. Experimental configuration variants: (a) baseline with characteristic dimensions (b) added masses, (c) extended elevator, (d) deflected aileron. Fig. 2. Experimental configuration variants: (a) baseline with characteristic dimensions (b) added masses, (c) extended elevator, (d) deflected aileron. TABLE I
PARAMETERS FOR SIMULATION AND PPO TABLE I
PARAMETERS FOR SIMULATION AND PPO TABLE I
PARAMETERS FOR SIMULATION AND PPO III. METHODS A. Neural-Network-Based Control in Aviation
(Simulation Studies) As the simulated environment with randomized parameters
canberegardedasatypeofpartiallyobservableMarkovdecision
process, its optimal policy in general depends on access to
the state’s history [36]. In this sense, it becomes essential to
use policies with some form of memory-augmentation along
with domain randomization. To address this point, models using
recurrent neural networks have been proposed, including Gated
Recurrent Unit (GRU) [37] and Long Short-Term Memory
(LSTM). Another method is to use policies without memory
functions and instead input multiple time steps to policies [33],
[35]. These successful previous studies suggest that domain
randomization should work if the policy captures time histories
by either method. This study follows recent success in using
LSTM with domain randomization [19], [20]. For nonlinear control, supervised learning has been applied
to flight control systems [1]. For example, gain schedules for
existing control systems [26], nonlinear transformations for
feedback linearisation [27], [28], and decision-making lookup
tables for collision avoidance [29], have all been approximated
by neural networks. Deep reinforcement learning, on the other hand, gener-
ates policies (controllers) without needing a baseline. Deep-
reinforcement-learning based controllers have shown a high
level of performance for complex tasks, such as trajectory plan-
ning and navigation [4], [8], fixed-wing aircraft landing under
wind disturbance [5], flocking control for fixed-wing UAVs [6],
aerobatic maneuvers [7], and attitude control for fixed-wing
UAVS [3] and quadrotors [2]. B. Controller Training The state and the dynamics parameter were received and processed by
128 LSTM cells and two 128 hidden units with elu activations. Fig. 5. Learning curves for the three controller types. The blue, red and green
lines correspond to the controller trained with linear model, friction model and
domain randomization, respectively. For each controller type, five independent
training ran with different random seeds. The solid lines and shaded areas
represent the mean and the minimum to maximum range, respectively. parameters were unknown to the controller. The LSTM layers
were employed to find an optimal history-dependent policy that
adapted to every randomized environment. In the absence of
direct knowledge of the real dynamics, a history of past states
and actions was expected to help capture the system dynamics. The value function was designed to observe the full conditions
because it was used only during training and the dynamic param-
eters of the simulator were known [19], [35]. The value function
estimates the value of the states in the randomized simulator. Fig. 5. Learning curves for the three controller types. The blue, red and green
lines correspond to the controller trained with linear model, friction model and
domain randomization, respectively. For each controller type, five independent
training ran with different random seeds. The solid lines and shaded areas
represent the mean and the minimum to maximum range, respectively. Three types of controller were trained: the first was trained
with the linear model (baseline), the second was trained with the
friction model (high-fidelity modeling approach), and the third
was trained with domain randomization (robust approach). The
linear model was used as the basis for the domain randomization. In the domain randomization, the aerodynamic coefficients were
randomly changed in each episode as listed in Table I. These
randomization factors were fed into the value function. wherex is time(ins), t0 is offset (in s), σ is thestandarddeviation
(1.67 s) and μ is the mean (−5.27 s). This is a log-normal
function with the peak at t0, which was found to best fit the
measured delay in the previous experiments [25]. The effective
delay was empirically set as t0 = 0.100 s. All controllers used the following common training con-
ditions, each episode had 30 s duration and 600 time steps,
and the control rate was set to 20 Hz. B. Controller Training Deep reinforcement learning was conducted using the Prox-
imal Policy Optimization (PPO) algorithm [39], which ensured 11738 IEEE ROBOTICS AND AUTOMATION LETTERS, VOL. 7, NO. 4, OCTOBER 2022 Fig. 4. Examples of the target pitch schedules. Each line shows the schedules
generated with different random seeds. The Ornstein–Uhlenbeck process [38],
with mean μ = 0, volatility σ = 2 and reverting rate towards the mean θ = 0.1,
was used to calculate 16 discrete points with random intervals, which are
indicated with circles. The adjacent points were linearly interpolated. The
initial point was randomly set within ±0.5 ◦, and the target pitch angles varied
approximately within ±10 ◦during each episode. Fig. 5. Learning curves for the three controller types. The blue, red and green
lines correspond to the controller trained with linear model, friction model and
domain randomization, respectively. For each controller type, five independent
training ran with different random seeds. The solid lines and shaded areas
represent the mean and the minimum to maximum range, respectively. Fig. 4. Examples of the target pitch schedules. Each line shows the schedules
generated with different random seeds. The Ornstein–Uhlenbeck process [38],
with mean μ = 0, volatility σ = 2 and reverting rate towards the mean θ = 0.1,
was used to calculate 16 discrete points with random intervals, which are
indicated with circles. The adjacent points were linearly interpolated. The
initial point was randomly set within ±0.5 ◦, and the target pitch angles varied
approximately within ±10 ◦during each episode. Fig. 4. Examples of the target pitch schedules. Each line shows the schedules
generated with different random seeds. The Ornstein–Uhlenbeck process [38],
with mean μ = 0, volatility σ = 2 and reverting rate towards the mean θ = 0.1,
was used to calculate 16 discrete points with random intervals, which are
indicated with circles. The adjacent points were linearly interpolated. The
initial point was randomly set within ±0.5 ◦, and the target pitch angles varied
approximately within ±10 ◦during each episode. Fig. 3. Schematic of the policy (top) and value (bottom) networks. The policy
received the state, which was processed by 128 LSTM cells and two 128 hidden
units with elu activations. The output layer specified the mean m of the action
distribution. The standard deviation Σ of the action distribution was specified
by a fixed diagonal matrix. The value function was modelled by a separate
network. B. Controller Training The wind speed was
randomly chosen for each episode ranging from 8 to 22 m/s,
and set constant over the episode. The target pitch schedules
were randomly assigned for each episode. Fig. 4 shows three
examples of the target pitch schedules with different random
seeds.Stateobservationnoisewasappliedateverytimestep.The
noise distributions for each observation parameter are shown in
Table I. These values were estimated from calibration tests of
the IMU and the air speed sensor. The elevator angle in the state
was the target value given in the previous time step. It was the
theoretical value, and therefore, the observation noise was not
applied. The reward function was defined as r = −(|eα| + 0.1|q| + 0.1|a| + 0.1|Δδe|),
(4) (4) where eα (rad) is the difference between target and observed
pitch angle, q (rad/s) is the pitch rate, a (rad/s) is the action
outputandΔδe (rad)isthedeviationofthecurrentelevatorangle
from the exponential moving average with 5 /cent smoothing
factor. The pitch rate, action and elevator penalties encouraged
smooth and stable pitch maneuvers. Fig. 5 shows the learning curves for the controllers considered
here. In the three training types, the total reward in the training
episodes reached saturation at around 1000 iterations and con-
verged for all trials, which indicated valid training results. Delay was randomly sampled and applied at every time step
based on the probability density function expressed as B. Generalized Performance Under Model Variations B. Generalized Performance Under Model Variations The three controllers successfully followed the target sched-
ules in both simulation scenarios, indicating valid training. How-
ever, the performance of the linear controller was unsatisfactory
for both wind speed experimental conditions. Two different fail-
ure modes were observed: an offset in pitch at 10 m/s (Fig. 6(a));
and oscillating behavior at 20 m/s (Fig. 6(b)). The offset in
pitch is thought to be related to friction effects, as at low wind
speeds, the elevator deflection did not produce enough moment
to overcome static friction and rotate the model. As for the
oscillating behavior, this could be explained by a combination of
friction and other factors such as system time delay. A previous
study [25] investigated the relationship between the simulated
delay in training and the effective delay in reality with this
experimental setup and concluded that if the delay used in the
training simulations was shorter than the actual experimental
delay then this induced oscillation at high wind speeds. This
behavior, i.e., the control performance not being guaranteed after
sim-to-real transfer, is typical of deep-reinforcement-learning-
based controllers when compensation measures are not taken. To examine the applicability of the sim-to-real transfer ap-
proaches in an extended context of large topology and dynamics
variations, control experiments were conducted with the wind
tunnel model variants as depicted in Figs. 2(b)–2(d), using the
same controllers presented in Section IV-A (Fig. 6). Fig. 7
presents a comparison of the pitch control experiments con-
ducted with the model variants and using controllers trained
with the linear model (Fig. 7(a)), the friction model (Fig. 7(b)),
and domain randomization (Fig. 7(c)). For the controller trained with the linear model, both failure
modes of pitch offset (for both wind speeds) and oscillating
behavior (for the higher wind speed) were observed for the added
masses and extended elevator variants (top and middle panels in
Fig. 7(a)). For the deflected aileron variant, pitch offset errors
were observed for both wind speeds (bottom panels in Fig. 7(a)). This is likely explained by the pitching-down moment produced
by the aileron deflection. For the controller trained with the friction model, the con-
troller was stable and showed good performance when applied to
the added masses and extended elevator variants for the 10 m/s
wind speed case. However, the oscillation failure mode was
observed at the 20 m/s wind speed case (top and middle-right
panels in Fig. WADA et al.: SIM-TO-REAL TRANSFER FOR FIXED-WING UNCREWED AERIAL VEHICLE 11739 Fig. 6. Pitch control histories when the nominal wind speed was: (a) 10 m/s and (b) 20 m/s. In each panel, the left and right graphs show simulated and
experimental results, respectively. The controllers were trained using the linear model (blue line), the friction model (red line) and domain randomization (green line). In contrast to the baseline performance (linear model), both the high-fidelity modeling approach (friction model) and the robust approach (domain randomization)
showed successful control in the experiments. Fig. 6. Pitch control histories when the nominal wind speed was: (a) 10 m/s and (b) 20 m/s. In each panel, the left and right graphs show simulated and
experimental results, respectively. The controllers were trained using the linear model (blue line), the friction model (red line) and domain randomization (green line). In contrast to the baseline performance (linear model), both the high-fidelity modeling approach (friction model) and the robust approach (domain randomization)
showed successful control in the experiments. dynamics model. It was anticipated that varying the aerody-
namic coefficients would express similar dynamics to that of the
friction model. It was interesting to note that a larger phase lag
was observed, which was particularly evident after the reference
transition from −5 ◦to 0 ◦at 10 m/s. The domain randomiza-
tion encouraged the controller to work across all the simulated
environments, and potentially made it conservative. brevity, we present a subset of these cases; these were chosen
as representative of the behavior observed for all experimental
conditions, with similar trends being observed through wind
speeds and repetitive trials. Fig. 6 shows a comparison of the pitch control time histories
for both simulation and real-world experiments. Fig. 6(a) shows
results for a wind speed of 10 m/s, while Fig. 6(b) shows results
for 20 m/s. In the simulation, the same system dynamics that
were used for training were used for cases presented here. To
ease comparison, a pitch angle doublet schedule was used as
reference for both simulations and experiments. A. Performance in Simulated and Real Environments f(x) =
1
(x −t0)σ
√
2π exp
−(log(x −t0) −μ)2
2σ2
The performance of the three types of trained controllers was
assessed through simulation and in experiments. In the experi-
ments, the wind speed range was 10 – 20 m/s at 2 m/s intervals. At each wind speed, control trials were repeated three times. For (3) /
/
At each wind speed, control trials were repeated three times. For V. DISCUSSION To highlight the adaptability of each controller type (or lack
of) to different conditions, the different control actions for each
controller type were studied. Fig. 9 shows the elevator deflection
time histories for the three controllers considered here. To study
the controller behaviors without oscillation, only the 10 m/s
wind speed case is presented. The test cases correspond to the
ones shown in Fig. 6(a) and 7. For the controller trained with the linear model, the four
elevator maneuvers were almost identical, even when a variety of
errors (such as positive and negative pitch offsets) were present. In the baseline linear dynamics simulation, the elevator and pitch
angles were uniquely related for a given wind speed, and training
under such dynamics resulted in a controller that simply learned
the simulation equilibrium conditions. Fig. 8. Root mean square errors for tracking the target pitch angle under model
variations. Error bar denotes standard deviation. Note that only the domain
randomization approach was able to maintain control performance across the
different model variations. On the other hand, for the controller trained with the friction
model, the equilibrium condition was path-dependent (i.e., the
elevator-pitch angle equilibrium relationship was not unique),
which encouraged the controller to react to model variations. This led to different behaviors in the elevator deflection histories
and a small degree of adaptability. However, when this controller
was applied to the deflected aileron variant, the elevator did
not show a significant difference in response, resulting in a
large pitch angle error. This is likely related to the effect of the
additional pitching moment, produced by the aileron deflection,
being larger than the one resulting from friction. The controller trained with domain randomization success-
fully followed the target in both wind speeds and when applied
to all three variants. A robust response was observed for all the
experimentalconditions,evenatthehigherwindspeedwherethe
oscillating behavior was observed for the other two controllers,
displaying only a small overshoot during the transitory phases
of the maneuver. When applied to the deflected aileron variant,
the starting pitch offset was rapidly corrected and remained
negligible throughout the duration of the tests (bottom panels
in Fig. 7(c)). To gain a statistical perspective, Fig. 8 shows the root mean
square error (RMSE) for the pitch tracking under model varia-
tions. IEEE ROBOTICS AND AUTOMATION LETTERS, VOL. 7, NO. 4, OCTOBER 2022 IEEE ROBOTICS AND AUTOMATION LETTERS, VOL. 7, NO. 4, OCTOBER 2022 11740 Fig. 7. Experimental results under a variety of model configuration changes and using controllers trained using: (a) the linear model (blue line), (b) the friction
model (red line) and (c) domain randomization (green line). Each panel shows results for two nominal wind speeds 10 m/s (left plots) and 20 m/s (right plots). Additionally, each panel shows results for the added masses (top plots), the extended elevator (middle plots) and the deflected aileron (bottom plots) variants. Under
the model variations, only the domain randomization approach was able to follow the target pitch schedule. Fig. 7. Experimental results under a variety of model configuration changes and using controllers trained using: (a) the linear model (blue line), (b) the friction
model (red line) and (c) domain randomization (green line). Each panel shows results for two nominal wind speeds 10 m/s (left plots) and 20 m/s (right plots). Additionally, each panel shows results for the added masses (top plots), the extended elevator (middle plots) and the deflected aileron (bottom plots) variants. Under
the model variations, only the domain randomization approach was able to follow the target pitch schedule. Fig. 8. Root mean square errors for tracking the target pitch angle under model
variations. Error bar denotes standard deviation. Note that only the domain
randomization approach was able to maintain control performance across the
different model variations. B. Generalized Performance Under Model Variations 7(b)). When applied to deflected aileron variant,
the controller failed to compensate the pitch offset (bottom
panels in Fig. 7(b)). In contrast, both the friction and domain randomization con-
trollers successfully followed the target pitch schedule in both
experimental conditions. The friction controller performance
was expected due to it being trained with a high-fidelity model. The domain randomization controller was effective as it was
trained to be robust to uncertainty in the parameters of the VI. CONCLUSION Here we studied sim-to-real transfer of pitch controllers for
a fixed-wing UAV. Three different approaches were studied
through controllers trained with a simple linear dynamics model,
a high-fidelity model and domain randomization. The perfor-
mance of these controllers was assessed via simulations and
experiments with a variety of configuration changes in the wind
tunnel. We found that only domain randomization was able to
perform consistently with variation in dynamic parameters. This
study highlights the importance of carrying out an experimental
investigation to evaluate the capabilities of controllers in a
real environment, and comparing different sim-to-real trans-
fer approaches to understand their respective applicability and
limitations. aileron variant, the action was significantly shifted towards
the negative elevator deflection side, which was necessary to
compensate for the pitching moment induced by the aileron. The theoretical elevator deflection equilibrium conditions can
be computed by solving (1) and 2 for δe, and by considering both
¨α = 0 and q = 0. This is given by ¯δe = −CM0 + CMα ¯α
CMδe
(5) (5) where ¯δe is the elevator deflection in equilibrium and ¯α is
the equilibrium pitch angle. Using (5) and the variant-specific
aerodynamic parameters (Table II), the elevator deflection equi-
librium conditions for 5 ◦pitch angle were −22.4 ◦, −18.6 ◦and
−32.6 ◦for the added mass, extended elevator and deflected
aileron configurations, respectively. When compared to the con-
troller actions shown in Fig. 9, only the controller actions of the
domain randomization controller (Fig. 9(c)) were close to these
values. To successfully apply deep reinforcement learning to fixed-
wing UAV flight control in real scenarios, future research could
look into extending the analysis presented here to multi-degree-
of-freedom situations under varying wind speeds, including
responsiveness and operation efficiency goals into the reward
function (aiming for both optimality and robustness), and study-
ing its applicability to inherently unstable aircraft or aircraft
with the ability to change stability (e.g. by means of geometry
morphing). The analysis presented here shows that the domain random-
ization approach produced a controller with a better degree
of adaptability. Table II gives the experimentally estimated
change in value for each of the parameters corresponding to
the configuration variants and with respect to the baseline values
(Table I). When compared with the randomization range as listed
in Table I, some parameters exceeded the values assumed in
training. V. DISCUSSION The mean and standard deviation of RMSE were calcu-
lated from 18 individual experiments (three controller types, at
six wind speeds, from 10 – 20 m/s with a 2 m/s interval, and
three trials at each condition). Considering the similar RMSE
and the small standard deviations, domain randomization was
shown to be robust to the model variations conducted. Note that
althoughthefrictionmodelmightseemtoshowonlyslightlarger
RMSEs than domain randomization, there was a significant
difference in the quality of control as seen in Fig. 7. The elevator deflection histories of the controller trained with
domain randomization exhibited markedly different responses,
whichhighlighteditsrobustnesstomodelparametererrors. Con-
centrating on the extended elevator variant, during the first half
of the experiments (0 s ≤t ≤15 s), the control action sat above
the actions corresponding to the other three cases, to compensate
for the change in the equilibrium point caused by the extended
elevator. Then during the transition from 5 ◦to −5 ◦in pitch
reference, overshoot was observed at t ≈15 s, transitioning to a
smaller value at 15 s ≤t ≤22.5 s. When applied to the deflected WADA et al.: SIM-TO-REAL TRANSFER FOR FIXED-WING UNCREWED AERIAL VEHICLE 11741 Fig. 9. Experimental elevator deflection time histories for the controllers trained with: (a) the linear model, (b) the friction model and (c) the domain randomization. The control actions when applied to the baseline configuration and three variants have been overlaid, with the baseline shown in black, the added masses shown in
blue, the extended elevator shown in orange and the deflected aileron shown in yellow. All the results correspond to the 10 m/s wind speed case. Note that only
the domain randomization controller adapted its output appropriately to changes in the model experimental parameters. Fig. 9. Experimental elevator deflection time histories for the controllers trained with: (a) the linear model, (b) the friction model and (c) the domain randomization. The control actions when applied to the baseline configuration and three variants have been overlaid, with the baseline shown in black, the added masses shown in
blue, the extended elevator shown in orange and the deflected aileron shown in yellow. All the results correspond to the 10 m/s wind speed case. Note that only
the domain randomization controller adapted its output appropriately to changes in the model experimental parameters. TABLE II
IDENTIFIED CHANGE IN DYNAMICS PARAMETERS FOR THREE VARIANTS flight conditions. This difference is likely to be significant and
possibly requires additional wind speed sensing and/or more
robust control capability against external disturbances. Future
work on sim-to-real approaches should examine varying wind
speed conditions. VI. CONCLUSION Considering the successful control results in experi-
ments, the domain randomization was able to encourage robust-
ness that was applicable to dynamics variation that were more
severe than assumed. Note that the true “working range” should
always be carefully examined as any case-specific evaluation
may not generalize for any combination of dynamics parameter
variations. ACKNOWLEDGMENT The authors would like to thank Mr. Lee Winter from the
University of Bristol Wind Tunnel Laboratory, for his invaluable
support and work during the assembly of the experimental
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http://eprints.staffs.ac.uk/5020/1/s12864-017-4270-9.pdf
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English
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A divide-and-conquer algorithm for large-scale de novo transcriptome assembly through combining small assemblies from existing algorithms
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BMC genomics
| 2,017
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cc-by
| 5,740
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© The Author(s). 2017 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 Background: While the continued development of high-throughput sequencing has facilitated studies of entire
transcriptomes in non-model organisms, the incorporation of an increasing amount of RNA-Seq libraries has made de
novo transcriptome assembly difficult. Although algorithms that can assemble a large amount of RNA-Seq data are
available, they are generally very memory-intensive and can only be used to construct small assemblies. Results: We develop a divide-and-conquer strategy that allows these algorithms to be utilized, by subdividing a large
RNA-Seq data set into small libraries. Each individual library is assembled independently by an existing algorithm, and
a merging algorithm is developed to combine these assemblies by picking a subset of high quality transcripts to form
a large transcriptome. When compared to existing algorithms that return a single assembly directly, this strategy
achieves comparable or increased accuracy as memory-efficient algorithms that can be used to process a large
amount of RNA-Seq data, and comparable or decreased accuracy as memory-intensive algorithms that can only be
used to construct small assemblies. Conclusions: Our divide-and-conquer strategy allows memory-intensive de novo transcriptome assembly algorithms
to be utilized to construct large assemblies. Keywords: Divide-and-conquer, RNA-Seq, de novo transcriptome assembly Although transcriptome assembly algorithms such as
SOAPdenovo-Trans [1] and Trans-ABySS [2] can be used
to process a large amount of RNA-Seq data, algorithms
such as Oases [3] and Trinity [4] that have higher accu-
racy are generally very memory-intensive, thus they can
only be used to construct small assemblies. We develop a
divide-and-conquer strategy that allows these algorithms
to be utilized. A large RNA-Seq data set is subdivided
into small libraries. Each individual library is assembled
independently, and a merging algorithm is employed to
combine the small assemblies into a large transcriptome
(Fig. 1). A divide-and-conquer algorithm for
large-scale de novo transcriptome assembly
through combining small assemblies from
existing algorithms Sing-Hoi Sze1,2*, Jonathan J. Parrott3 and Aaron M. Tarone3 From 6th IEEE International Conference on Computational Advances in Bio and Medical Sciences (ICCABS)
Atlanta, GA, USA. 13-15 October 2016 From 6th IEEE International Conference on Computational Advances in Bio and Medical Sciences (ICCABS)
Atlanta, GA, USA. 13-15 October 2016 From 6th IEEE International Conference on Computational Advances in Bio and Medical Sciences (ICCABS)
Atlanta, GA, USA. 13-15 October 2016 *Correspondence: shsze@cse.tamu.edu
1Department of Computer Science and Engineering, Texas A&M University,
College Station, Mexico, TX, 77843 USA
2Department of Biochemistry & Biophysics, Texas A&M University, College
Station, Mexico, TX, 77843 USA
Full list of author information is available at the end of the article Sze et al. BMC Genomics 2017, 18(Suppl 10):895
DOI 10.1186/s12864-017-4270-9 Sze et al. BMC Genomics 2017, 18(Suppl 10):895
DOI 10.1186/s12864-017-4270-9 Picking high quality transcripts transcripts, which are more highly expressed and better
assembled. A de Bruijn graph is constructed to extend
some of these transcripts at the left end and at the right
end when there are no ambiguities. To reduce redun-
dancy, lower ranked transcripts with all their correspond-
ing nodes in the de Bruijn graph covered by higher ranked
transcripts are removed. Since each transcript corresponds to a path in the de
Bruijn graph and there may be extra bases at the begin-
ning node and the ending node of the path that are not
included within the transcript, these bases form unam-
biguous extensions of the transcript and are added to
the transcript. To reduce noise, we only retain a tran-
script if its length divided by the number of nodes in
its path is above the average length cutoff c1, where c1
is a given parameter. To form the transcriptome from
these extended transcripts, our algorithm picks a sub-
set of high quality transcripts while preferring longer
transcripts. We validate our algorithm by performing Schizosaccha-
romyces pombe, Drosophila melanogaster and Arabidopsis
thaliana transcriptome assemblies using publicly avail-
able RNA-Seq libraries. We demonstrate our algorithm by
assembling a large set of 93 Cochliomyia macellaria RNA-
Seq libraries that we have constructed, which is about
298 G in size. Since the longest transcripts are not always correct and
may have translocations, and we have to make sure that
redundant subsequences of a transcript are not included Background While high-throughput sequencing has made it possible
to perform studies of entire transcriptomes in non-model
organisms, applying de novo transcriptome assembly algo-
rithms has been increasingly difficult due to an increasing
amount of RNA-Seq libraries that include many experi-
mental conditions or developmental stages with replicated
experiments. *Correspondence: shsze@cse.tamu.edu
1Department of Computer Science and Engineering, Texas A&M University,
College Station, Mexico, TX, 77843 USA
2Department of Biochemistry & Biophysics, Texas A&M University, College
Station, Mexico, TX, 77843 USA
Full list of author information is available at the end of the article The merging algorithm picks a subset of high quality
transcripts to form a transcriptome by preferring longer Page 44 of 88 Page 44 of 88 Sze et al. BMC Genomics 2017, 18(Suppl 10):895 Fig. 1 Illustration of the divide-and-conquer strategy. A large
RNA-Seq data set is subdivided into small libraries. Each individual
library is assembled independently, and a merging algorithm is
employed to combine the small assemblies into a large transcriptome independently using an existing algorithm, our algorithm
constructs a de Bruijn graph from the set of all pre-
dicted transcripts. Note that the k-mer coverage cutoff c is
only applied during individual library assemblies and not
during the merging step. To construct the de Bruijn graph, we follow the iterative
one-letter extension strategy in [7] to identify all k-mers. Given a sorted array that contains all n k′-mers in either
the forward or the reverse complementary direction for
k′ < k, an array of size 4n is created that contains four
slots for each k′-mer. For each (k′ + 1)-mer, binary search
is applied to locate its k′-prefix within the array and one
of the four slots that corresponds to its last nucleotide
is updated. Slots with zero counts are removed to obtain
all (k′ + 1)-mers. Edges in the de Bruijn graph are con-
structed by locating the corresponding k-mers through
binary search. Since this step is performed on the tran-
scripts and not on the reads, it is not time consuming and
the memory requirement has a multiplicative constant of
four per k-mer. Fig. 1 Illustration of the divide-and-conquer strategy. A large
RNA-Seq data set is subdivided into small libraries. Each individual
library is assembled independently, and a merging algorithm is
employed to combine the small assemblies into a large transcriptome De Bruijn graph construction Given a set of reads and a parameter k that denotes the
k-mer length, a de Bruijn graph is defined by taking each
k-mer that appears in the reads as a vertex, and connecting
two k-mers s1s2 · · · sk and s2 · · · sksk+1 by a directed edge
if the (k −1)-suffix of the first k-mer is the same as the
(k −1)-prefix of the second k-mer and the (k + 1)-mer
s1s2 · · · sksk+1 appears in the reads. Table 1 Data sets used in the evaluation, with organism
denoting the organism, type denoting whether the organism is
model or non-model, libraries denoting the total number of
libraries with the number in parentheses denoting the number of
libraries after combining the biological replicates for
independent assembly in our algorithm, size denoting the total
number of bases in all the reads after quality trimming, and
reference denoting the publication that describes the libraries
Organism
Type
Libraries
Size
Reference
Schizosaccharomyces pombe
Model
32
16.9 G
[4]
Drosophila melanogaster
Model
13
9.4 G
[18]
Arabidopsis thaliana
Model
5
16.1 G
[19]
Drosophila melanogaster
Model
245 (34)
158 G
[20]
Cochliomyia macellaria
Non-model
93 (31)
298 G
New data Since the de Bruijn graph implicitly represents an
assembly of the reads, it is employed by short read assem-
bly algorithms [5, 6]. To reduce noise, a k-mer coverage
cutoff c is imposed to remove k-mers that appear less than
c times. Each maximal non-branching linear path is col-
lapsed into a single node, thus each node can contain a
longer string that is formed from concatenating succes-
sive k-mers that overlap by k −1 letters between each
adjacent pair. After each individual library is assembled Page 45 of 88 Sze et al. De Bruijn graph construction BMC Genomics 2017, 18(Suppl 10):895 Table 2 Comparisons of Schizosaccharomyces pombe transcriptome assemblies of Oases, Trinity and kCombine with k = 25 over
different values of k-mer coverage cutoff c, with transcripts denoting the number of predicted transcripts, n50 denoting the N50 value
of the length of predicted transcripts, blast denoting the number of hits from nucleotide BLAST search of predicted transcripts to
different transcripts of the known transcriptome with e-value below 10−100, full denoting the number of predicted transcripts that are
full length transcripts in which an entire coding region is included in a BLAST alignment, spec denoting the percentage of positions in
the predicted transcripts that are included in a BLAST alignment, unique denoting the number of predicted transcripts that are
uniquely mapped as reported by GMAP with the percentage in parentheses, transloc denoting the number of predicted transcripts
that are translocated as reported by GMAP with the percentage in parentheses, and memory denoting the physical memory
requirement as a power of 2 with Trinity using 32 CPU q
p
y
g
c
Transcripts
n50
Blast
Full
Spec
Unique
Transloc
Memory
Oases
10
8244
6520
6515
5623
91.9%
7268 (88.2%)
1084 (13.1%)
128 GB
20
6509
5412
6458
4597
93.1%
5959 (91.6%)
672 (10.3%)
128 GB
kCombine(Oases)
5
8585
2705
6227
5223
94.8%
8019 (93.4%)
646 (7.5%)
8 GB
10
6603
2620
5767
4443
95.2%
6173 (93.5%)
472 (7.1%)
8 GB
Trinity
5
8568
3368
6490
5279
93.7%
8228 (96.0%)
150 (1.8%)
512 GB
10
7313
2604
6415
4271
95.1%
7064 (96.6%)
156 (2.1%)
512 GB
kCombine(Trinity)
3
6862
2319
6152
3858
96.5%
6628 (96.6%)
262 (3.8%)
8 GB
5
6618
1938
5898
3182
97.1%
6415 (96.9%)
166 (2.5%)
8 GB length above the node length cutoff c2, where c2 is another
given parameter. We only retain a lower ranked tran-
script when it contains a long node that is not cov-
ered by higher ranked transcripts. We group transcripts
into a locus when they share at least one long node. This condition is applied transitively to collect all related
transcripts so that each transcript in a locus shares
at least one long node with another transcript in the
same locus. multiple times, we rank the transcripts in decreasing order
of the number of nodes that form each transcript in the
de Bruijn graph. De Bruijn graph construction BMC Genomics 2017, 18(Suppl 10):895 Page 46 of 88 Table 4 Comparisons of Arabidopsis thaliana transcriptome assemblies of SOAPdenovo-Trans, Trans-ABySS and kCombine with k = 25
over different values of k-mer coverage cutoff c
c
Transcripts
n50
Blast
Full
Spec
Unique
Transloc
Memory
SOAPdenovo-Trans
20
88808
841
27325
1583
78.7%
76542 (86.2%)
44 (0.0%)
4 GB
50
59638
887
24554
1302
82.9%
51402 (86.2%)
38 (0.1%)
4 GB
kCombine(SOAPdenovo-Trans)
3
88611
1103
29057
2666
81.7%
81576 (92.1%)
1324 (1.5%)
4 GB
5
70854
1125
28020
2354
84.1%
65574 (92.5%)
972 (1.4%)
4 GB
Trans-ABySS
20
124125
830
28414
4372
88.0%
119120 (96.0%)
1574 (1.3%)
2 GB
50
62093
1006
25533
3109
91.9%
59420 (95.7%)
1010 (1.6%)
2 GB
kCombine(Trans-ABySS)
3
90109
1147
30333
3261
82.2%
83037 (92.2%)
4242 (4.7%)
4 GB
5
59411
1315
29266
3303
88.0%
54903 (92.4%)
3464 (5.8%)
4 GB
Notations are the same as in Table 2 Table 4 Comparisons of Arabidopsis thaliana transcriptome assemblies of SOAPdenovo-Trans, Trans-ABySS
over different values of k-mer coverage cutoff c Table 4 Comparisons of Arabidopsis thaliana transcriptome assemblies of SOAPdenovo-Trans, Trans-ABySS and kCombine with k = 25
over different values of k-mer coverage cutoff c single library for independent assembly in our algorithm. We trimmed each read by removing bases starting from
the first position that has a quality score of less than 15. We applied Oases and Trinity to the small data sets, and
SOAPdenovo-Trans and Trans-ABySS to the large data
sets. De Bruijn graph construction Since transcripts that are formed from
a larger number of nodes in the de Bruijn graph tend to
be longer, this strategy has a preference towards longer
transcripts while at the same time reduces the number of
translocated transcripts. To remove redundant transcripts, we consider long
nodes in the de Bruijn graph that contain a string of Table 3 Comparisons of small Drosophila melanogaster transcriptome assemblies of Oases, Trinity and kCombine with k = 25 over
different values of k-mer coverage cutoff c
c
Transcripts
n50
Blast
Full
Spec
Unique
Transloc
Memory
Oases
10
41867
1743
24187
8444
91.6%
36080 (86.2%)
7026 (16.8%)
128 GB
20
38377
1164
21606
4922
88.1%
34926 (91.0%)
3830 (10.0%)
128 GB
kCombine(Oases)
5
52282
1144
23573
7152
89.9%
44118 (84.4%)
9938 (19.0%)
8 GB
10
36813
939
20389
4430
88.3%
32591 (88.5%)
5272 (14.3%)
8 GB
Trinity
5
35190
1313
24434
6177
95.7%
32481 (92.3%)
2664 (7.6%)
512 GB
10
29237
970
22388
3986
96.9%
27883 (95.4%)
978 (3.3%)
512 GB
kCombine(Trinity)
3
34150
884
22746
4097
96.0%
31540 (92.4%)
2558 (7.5%)
8 GB
5
26535
753
20525
2932
96.4%
24994 (94.2%)
1214 (4.6%)
8 GB
Notations are the same as in Table 2 Table 3 Comparisons of small Drosophila melanogaster transcriptome assemblies of Oases, Trinity and kComb
different values of k-mer coverage cutoff c Sze et al. Notations are the same as in Table 2 Results and discussion
Data sets We applied our algorithm kCombine to perform tran-
scriptome assemblies using publicly available RNA-Seq
libraries from the sequence read archive [8], including one
set of Schizosaccharomyces pombe libraries, two sets of
Drosophila melanogaster libraries with one small set and
one large set, and one set of Arabidopsis thaliana libraries
(Table 1). We fixed the k-mer length to 25 and varied the k-mer
coverage cutoff c when applying each algorithm. We used
the same value of k to construct the de Bruijn graph in our
algorithm, and set the average length cutoff c1 to 25 and
the node length cutoff c2 to 50. These parameters were
determined by trying a few combinations and choosing
the values that give satisfactory performance. Since the
performance of each algorithm is highly dependent on the
k-mer coverage cutoff c and different values are needed We compare the performance of our algorithm that uti-
lizes an existing algorithm to assemble each individual
library independently to the same algorithm that returns
a single assembly directly from all libraries, with each
library corresponding to one sequencing run of a biolog-
ical sample and all biological replicates combined into a Table 5 Comparisons of large Drosophila melanogaster transcriptome assemblies of SOAPdenovo-Trans, Trans-ABySS and kCombine
with k = 25 over different values of k-mer coverage cutoff c
c
Transcripts
n50
Blast
Full
Spec
Unique
Transloc
Memory
SOAPdenovo-Trans
20
92193
1272
27258
6926
72.1%
81426 (88.3%)
186 (0.2%)
16 GB
50
65273
1422
26671
6832
81.8%
58101 (89.0%)
90 (0.1%)
16 GB
kCombine(SOAPdenovo-Trans)
3
84307
1944
27391
12269
79.6%
77649 (92.1%)
1222 (1.4%)
4 GB
5
66098
2037
27153
11710
84.0%
61227 (92.6%)
976 (1.5%)
4 GB
Trans-ABySS
20
99492
1581
27631
11103
75.7%
90854 (91.3%)
5118 (5.1%)
8 GB
50
62731
1980
27262
12151
86.0%
57508 (91.7%)
2786 (4.4%)
8 GB
kCombine(Trans-ABySS)
3
119772
2628
27612
22113
79.3%
99021 (82.7%)
22638 (18.9%)
4 GB
5
80966
3009
27612
23110
84.5%
64161 (79.2%)
22012 (27.2%)
4 GB
Notations are the same as in Table 2 Table 5 Comparisons of large Drosophila melanogaster transcriptome assemblies of SOAPdenovo-Trans, Trans
with k = 25 over different values of k-mer coverage cutoff c Sze et al. BMC Genomics 2017, 18(Suppl 10):895 Page 47 of 88 Fig. Results and discussion
Data sets 2 Comparisons of the cumulative distribution of the FPKM expression estimates of predicted transcripts that are full length transcripts in
Schizosaccharomyces pombe transcriptome assemblies of Oases, Trinity and their respective applications of kCombine, with k = 25 over different
values of k-mer coverage cutoff c and the range of FPKM values in each assembly divided into 20 intervals of equal width Fig. 2 Comparisons of the cumulative distribution of the FPKM expression estimates of predicted transcripts that are full length transcripts in
Schizosaccharomyces pombe transcriptome assemblies of Oases, Trinity and their respective applications of kCombine, with k = 25 over different
values of k-mer coverage cutoff c and the range of FPKM values in each assembly divided into 20 intervals of equal width percentage of translocated transcripts is about the same,
and kCombine had a higher percentage of translocated
transcripts when the other performance is about the
same. Tables 4 and 5 show that kCombine had improved
performance when compared to SOAPdenovo-Trans and
Trans-ABySS at the expense of having more translocated
transcripts. when applying an existing algorithm during the divide-
and-conquer strategy as opposed to obtaining a single
assembly directly, we report the results that give the most
comparable performance. To assess the extent of translocations in predicted tran-
scripts, we applied GMAP [9] to map the predicted tran-
scripts to the known genome. To investigate whether
our algorithm may systematically remove certain types of
RNA, we applied eXpress [10] to the reads in each data
set with respect to all the predicted transcripts that are
full length transcripts in each assembly to obtain FPKM
expression estimates. In terms of memory requirement, while Oases and Trin-
ity required much more memory than kCombine, the
memory requirement of SOAPdenovo-Trans and Trans-
ABySS was comparable to kCombine. More memory was
needed during the merging step by kCombine than the
independent assembly of each individual library by each
algorithm. Model organisms Figures 2 and 3 show that kCombine recovered com-
parable proportion of full length transcripts with low
expression levels as Oases and Trinity, with slightly
higher proportion than Oases in Schizosaccharomyces
pombe and lower proportion than Trinity in Drosophila
melanogaster. Figures 4 and 5 show that kCombine recov-
ered comparable proportion of full length transcripts
with low expression levels as SOAPdenovo-Trans and Tables 2 and 3 show that kCombine generally had
decreased performance when compared to obtaining sin-
gle assemblies directly from Oases or Trinity. When
compared to Oases, kCombine was able to obtain less
translocated transcripts in Schizosaccharomyces pombe
and comparable percentages of translocated transcripts
in Drosophila melanogaster. When compared to Trin-
ity, kCombine had decreased performance when the Fig. 3 Comparisons of the cumulative distribution of the FPKM expression estimates of predicted transcripts that are full length transcripts in small
Drosophila melanogaster transcriptome assemblies of Oases, Trinity and their respective applications of kCombine, with k = 25 over different values
of k-mer coverage cutoff c and the range of FPKM values in each assembly divided into 20 intervals of equal width Fig. 3 Comparisons of the cumulative distribution of the FPKM expression estimates of predicted transcripts that are full length transcripts in small
Drosophila melanogaster transcriptome assemblies of Oases, Trinity and their respective applications of kCombine, with k = 25 over different values
of k-mer coverage cutoff c and the range of FPKM values in each assembly divided into 20 intervals of equal width Sze et al. BMC Genomics 2017, 18(Suppl 10):895 Page 48 of 88 Fig. 4 Comparisons of the cumulative distribution of the FPKM expression estimates of predicted transcripts that are full length transcripts in
Arabidopsis thaliana transcriptome assemblies of SOAPdenovo-Trans, Trans-ABySS and their respective applications of kCombine, with k = 25 over
different values of k-mer coverage cutoff c and the range of FPKM values in each assembly divided into 20 intervals of equal width Fig. 4 Comparisons of the cumulative distribution of the FPKM expression estimates of predicted transcripts that are full length transcripts in
Arabidopsis thaliana transcriptome assemblies of SOAPdenovo-Trans, Trans-ABySS and their respective applications of kCombine, with k = 25 over
different values of k-mer coverage cutoff c and the range of FPKM values in each assembly divided into 20 intervals of equal width Trans-ABySS,
with
slightly
lower
proportion
than
SOAPdenovo-Trans
and
higher
proportion
than
Trans-ABySS in Arabidopsis thaliana. Model organisms stages (feeding instar, early post feeding, late post feed-
ing, early pupae, early middle pupae, late middle pupae,
and late pupae). We also include additional libraries that
were selected for fast and slow development and a control
sample. Non-model organism Since our goal is to obtain a transcriptome that is as
accurate and complete as possible, we applied kCombine
based on Oases and Trinity due to their generally higher
accuracy in terms of specificity. Table 6 shows that the
assemblies were of high quality. In both cases of kCom-
bine based on Oases and Trinity, the assembly based on
the middle value of the k-mer coverage cutoff c pro-
vides a balanced result between sensitivity and correct-
ness. In terms of memory requirement, Oases and Trinity
required a large amount of memory during the indepen-
dent assembly of each individual library. Since the total
size of libraries is very large, the assembly of each indi-
vidual library is difficult even when the data set is divided
into 31 libraries after combining the biological replicates. The merging step by kCombine required comparably little
memory. We applied our algorithm to assemble the transcriptome
of the blow fly Cochliomyia macellaria from a set of
RNA-Seq libraries that we have constructed (Table 1), in
which the full transcriptome was not available before. The
blow fly Cochliomyia macellaria is a primary colonizer
of human and animal remains, and is important in nutri-
ent cycling [11, 12] and forensic investigations of deaths
[13, 14]. As an agent of myiasis [15, 16] and as a filth
feeding fly, this species can be a veterinary and medical
pest by causing direct damage to hosts and by spreading
pathogenic bacteria [17]. Genomic tools for this blow fly
can be expected to improve the benefits of Cochliomyia
macellaria biology and to ameliorate the negative aspect. Three biological replicates were generated for each Three biological replicates were generated for each
combination of one of four temperatures (20 °C, 25 °C,
30 °C, and fluctuated) and one of seven developmental Fig. 5 Comparisons of the cumulative distribution of the FPKM expression estimates of predicted transcripts that are full length transcripts in large
Drosophila melanogaster transcriptome assemblies of SOAPdenovo-Trans, Trans-ABySS and their respective applications of kCombine, with k = 25
over different values of k-mer coverage cutoff c and the range of FPKM values in each assembly divided into 20 intervals of equal width Fig. Non-model organism 5 Comparisons of the cumulative distribution of the FPKM expression estimates of predicted transcripts that are full length transcripts in large
Drosophila melanogaster transcriptome assemblies of SOAPdenovo-Trans, Trans-ABySS and their respective applications of kCombine, with k = 25
over different values of k-mer coverage cutoff c and the range of FPKM values in each assembly divided into 20 intervals of equal width Page 49 of 88 Sze et al. BMC Genomics 2017, 18(Suppl 10):895 Table 6 Transcriptome assemblies of kCombine based on Oases and Trinity in Cochliomyia macellaria with k = 25 over different values
of k-mer coverage cutoff c, with locus denoting the number of predicted locus, transcripts denoting the number of predicted
transcripts, n50 denoting the N50 value of the length of predicted transcripts, blastx denoting the number of hits from translated
BLAST search of predicted transcripts to different transcripts of the known Drosophila melanogaster transcriptome with e-value below
10−20, and memory denoting the physical memory requirement as a power of 2, with the value to the left of “+” indicating the
memory requirement of the independent assembly of each individual library by Oases and Trinity, and the value to the right of “+” g
y
q
g g
p y
c
Locus
Transcripts
n50
Blastx
Memory
kCombine(Oases)
10
60477
140681
2502
20385
256 GB + 4 GB
20
42334
101421
2349
20579
256 GB + 4 GB
50
30858
67217
1924
20101
256 GB + 4 GB
kCombine(Trinity)
5
46342
153790
1961
22996
512 GB + 16 GB
10
34614
111379
1934
22874
512 GB + 16 GB
20
28756
85093
1567
22595
512 GB + 16 GB Funding Funding
AMT is supported by start-up funds from the College of Agriculture and Life
Sciences at Texas A&M University and Texas AgriLife Research. This work was
supported by the National Institute of Justice grants 2012-DN-BX-K024 and
2015-DN-BX-K020. Points of view in this document are those of the authors
and do not necessarily represent the official position or policies of the U.S. Department of Justice. Publication costs for this work were funded by the
Open Access to Knowledge (OAK) Fund at the Texas A&M University Libraries. g
AMT is supported by start-up funds from the College of Agriculture and Life
Sciences at Texas A&M University and Texas AgriLife Research. This work was
supported by the National Institute of Justice grants 2012-DN-BX-K024 and
2015-DN-BX-K020. Points of view in this document are those of the authors
and do not necessarily represent the official position or policies of the U.S. D
t
t f J
ti
P bli
ti
t f
thi
k
f
d d b th We have developed a divide-and-conquer strategy that
allows memory-intensive de novo transcriptome assem-
bly algorithms to be utilized to construct large assemblies. We have developed a divide-and-conquer strategy that
allows memory-intensive de novo transcriptome assem-
bly algorithms to be utilized to construct large assemblies. After the individual libraries are assembled independently,
the merging algorithm consumes little computational
time and memory. In all our tests, the independent assem-
bly of each individual library can be completed in a few
days when performed in parallel on a computing clus-
ter. The merging step then takes up to a few days for the
largest data sets. After the individual libraries are assembled independently,
the merging algorithm consumes little computational
time and memory. In all our tests, the independent assem-
bly of each individual library can be completed in a few
days when performed in parallel on a computing clus-
ter. The merging step then takes up to a few days for the
largest data sets. Availability of data and materials A software program that implements our algorithm (kCombine) is available at
http://faculty.cse.tamu.edu/shsze/kcombine. Author details Author details
1Department of Computer Science and Engineering, Texas A&M University,
College Station, Mexico, TX, 77843 USA. 2Department of Biochemistry &
Biophysics, Texas A&M University, College Station, Mexico, TX, 77843 USA. 3Department of Entomology, Texas A&M University, College Station, Mexico,
TX, 77843 USA. About this supplement The choice of which algorithm to use depends on
the goal of the assembly. While the memory require-
ment can still be high even after applying the divide-
and-conquer strategy on memory-intensive algorithms for
very large data sets, they are generally more accurate, with
Oases returning more and longer transcripts and Trin-
ity returning more transcripts with low expression levels
and with less translocations. Among the memory-efficient
algorithms, SOAPdenovo-Trans returns transcripts with
less translocations while Trans-ABySS returns more and
longer transcripts with higher specificity. This article has been published as part of BMC Genomics Volume 18
Supplement 10, 2017: Selected articles from the 6th IEEE International
Conference on Computational Advances in Bio and Medical Sciences
(ICCABS): genomics. The full contents of the supplement are available online
at https://bmcgenomics.biomedcentral.com/articles/supplements/volume-
18-supplement-10. Consent for publication Not applicable. Authors’ contributions
S HS
d AMT d
i
d S-HS and AMT designed the computational work. JJP and AMT designed the
molecular experiments. JJP performed the molecular experiments. All authors
read and approved the final manuscript. Competing interests Competing interests
The authors declare that they have no competing interests. Ethics approval and consent to participate Ethics approval and consent to participate Ethics approval and consent to participate
Not applicable. Not applicable. Since there is a decrease in performance in the divide-
and-conquer strategy as the number of libraries increases,
it is better to subdivide into smaller number of libraries
as long as there are enough computational resources to
assemble them independently. To optimize the perfor-
mance, different values of the k-mer coverage cutoff c can
be used on libraries of different sizes. References 1. Xie Y, Wu G, Tang J, Luo R, Patterson J, Liu S, Huang W, He G, Gu S, Li S,
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Li W, Gibbs R, Chen R. The Drosophila melanogaster transcriptome by
paired-end RNA sequencing. Genome Res. 2011;21:315–24. • We accept pre-submission inquiries
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p y
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p 19. Marquez Y, Brown JWS, Simpson C, Barta A, Kalyna M. Transcriptome
survey reveals increased complexity of the alternative splicing landscape
in Arabidopsis. Genome Res. 2012;22:1184–95. 20. Graveley BR, Brooks AN, Carlson JW, Duff MO, Landolin JM, Yang L,
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Oliver B, Celniker SE. The developmental transcriptome of Drosophila
melanogaster. Nature. 2011;471:473–9.
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English
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A SOCIOLOGICAL ANALYSIS OF CYBER CRIME SECURITY AWARENESS AMONG TEENAGERS.
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International journal of advanced research
| 2,016
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cc-by
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A SOCIOLOGICAL ANALYSIS OF CYBER CRIME SECURITY AWARENESS AMONG TEENAGERS. Dr. Grace Varghese, MA, MPhil, PhD. Dr. Grace Varghese, MA, MPhil, PhD. Dr. Grace Varghese, MA, MPhil, PhD. Pullannivelical House Mylapra Town P.O Pathanamthitta Dist Kerala, India. …………………………………………………………………………………………………….... Manuscript Info
Abstract
……………………. ………………………………………………………………
Manuscript History
Received: 23 October 2016
Final Accepted: 21 November 2016
Published: December 2016
Key words:-
Education, Internet, Cybercrime,
Disadvantages, Modern, Students. The advent of e-technology has brought variety of opportunities and
some of these, not surprisingly, are of a criminal nature. The
Cyberspace created by computer technology provides a medium for
doing many things in efficient manner. The automations use of
machine replacing human hands provided great opportunities and
options. The connected computer machines have created a different
world called Cyber world or cyber space. It is a different world
altogether, quite different from our real world! Due to special nature
of this Cyberspace the cyber criminals get maximum opportunity to
commit crime. Cybercrimes are committed by using mobile phones,
computers, scanners, digital cameras and other electronics devices. In the modern educational field rapid changes are happening in a vast
country like India. Enormous developmental work is now being done
in the field of education so as to enable students to get a better vision
of life in other parts of the world and scientific development around
the globe. The “Internet” in India is growing rapidly; it has given rise
to new opportunities in every field: - be it entertainment, business or
education. Internet also has its own disadvantages; one of the major
disadvantages is “Cyber-crime” i.e, illegal activity committed on the
internet. Copy Right, IJAR, 2016,. All rights reserved. …………………………………………………………………………………………………….... g
Pullannivelical House Mylapra Town P.O Pathanamthitta Dist Kerala, India. ………………………………………………………………
The advent of e-technology has brought variety of opportunities and
some of these, not surprisingly, are of a criminal nature. The
Cyberspace created by computer technology provides a medium for
doing many things in efficient manner. The automations use of
machine replacing human hands provided great opportunities and
options. The connected computer machines have created a different
world called Cyber world or cyber space. It is a different world
altogether, quite different from our real world! Due to special nature
of this Cyberspace the cyber criminals get maximum opportunity to
commit crime. Cybercrimes are committed by using mobile phones,
computers, scanners, digital cameras and other electronics devices. In the modern educational field rapid changes are happening in a vast
country like India. ISSN: 2320-5407 ISSN: 2320-5407 Int. J. Adv. Res. 4(12), 1048-1054 1048
Corresponding Author:- Dr. Grace Varghese.
Address:- Pullannivelical House Mylapra Town P.O Pathanamthitta Dist Kerala, India. A SOCIOLOGICAL ANALYSIS OF CYBER CRIME SECURITY AWARENESS AMONG TEENAGERS. Enormous developmental work is now being done
in the field of education so as to enable students to get a better vision
of life in other parts of the world and scientific development around
the globe. The “Internet” in India is growing rapidly; it has given rise
to new opportunities in every field: - be it entertainment, business or
education. Internet also has its own disadvantages; one of the major
disadvantages is “Cyber-crime” i.e, illegal activity committed on the
internet. Copy Right, IJAR, 2016,. All rights reserved. Copy Right, IJAR, 2016,. All rights reserved. …………………………………………………………………………………………
Introduction:-
Information Technology has made revolutionary changes in the dissemination of information and
line transactions on banking, reservation of tickets, downloading of certificates, procurement of go
electronic mails are advantageous and save time, energy and money. But with the increase in the u
internet and mobile phones in our daily life, there is also a corresponding increase in Cybercrimes
high punishments prescribed by law, such crimes are on the rise. Cybercrime is the deadliest type of crime. It can disturb existing set-up within the fraction of a sec
criminality has potentiality to show its effect across the globe. The problem of Cybercrime can b
understanding Cyber technology. The world is witnessing the situation of ever growing field of C
The technological growth rate is too fast. In last decade itself, the number of citizens increased b
Technological adoption is at its peak. However, the problem of Cyber criminality oozing out
adoption is not properly tackled along the line of its growth. Thus, the period has been witnessing t
of development between Cyber technology and infrastructure in preventing Cybercrime. Within
Corresponding Author:- Dr. Grace Varghese. Address:- Pullannivelical House Mylapra Town P.O Pathanamthitta Dist Kerala, India. Education Awareness about Cyber Crime:- Education Awareness about Cyber Crime:- The educational curriculum of these courses on computer technology hardly contains the awareness about
Cybercrime. Students are not aware of the consequences of Cybercrime and its punishment. That is a reason why
they fall in to such traps. So the Parliament of India enacted the Information technology Act in 2000, framing a
uniform regulation in the field of electronics governance, e-commerce and Information technology. Under the act
certain violations are treated as serious crimes and offenders are liable to penal actions. In this context it is important
that awareness is spread on the existing problem and laws related to it. The Government has an important role to play in cyber security assurance in the form of long-term strategies. A
cyber security strategy has been outlined by the Department of Information Technology, Government of India, to
address the strategic objectives for securing country‟s cyber space and is being implemented through the following
major initiatives. It focuses on creation, establishment and operation of cyber security assurance framework aimed at
assisting government, critical infrastructure organizations and other key users of nation‟s economy. Protection of
Critical Information Infrastructure, augmentation of facilities at Computer Emergency Response Team (CERT),
creation of sectoral CERTs, enhancing global co-operation among security agencies, promoting national awareness
programs and training on cyber security, security research and development are some of the strategies adopted by
the Government. It also focuses on creation of national cyber alert system for rapid identification and response to
security incidents and information exchange to reduce the risk of cyber threat and resultant effects. There is no serious provision for creating awareness and putting such initiatives in place in the Act. The government
or the investigating agencies like the Police department (whose job has been made comparatively easier and focused,
thanks to the passing of the IT Act), have taken any serious steps to create public awareness about the provisions
contained in these legislations, which is absolutely essential considering the fact that this is a new area and
technology has to be learnt by all the stake-holders like the judicial officers, legal professionals, litigant public and
the public or users at large. Especially, provisions like scope for adjudication process are never known to many
including those in the investigating agencies. Introduction:- Information Technology has made revolutionary changes in the dissemination of information and knowledge. On
line transactions on banking, reservation of tickets, downloading of certificates, procurement of goods and services,
electronic mails are advantageous and save time, energy and money. But with the increase in the use of computers,
internet and mobile phones in our daily life, there is also a corresponding increase in Cybercrimes. In spite of very
high punishments prescribed by law, such crimes are on the rise. Cybercrime is the deadliest type of crime. It can disturb existing set-up within the fraction of a second. Thus, Cyber
criminality has potentiality to show its effect across the globe. The problem of Cybercrime can be understood by
understanding Cyber technology. The world is witnessing the situation of ever growing field of Cyber technology. The technological growth rate is too fast. In last decade itself, the number of citizens increased by 100% in India. Technological adoption is at its peak. However, the problem of Cyber criminality oozing out of technological
adoption is not properly tackled along the line of its growth. Thus, the period has been witnessing the different pace
of development between Cyber technology and infrastructure in preventing Cybercrime. Within a short span of 104
Corresponding Author:- Dr. Grace Varghese. Address:- Pullannivelical House Mylapra Town P.O Pathanamthitta Dist Kerala, India. 1048
Corresponding Author:- Dr. Grace Varghese. Address:- Pullannivelical House Mylapra Town P.O Pathanamthitta Dist Kerala, India. 1048 ISSN: 2320-5407 Int. J. Adv. Res. 4(12), 1048-1054 decades, a huge gap between Cyber technology and Cyber criminality has happened without any hope of bridging
the gap. A common man who is a user of computer and internet and cell phone is unaware of the traps set by clever criminals
in the Cyber space and the ways to get rid of them. Computers evolved as a result of man‟s search for fast and
accurate calculating devices. Forging documents is one of the best examples of this kind of a Cybercrime. Image
morphing, circulation of defamatory comments and threats through mobile phones, taking photographs without
consent and consistent blackmailing may be some other examples. With the rise in the internet users Cyber criminals are also increasingly targeting cyber space to commit their illegal
designs. Majority of the victims of Cybercrimes are children of adolescent age. Introduction:- Recently in Kerala an incident was reported in which three girl students of which one committed suicide and the
other two are surviving remind victims of Cybercrimes. The nude pictures of these girls were circulated on the
internet by their own boyfriends who were later arrested by police.( Mathrubhumi, 2014) In another incident in Kerala, teachers were blackmailed by one of their students by morphing the body of another
person and uploading their photos in Face book. This is how students use the internet illegally and commit
Cybercrime. (Malayala Manoramma, 2014) Nowadays people have started to think that hacking is only hijacking Facebook accounts. A relevant example is of
the official website of the Pakistan People‟s Party (PPP) that was hacked earlier this month by a 16year old Indian
boy, apparently due to Bilawal Bhutto‟s provocative speech on Kashmir.( Indian Express 2014) Need and significance of the study:- It is important that initiatives are taken in the education sector itself for understanding the issues involved in
Cybercrime. One important step forward in this direction is to spread awareness among adolescent students against
the crime and encouraging them to utilize the positive side of the internet. Every person in the world is dependent
on Cyberspace in one way or another, directly or indirectly. Cyberspace is open to participation by all. And
interactions between students, scholars and teachers across continents are made possible by the internet. If internet is
used wisely it can help in minimizing the gap between the „haves‟ and „have-nots‟ of the world. This study aims to test the effectiveness of an instructional module that is meant to be distributed among students
with the aim to spread awareness on Cyber crime and the laws against it. It is the need of the hour that such a study
is conducted in schools as newspaper reports suggest that adolescent people, especially those who go to higher
secondary school fall prey to such activities the modern thief can steal more with a computer than with a gun. Tomorrow‟s terrorists may be able to do more damage with a keyboard than with a bomb. Information and
technology are all encompassing. They have brought transformation from paper to paperless world. Internet has
dramatically changed the way we think, the way we live, the way we govern, the way we do commerce and the way
we perceive ourselves. Information Technology encompasses all walks of life, all over the world. Cyberspace is full of opportunities. However like in real life, good and bad practices exist in this sphere too. Moral,
civil and criminal wrongs are committed in internet. It has now given a new way to express criminal tendencies. It
has become a space for power play between nations. Instead of utilizing the good side of the internet, new ways to
intimidate and threaten the perceived enemies are created. Fighting bad practices spread through internet is found to
be a challenge by educational institutions all over the world. It is in the interest of the educational sector all over the world that innovative and enabling legal infrastructure in
tune with the times is put in place. Students who are gullible due to their tender age and inexperience are particularly
vulnerable as far as criminal tendencies in internet are concerned. Education Awareness about Cyber Crime:- Cyber law is important because it touches almost all aspects of transactions and activities on and concerning the
Internet, the World Wide Web and Cyberspace. Initially it may seem that Cyber laws are a very technical field and
that it does not have any bearing to most activities in Cyberspace. But the actual truth is that nothing could be
further than the truth. Whether we realize it or not, every action and every reaction in Cyberspace has some legal
and Cyber legal perspectives. 1049 Int. J. Adv. Res. 4(12), 1048-1054 Int. J. Adv. Res. 4(12), 1048-1054 ISSN: 2320-5407 Cyber law Awareness program will cover:- Cyber law Awareness program will cover:- 1. The basics of Internet Security. 2. Basic information on Indian Cyber Law. 3. Impact of technology aided crime. 4. Indian IT Act on covering the legal aspects of Online Activities 5. Types of Internet policies required for an Organization. 5. Types of Internet policies required for an Organization. Information is a critical asset. Therefore, it must be protected from unauthorized modification, destruction and
disclosure. This brochure describes information security concepts and defines steps required to properly safeguard
information. It is the responsibility of everyone employee and home user to become familiar with good security
principles and to follow the information protection tips. p
p
vestigator, in the case of this study, tries to focus on the creation of a module that will spread
Cybercrime and the laws that are related to the crime. Therefore the investigator, in the case of this study, tries to focus on the creation of a module
awareness on the Cybercrime and the laws that are related to the crime. Need and significance of the study:- New legal tools developed against exploitation of
vulnerable groups in cybercrime should focus on students. It is a fact that cybercrime is here to stay and it has to be
treated as a „necessary evil‟. As far as education sector is concerned, welfare of the students should be compelling
enough for us to move forward regardless of the problems and we should move ahead by creating legal tools for
utilizing the positive sides of internet while minimizing the harm. We are in the period of „Information Explosion‟. The electronic and digital media have completely changed the
Indian social scenario. All types of information reach home easily. This has not only changed the psychology of
people but also changed their behaviour and life style. Even though information explosion has affected all sections
of society, it is the youth and adolescents who are most influenced by the technological spurt. Information
Revolution has given impetus to the process of Globalisation. Due to information explosion all types of control on
the flow of information are impossible. Newspapers are read online and mobile use is expanding. Internet is the heart of the Information revolution. Youth
and adolescents are more diligent Cyberspace consumers. Internet occupies a central position in present day mass
media society and internet activities seem to be a natural element in leisure. Internet use today serves not only as a
mirror of society but also as an instrument of social change .The use of Cyberspace is now more and more 1050 Int. J. Adv. Res. 4(12), 1048-1054 Int. J. Adv. Res. 4(12), 1048-1054 Int. J. Adv. Res. 4(12), 1048-1054 ISSN: 2320-5407 ISSN: 2320-5407 individualized. Furthermore, in the contemporary era the geographical distances play a vague role. Social relations
are lifted from the physical context. This is all because of technological development. In network society, we
organise our lives around mass and social networking society. It seems natural for us to see the media as anchored in
the private spheres. A radical change is evident in the norms and lifestyle of adolescents in modern India. The
impact of westernization and globalization has made young generation techno survey and leisure oriented. One of the most noticeable changes is the adolescent‟s preoccupation with electronic gadgets such as mobiles, I
pads, laptops or in other words their heightened “technical consciousness”. The past ten years have witnessed the
emergence of a new phenomenon i.e. Social Networking sites. Need and significance of the study:- Networking is as old as Human Society. Human
beings have always sought to live in a social environment. However, now networking has shifted towards Internet to
significant extent. This is an activity in which millions of internet users are involved both in their leisure time and at
work. However, there has been very little research on the impact of these sites on adolescents in the Indian context. The world of Internet continues to expand as does the research about its use by adolescents. Thus, with this
background an attempt is made in this study to analyse the impact of networking on adolescents and their life style
.An attempt has been made to find out their activities on social networking sites and whether they provide a new
venue for adolescents to behave in a deviant manner. The study also attempts to identify why adolescents are more likely to involve in deviant behaviour through social
networking sites. For this individual attributes such as age, sex, education and parent‟s income are used to construct
a predictive model. Further, this study also analyses various types of Cybercrimes on internet sites in which
adolescents are either victims or perpetrators. For an understanding of the ideas, values, attitudes and aspirations of
the adolescents, certain sociological variables are taken into consideration to know how they influence the behaviour
of the adolescents. The variables such as age, sex composition, family income, and school background provide the
profile of adolescents. Adolescents form an important group in the use of networking sites. The study covers the adolescents ranging from 13 years to 17 years. Information technology has spread throughout
the world. The computer is used in each and every sector wherein Cyberspace provides equal opportunities to all for
economic growth and human development. As the user of Cyberspace grows increasingly diverse and the range of
online interaction expands, there is expansion in the Cybercrimes i.e. breach of online contracts, perpetration of
online torts and crimes etc. Due to these consequences there was need to adopt a strict law by the Cyber space
authority to regulate criminal activities relating to Cyberspace and to provide better administration of justice to
victims of Cybercrime. In the modern cyber technology world, it is very much necessary to regulate Cybercrimes
and most importantly cyber law should be made stricter in the case of Cyber terrorism and hackers. Module:- Module explores some key educational concepts and applies them to clinical teaching and learning situation (www,
webcrawler.com 2014) A unit of education or instruction with a relatively low student to teacher ratio, in which a single topic or a small
section of a broad topic is studied for a given period of time. A unit of education or instruction with a relatively low student to teacher ratio, in which a single topic or a small
section of a broad topic is studied for a given period of time. Need and significance of the study:- Mostly people don‟t know about Cybercrime and Cyber laws. So today‟s need to aware the society about
Cybercrimes and Cyber laws. The present studies the investigator try to give the awareness programme about
Cybercrime and its law. Cyber Law:- Cyber law is the area of law that deals with the Internet's relationship to technological and electronic elements,
including computers, software, and hardware information systems. (National Research Council,2013) In the present study cyber law is the area of law that deals with the internet‟s relationship to technological and
electronic element, including computers, software, and information systems. Cybercrime:- y
Cyber-crime is a crime involving, using or relating to computers especially the internet. Crimes involving use of
information technology or usage of electronic means in furtherance of crime are covered under the scope of Cyber-
crime. The ambit of the term includes all kinds of objectionable or unlawful activities, misuse or abuse taking place
in cyber world, through or against the computer, internet, and telecommunication networks run with computer
system or technology. The scope of Cyber-crime is bound to increases in view of the ever increasing technological
advancement in the area. Security Awareness:- y
Awareness as the quality on state of being aware consciousness aware is defined as informed, cognizant, conscious,
and sensible. 1051 Int. J. Adv. Res. 4(12), 1048-1054 Int. J. Adv. Res. 4(12), 1048-1054 Int. J. Adv. Res. 4(12), 1048-1054 ISSN: 2320-5407 ISSN: 2320-5407 In this study the purpose of cyber security awareness presentation is simply to focus attention on cyber security. Awareness presentation is intended to allow individuals to recognize information technology security concerns and
respond. Investigator go review seriously so many literature regarding the chapter Cybercrime and related concepts,
investigator didn‟t identify any study relating the awareness of an testing of an instruction that develop the
awareness among secondary school students, that is motivating her to take up study on the area of Cybercrime
especially among the school children more one last of experience a school teacher she handled so many issues
relating the Cybercrime among the children that also learn to develop an instructional module for developing
information security awareness. In the present study the module deals with basic awareness of various Cyber related Crimes, p
y
y
1. Consists of information regarding various Cyber gadgets, sources of Cyber manipulation, creation of various
fake information. 1. Consists of information regarding various Cyber gadgets, sources of Cyber manipulation, creation of various
fake information. 2
d
i h 2. Laws and punishments 3. Educating the students for the purposeful use of Cyber gadget. 3. Educating the students for the purposeful use of Cyber gadg 4. Spreading awareness on different legal acts that may apply in the case of Cybercrimes. 4. Spreading awareness on different legal acts that may apply in the case of Cybercrimes. Different Types of Cybercrimes:- Cyber-crimes are new generation crimes where the achievements of information technology are misused for
criminal activities. Such crimes may be committed against the governments, individuals, and institutions. Generally
most of the Cyber-crimes are adversely affect individuals, and society at large. The common types of Cyber-crimes
are discussed under the following heads: Conclusion:- The present study was concluded in such a way that the Module on Cyber laws is more effective, The investigator
should develop the module about different types of Cybercrimes, and its consequences. It helps to develop the
curiosity among the students to know about the crimes in our locality. Such controls include personal security and
incident handling measures to prevent fraud and security breaches. The study observes that majority of the adolescents are online daily for along period of time due to which their
studies are affected negatively resulting in poor academic grades. It is also observed that students who use social
networking sites on a regular basis tend to have negative effect on health such as stomach aches, poor sleep patterns,
eye strain, anxiety and depression. The study reflected that the adolescence are having on online addiction in which
an individual forgets his/her real life and finds solace in virtual life. This trend makes a person avoid his real life
problems and indulge in cyber space. This is having adverse effect to the personality of adolescents who have
framed their dual identity one in the real world and another in the virtual world. The present study proves that the Module on Cyber law is highly effective for the development of cybercrime
awareness among higher secondary school students. The role of an educator is not merely teaching students theories and facts.To enhance the learning experience of
every students, the educator has to cater to many different learning styles and capacities. The présent study provide sastrong base for analyzing the technological implications on adolescent behaviour in the
domain of society. In this study is to shed light on the role of social networking sites on adolescent internet users and
their effects on social, physical and psychological aspects of adolescents. The present studies shows that adolescent
users belonging to 16-17 years of age group have easy access to internet in comparison with other age groups and
they easily convince their parents for the same. Here the studies have suggested that the unique features of Cyber law for developing information security awareness
is a powerful technique device. This technique once learnt, can be utilized throughout an individual‟s academic life
and modified to be used in working life as well. Conclusion:- As an educator, it is imperative that all potentially beneficial
teaching and learning techniques are explored and passed on to students in order to cultivate realization that the
learning process is an infinite continuum. Cyber Terrorism:- Cyber terrorism is the convergence of cyberspace and terrorism. It refers to unlawful attacks and threads of attacks
against computers, networks and the information stored therein that are carried out to intermediates or coerce a
country‟s government or citizens in furtherance of political or social objectives. Serial attacks against crucial
infrastructures could count as acts of cyber terrorism. The cyber terrorism attacks and threats includes interfering
and disrupting information and transportation systems, emergency services and government services,
communication networks, infrastructure systems, banking and fiancé system. Different types of cybercrimes, it act and its punishments:-
Cybercrimes
IT Act
Punishments
Impersonation (fake) profiles, in community
networks, fake e-mail Ids etc…)
66 D of IT Act
419 IPC
3 years and fine up to 1 Lakh
Cheating (lottery scam using e mails and SMS, fake
websites, mail asking financial helps etc…)
66 A, 66A (b) (c),66 D 420
IPC
3 Years and fine
Software theft (using the source code of software
and creating another). 66 A r/w 43 (j)
3 years or fine up to 5 lakhs
or both
Morphing ( editing the photo with nude photo and
publishing )
66 C r/w 43 (i),66 A(b),509
IPC
3 years or fine up to five lakh
or both
Cyber terrorism (crimes against the Nations by
means of computer /mobile/internet etc. 66 F of IT Act
Up to life imprisonment. Spoofing (hiding identity and using others identity)
66D of IT Act
3 years and fine up to 1 lakh
Website Hacking
66C r/w 43(a)
3 years or fine up to 5 lakh or
both 1052 Int. J. Adv. Res. 4(12), 1048-1054 Int. J. Adv. Res. 4(12), 1048-1054 ISSN: 2320-5407 Email Hacking (methods are phishing, new website. Account registration) etc
66 E r/ w 43(a)
3 years or fine up to 5 lakh or
both
Pornography (makes people especially children
leading psychic states that cause rape, child
molestation, sex, exhibitionism, sexual violence
etc…)
67,and 67A of IT Act
5(7) years and fine up to 10
lakh
Child pornography(hidden in pornographic sites)
67 B of IT Act
5(7) years and fine up to
Credit / ATM card Frauds (using scanner and
camera)
420 IPC 66 D 0F IT ACT
3 years and fine up to 1 lakh
Taking video /photo of women or spreading it
119 (b) of KP ACT
3 years or RS.10,000 or both References:
1. Akers, Ronald L. (1998), Social Learning and Social Structure: A General Theory of Crime and Deviance,
North Eastern University Press, Boston.
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Crime and Militancy. The University of Michigan press, USA
3. Baudrillard, J. (1984), Simulations. Semiotext (e), New York
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5. Carey, James W. (1993). Everything that rises must diverge: Notes on Communication, Technology & the
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6. Das, Biswajet and Sahoo, Jyoti (2011), Social Networking Sites. A Critical Analysis of its impact on Personal
and Social life. International Journal of Business and Social Science, Vol.2 (14)
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New York. 1. Akers, Ronald L. (1998), Social Learning and Social Structure: A General Theory of Crime and Deviance,
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Criminology ,Vol 1 Issue 2
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1. http://www.legalserviceindia.com/article/1129-Torts-In-India.html ( 20/5/2015)
2. http://www.cyberlawsindia.net/black-hatml.7/4/2015
3. http://searchsecurity.techtarget.com/definition/adware.27/5/2015. 4. http:// www.mit.gov.in/content/strategic-approch.20/2/2015. 5. http://www.interpol.int/public/technologycrime/crimeprev/itsecurity.asp#21/4/2015 8. Jaishankar,K (2007),Establishing a Theory of Cyber Crime ,Pg 7-9International Journal of Cyber
Criminology ,Vol 1 Issue 2 8. Jaishankar,K (2007),Establishing a Theory of Cyber Crime ,Pg 7-9International Journal of Cyber
Criminology ,Vol 1 Issue 2 gy ,
9. Mohan Rama.U and Barkha(2014).Cyber Law and Crimes.Harita Graphics,Hyderabad (
)
y
p
y
10. Thomas, Douglas and Loader Brian (2000),Cybercrime Law Enforcement, Security and Surveillance in the
Information Age, Pg 8,Routledge, London. f
g
g
g
11. Young, Kimberly (1998), Internet Addiction: The Emergence of anew clinical Disorder, Cyber Psychology and
Behavior, Vol. 1-No.3, Pg. 237-244. p
g
g
pp
5. http://www.interpol.int/public/technologycrime/crimeprev/itsecurity.asp#21/4/2015 References:- 1. Akers, Ronald L. (1998), Social Learning and Social Structure: A General Theory of Crime and Deviance,
North Eastern University Press, Boston. y
y
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Prevalence of clarithromycin resistance in Helicobacter pylori in Santiago, Chile, estimated by real-time PCR directly from gastric mucosa
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BMC gastroenterology
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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. Gonzalez-Hormazabal et al. BMC Gastroenterology (2018) 18:91
https://doi.org/10.1186/s12876-018-0820-0 Gonzalez-Hormazabal et al. BMC Gastroenterology (2018) 18:91
https://doi.org/10.1186/s12876-018-0820-0 Open Access Abstract Background: Current available treatments for Helicobacter pylori eradication are chosen according to local
clarithromycin and metronidazole resistance prevalence. The aim of this study was to estimate, by means of
molecular methods, both clarithromycin and metronidazole resistance in gastric mucosa from patients infected
with H.pylori. Methods: A total of 191 DNA samples were analyzed. DNA was purified from gastric mucosa obtained from
patients who underwent an upper gastrointestinal endoscopy at an university hospital from Santiago, Chile,
between 2011 and 2014. H.pylori was detected by real-time PCR. A 5’exonuclease assay was developed to detect
A2142G and A2143G mutations among H.pylori-positive samples. rdxA gene was sequenced in samples harboring
A2142G and A2143G mutations in order to detect mutations that potentially confer dual clarithromycin and
metronidazole resistance. Results: Ninety-three (93) out of 191 DNA samples obtained from gastric mucosa were H.pylori-positive (48.7%). Clarithromycin-resistance was detected in 29 samples (31.2% [95%CI 22.0–41.6%]). The sequencing of rdxA gene
revealed that two samples harbored truncating mutations in rdxA, one sample had an in-frame deletion, and 11
had amino acid changes that likely cause metronidazole resistance. Conclusions: We estimated a prevalence of clarithomycin-resistance of 31.8% in Santiago, Chile. Three of them
harbor inactivating mutations in rdxA and 11 had missense mutations likely conferring metronidazole resistance. Our results require further confirmation. Nevertheless, they are significant as an initial approximation in re-
evaluating the guidelines for H.pylori eradication currently used in Chile. Keywords: Helicobacter pylori, Clarithromycin, Metronidazole, 23S rRNA, rdxA, Mutation Keywords: Helicobacter pylori, Clarithromycin, Metronidazole, 23S rRNA, rdxA, Mutation Prevalence of clarithromycin resistance in
Helicobacter pylori in Santiago, Chile,
estimated by real-time PCR directly from
gastric mucosa Patricio Gonzalez-Hormazabal1*
, Maher Musleh2,3, Susana Escandar2, Hector Valladares2,3, Enrique Lanzarini2,3,
V. Gonzalo Castro1, Lilian Jara1 and Zoltan Berger2 Background lymphoma, and iron deficiency anemia. The evidence
strongly suggests that H.pylori eradication is beneficial
in treatment or prevention of those diseases [1]. One
treatment for H.pylori eradication is the triple-therapy
that comprises clarithromycin, amoxicillin, and proton
pump inhibitor (PPI), nevertheless, triple-therapy is not
recommended in regions with high clarithomycin resist-
ance rate [1]. Global claritrhomycin-resistance rates in
adults are contrasted among various countries, from 1%
in The Netherlands to 37.6% in Italy [2]. In light of this
observation, the last Maastricht V/Florence consensus Helicobacter pylori is a gram-negative bacillus that colo-
nizes the gastric mucosa. The infection is associated
with both gastric and extragastric diseases such as peptic
ulcer disease, gastric atrophy, gastric adenocarcinoma,
gastric
MALT
(mucosa-associated
lymphoid
tissue) * Correspondence: patriciogonzalez@uchile.cl * Correspondence: patriciogonzalez@uchile.cl
1Human Genetics Program, Institute of Biomedical Sciences, School of
Medicine, University of Chile, Av. Independencia 1027, 8380453 Santiago, CL,
Chile * Correspondence: patriciogonzalez@uchile.cl
1Human Genetics Program, Institute of Biomedical Sciences, School of
Medicine, University of Chile, Av. Independencia 1027, 8380453 Santiago, CL,
Chile Full list of author information is available at the end of the article Full list of author information is available at the end of the article * Correspondence: patriciogonzalez@uchile.cl
1Human Genetics Program, Institute of Biomedical Sciences, School of
Medicine, University of Chile, Av. Independencia 1027, 8380453 Santiago, CL,
Chile Subjects Subjects invited to participate were patients who under-
went a physician-requested upper gastrointestinal endos-
copy, at the Department of Gastroenterology at the
University of Chile Clinical Hospital between July 2011
and July 2014. Participants were asked to donate a sam-
ple of gastric mucosa obtained from antrum and corpus. This study was approved by the Ethical Committee of
the following institutions: University of Chile School of
Medicine (#023/2011), and University of Chile Clinical
Hospital (#029/2011). The study was performed in
accordance with the Declaration of Helsinki. DNA was iso-
lated from gastric mucosa using FavorPrep Tissue DNA Ex-
traction Mini Kit (Favorgen Biotech Corp, Taiwan, China). Samples were identified as H.pylori-positive or H.pylori-ne-
gative by detection of the 16S rRNA gene by real-time PCR
as described by Kobayashi et al. [10]. Gonzalez-Hormazabal et al. BMC Gastroenterology (2018) 18:91 Page 2 of 5 Chile. To do so, we developed a test to detect A2142G
and A2143G mutations in 23S gene by 5’nuclease assay. We also aimed to detect mutations in the rdxA gene
among ClaR isolates. on management of H.pylori [1] recommends that in
high-clarithromycin resistance regions (> 15% resistance
prevalence), quadruple therapy is necessary. If dual clari-
thromycin
and
metronidazole
resistance
is
> 15%,
bismuth-containing quadruple therapy is the recom-
mended first-line treatment. Analysis of 23S rRNA mutations y
A segment of the 23S rRNA gene from position 1911 to
2200 was amplified by PCR using primers described by
Agudo et al. [11], and sequenced by Sanger sequencing
(Service provided by Macrogen Inc., Korea). A 5’exo-
nuclease assay was developed to detect A2142G and
A2143G mutations. We designed primers and probes for
each allele in highly conserved regions from position
1911 to 2200, comparing sequences obtained from 44
randomly selected H.pylori-positive subjects recruited for
this study. Both mutations were tested in separate assays. Primers were: F: 5’-GAGCTGTCTCAACCAGAG-3′ and
R: 5’-GCGCATGATATTCCCATTA-3′. Probes detecting
2142G (5’-CAAGACGGGAAGACCCC-3′) and 2143G
(5’-CAAGACGGAGAGACCCC-3′) alleles were dual-labeled
with 5’FAM-3’BHQ1, and the probe complementary to the
2142A - 2143A allele (5’-CAAGACGGAAAGACCCCG-3′)
was dual-labeled with 5’HEX-3’BHQ1. Primers and
probes were synthesized at Macrogen Inc. (Korea). The
5’exonuclease was carried out in a StepOne Real Time
PCR system (Applied Biosystems, USA) using 5X HOT
FIREPol Probe qPCR Mix Plus (ROX) (Solis BioDyne,
Estonia) according to manufacturer’s directions. Metronidazole is a nitroimidazole synthesized as an in-
active antibiotic prodrug. The bactericidal effect of the drug
is mainly explained by the cytotoxic effect of molecules
resulting from a reduction of metronidazole intracellularly
in facultative anaerobic bacteria. Reduction of metronida-
zole in H.pylori is mediated mainly by a nonessential
oxygen-insensitive NADPH nitroreductase encoded by the
rdxA gene. NADPH-flavin-oxidoreductase (FrxA) is also
involved in the reduction of metronidazole [3]. Various
authors found that metronidazole resistance arises from
inactivating mutations in rdxA found only in resistant
isolates, and missense mutations have also been involved in
metronidazole resistance [8]. On the other hand, the role of
inactivating mutations in the frxA gene is controversial
since truncating mutations have been found in sensitive iso-
lates, and some studies report that frxA mutations alone
may not be enough to confer resistance [8]. Vallejos et al. [9] studied resistance to clarithromycin and metronidazole
in 50 isolates obtained from patients in Santiago, Chile. Forty-five and twenty percent respectively were resistant,
resulting in 13.2% of the prevalence of dual clarithromycin
and metronidazole resistance. Methods Clarithromycin belongs to the class of macrolide antibi-
otics. The mechanism of action is the inhibition of protein
synthesis of bacteria by binding to the 23S ribosomal sub-
unit (23S rRNA). Mutations in the peptidyltransferase re-
gion encoded in domain V of 23S rRNA are responsible
for macrolide resistance [3]. Three mutations in the 23S
rRNA gene account for nearly all clarithromycin-resistant
(ClaR) strains: A2143G, A2142G and A2142C to a small
extent [3]. Standard method (antibiogram), after culture
or molecular tests, can be used to detect H.pylori and clar-
ithromycin resistance directly from the gastric biopsy [1]. Epidemiological studies evaluating regional clarithromycin
resistance have been conducted based on detection of
A2142G and A2143G in DNA isolated from gastric biop-
sies [4, 5]. Commercial tests detecting both mutations are
available [3], performing at a sensitivity rate of > 94% and
a specificity rate of > 98.5% compared to the culture sus-
ceptibility test. Recently, Tamayo et al. [6] found a high
concordance between Etest for clarithromycin and 23S
rRNA mutations. To date, only Garrido and Toledo [7]
have evaluated primary resistance of H.pylori to clarithro-
mycin in clinical isolates from Santiago, Chile. They found
primary resistance in 10 out of 50 isolates by agar dilution. Resistant isolates harbor A2142G or A2143G mutations. Results A total of 191 subjects were included in this study,
124 were female (64.9%) and 67 male (35.1%). The
median age was 46 years (range 19 years to 82 years). None of them was treated for H.pylori eradication. Table 1 Missense changes and truncating mutations in the rdxA gene among 28 clarithromycin-resistant samples
Amino acid positiona
Sample
6
16
31
49
53
62
67
73
75
88
90
97
98
118
123
151
162
166
172
175
183
26695b
Q
R
T
T
H
L
A
N
E
S
R
H
G
A
V
V
G
P
V
E
A
EN02
H
. E
. . . . . . . K
T
. . . . . . I
Qd
. EN05
. . E
. . . . . . . . . . . . . . A
I
. . EN07
H
. E
. . . Vd
. . . K
. . T
. . . . . . . EN11
. . . . . . . . . . K
. . . . . . . I
. . EN15
H
. . . R
V
. . . P
. . S
. . L
. . I
. . EN16
H
. . . R
V
. . . P
. T
. . . . . . . . . EN20
. H
. . . . . . . . . . . T
. . . . I
. . EN30
. . . . . . . . . . . Y
. . . . . S
. . V
EN33
H
. . . . . . . Q
. . . . . . . . . . . . EN36
H
. . . X d
. . . . . . . . . . . . . . . . EN42
. . . . . . . . . . . . S
. . . . . I
Q
. EN61
. . E
. . . . . . . K
. . . . . . . I
Q
. EN70
H
. E
. . . . . . . K
. . . . . . . I
Q
V
EN82 c
. . . . . . . . . . . Detection of mutations in rdxA gene The sequence of rdxA was obtained from samples harbor-
ing 23S 2142G or 2143G. Primers for PCR amplification
of rdxA (F: 5’-CRTTAGGGATTTTATTGTATGC-3′ R:
5’-CTCTTRCCCAAWGCGATC-3′) were designed with
AliView 1.18 for Linux
in
conserved
regions
after The aim of the present study was to estimate claritho-
mycin resistance in 93 clinical isolates from Santiago, Gonzalez-Hormazabal et al. BMC Gastroenterology (2018) 18:91 Page 3 of 5 Page 3 of 5 comparing 83 sequences of H.pylori obtained from
GenBank. PCR was performed using Q5 Hot Start High
Fidelity DNA Polymerase (New England Biolabs, USA)
according to manufacturer’s directions, using an annealing
temperature of 56 °C. Electrophoretograms were obtained
by Sanger sequencing (Service provided by Macrogen
Inc., Korea). Ninety-three (93) out of 191 DNA samples obtained
from gastric mucosa were H.pylori-positive (48.7%). Clarithomycin resistance was detected using a 5’exonucle-
ase assay in 29 samples (31.2% [95%CI 22.0–41.6%]). Of
them, 9 harbored 2142G (31.0%) and 20 (69.0%) harbored
2143G. All ClaR samples were confirmed by Sanger se-
quencing. Both Sanger sequencing and 5’exonuclease
allowed us to detect heteroresistance, i.e., the coexistence
of resistant and susceptible bacterial strains in the
same patient [12]. Sixteen (16) out of 93 H.pylori sub-
jects were homoresistant to clarithromycin (17.2%). In
addition, we detected heteroresistance in 13 out of 93
patients (14.0%). Ninety-three (93) out of 191 DNA samples obtained
from gastric mucosa were H.pylori-positive (48.7%). Clarithomycin resistance was detected using a 5’exonucle-
ase assay in 29 samples (31.2% [95%CI 22.0–41.6%]). Of
them, 9 harbored 2142G (31.0%) and 20 (69.0%) harbored
2143G. All ClaR samples were confirmed by Sanger se-
quencing. Both Sanger sequencing and 5’exonuclease
allowed us to detect heteroresistance, i.e., the coexistence
of resistant and susceptible bacterial strains in the
same patient [12]. Sixteen (16) out of 93 H.pylori sub-
jects were homoresistant to clarithromycin (17.2%). In
addition, we detected heteroresistance in 13 out of 93
patients (14.0%). a(.): same amino acid as for reference strain
bCorresponds to amino acid sequence of RdxA from strain 26,695
cIn-frame deletion of 39 nucleotides which causes the deletion of amino acids 80 to 92
dCoexistence of both nucleotide substitutions in the same sample. X: Stop codon a(.): same amino acid as for reference strain
b In frame deletion of 39 nucleotides which causes the deletion of amino acids 80 to 92
dCoexistence of both nucleotide substitutions in the same sample. X: Stop codon Results . . . . . . . . . . EN89
. . E
. . . . . . . . Y
. T
. . . . . . . EN97
H
. . . . . . . . . K
. S
. T
. . . I
Q
. EN115
H
. . . . . Vd
. . . . . . . . . . . . . . EN117
. . E
. . . . . . . . . . . . . . . I
Q
. EN125
. . . . . . . . . . . . . T
. . . . I
Q
. EN131
. . . . . . . . . . . . . . . . . . . . . EN137
. . E
. . . . . . . . Y
. . T
. . . . . . EN148
. . E
. . . . . . . . . . . . . . . I
Q
V
EN155
H
. . . . . . X d
. . . . . . . . . . . . . EN161
H
. . . R
V
. . . P
. . S
. . L
. . I
. . EN167
H
. E
. . . . . . . . Y
. . T
. Rd
. . . . EN179
H
. E
. . . . . . . . . . T
. . . . I
Q
V
EN187
. . . M
. . . . . . . . . . . . . . . . . EN190
H
H
E
. . . . . . . K
. . T
. . . . . . . a(.): same amino acid as for reference strain
bCorresponds to amino acid sequence of RdxA from strain 26,695
cIn-frame deletion of 39 nucleotides which causes the deletion of amino acids 80 to 92
dCoexistence of both nucleotide substitutions in the same sample. X: Stop codon 1 Missense changes and truncating mutations in the rdxA gene among 28 clarithromycin-resistant samples Gonzalez-Hormazabal et al. BMC Gastroenterology (2018) 18:91 Gonzalez-Hormazabal et al. Competing interests Competing interests
The authors declare that they have no competing interests. p
g
The authors declare that they have no competing interests. The authors declare that they have no competing interests. Results BMC Gastroenterology (2018) 18:91 Page 4 of 5 The rdxA gene was sequenced in 28 ClaR samples. One ClaR sample was not available at the time of ana-
lysis. A nonsense substitution (H53stop) was found in
one sample; and an insertion of A at nucleotide 186,
leading to a frameshift resulting in N73stop, was found
in a different sample. Therefore, two samples harbored
truncating mutations in rdxA. One sample had an
in-frame deletion of 39 nucleotides which causes the de-
letion of amino acids 80 to 92 (ASALMVVCSLKPS). Table 1 shows the amino acid variations in rdxA found
in ClaR samples. stability), P166S = −1.50 (large decrease in stability). Thus,
we propose that E75Q (1 sample), P166A (1 sample), and
P166S (1 sample) could confer MtzR. However, a pheno-
typic testing shloud be carry out to confirm it. Funding This work was supported by Fondo Nacional de Desarrollo Científico y
Tecnológico -Chile- (FONDECYT) [1151015]. The authors declare that the
funding body had no participation in the design of the study and collection,
analysis, and interpretation of data and in writing the manuscript. Abbreviations
ClaR: Clarithromycin-resistant; PPI: Proton pump inhibitor Abbreviations
ClaR: Clarithromycin-resistant; PPI: Proton pump inhibitor Among 28 ClaR samples, two of them harbored trun-
cating mutations and one an in-frame deletion in rdxA,
and therefore are likely resistant to metronidazole. A
total of 22 amino acid substitutions was found. Thirteen
of them (Q6H, T31E, H53R, L62 V, R90K, H97T, H97Y,
G98S, A118T, V123 T, V172I, E175Q, A183V) have been
previously observed in metronidazole-resistant (MtzR)
as well as in metronidazole-sensitive strains [13–17]. Therefore, those substitutions probably do not confer re-
sistance to metronidazole. Four substitutions (R16H, A67V,
S88P and G162R) were found in 8 samples. R16H and S88P
have been observed only in MtzR strains [13–15]. In fact,
Arg 16 is one of the residues of RdxA that interacts with the
cofactor flavin mononucleotide (FMN) [18]. A67V corre-
sponds to the replacement of a small alanine by a bulky val-
ine in the protein core, and is associated with resistance [8,
19]. Analysis from the crystallographic data of RdxA reveals
that G162 is involved in FMN binding [18]. Hence, missense
mutations R16H, A67V, S88P and G162R likely cause MtzR. Nevertheless, phenotypic testing is needed to confirm that
those mutations actually cause MtzR. 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. Conclusions We estimated a prevalence of ClaR of 31.2% in Santiago,
Chile, which is above the suggested 15% threshold to
abandon triple-therapy, according to the recommendation
of Maastricht V/Florence consensus. Three out of 28 ClaR
samples harboring inactivating mutations in rdxA may be
clearly MtzR, and 11 of them had missense mutations in
rdxA likely confering MtzR. Further phenotypic testing is
needed to obtain a clinically significant conclusion from
rdxA sequencing results. Nevertheless, the estimated
prevalence of ClaR is significant as an approximation in
re-evaluating the triple-therapy for H.pylori eradication
currently used in Chile. Acknowledgements
ld l k
k We would like to acknowledge Paul Zuckerman for his help in proofreading
the manuscript. Availability of data and materials The data that support the findings of this study are available from the
corresponding author upon reasonable request. Ethics approval and consent to participate pp
p
p
This study was approved by the Ethical Committee of the following
institutions: University of Chile School of Medicine (#023/2011), and
University of Chile Clinical Hospital (#029/2011). The study was performed in
accordance with the Declaration of Helsinki. All subjects are adults and gave
written informed consent. Five amino acid changes in RdxA (T49 M, E75Q,
V151 L, P166A and P166S) have not been described previ-
ously in the literature. None of them have been observed
as an important amino acid for the RdxA function. We
predicted the effect of those substitutions using the crys-
tallographic data of RdxA protein (Protein Data Bank
accession number 3QDL) in the I-Mutant Suite server
[20], which estimates protein stability changes (expressed
as delta delta G values) upon single-point mutations from
the protein structure. Delta-delta G values and the predic-
tion were as follows: T49 M = −0.41 (neutral stability),
E75Q = −0.78 (large decrease of stability), V151 L = −0.44
(neutral
stability),
P166A = −1.28
(large
decrease
in Authors’ contributions PGH conceived, designed the study and drafted the manuscript. PGH, MM
and ZB were responsible for analysis and interpretation of data. VGC and LJ
performed molecular analysis. MM, SE, HV and EL were involved in
acquisition of samples and data. All authors read and approved the final
manuscript. Discussion A total of 29 out of the 93 H.pylori positive samples re-
sulted resistant to clarithomycin (31.2%). This estimation
is higher than the 20% of ClaR reported by Garrido and
Toledo in Chile [7]. Taken together, ClaR resistance re-
ported for Chile is above the suggested 15% threshold to
abandon triple-therapy, according to the recommenda-
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1 1Human Genetics Program, Institute of Biomedical Sciences, School of
Medicine, University of Chile, Av. Independencia 1027, 8380453 Santiago, CL,
Chile. 2Department of Gastroenterology, University of Chile Clinical Hospital,
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Microstructural Evolution And Mechanical Properties of FCAW Joints In 9% Ni Steel Prepared With Two Types of Ni-Based Weld Metals
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Microstructural Evolution And Mechanical Properties of FCAW
Joints In 9% Ni Steel Prepared With Two Types of Ni-Based Weld
Metals Jiyong Hwang
Korea Institute of Industrial Technology
Kwangsu Choi
Korea Institute of Industrial Technology
Sang Min Lee
Korea Institute for Defense Analyses
Hyo Yun Jung
(
hjung@kitech.re.kr
)
Korea Institute of Industrial Technology Introduction For this, a metallographic analysis of the microstructure was performed in each of
the joint interfaces formed by single beads of Hastelloy 609 and Inconel 625 weld metal, which were welded under identical
conditions. Introduction In 2020, the International Maritime Organization (IMO) imposed a new upper limit of 0.5% sulfur content in the shipping fuel (reduced
from 3.5%) [1]. The new regulation aims to reduce the harmful sulfur oxide emissions from ships and prompts the shipping industry
to adopt environmentally friendly fuels. Liquefied natural gas (LNG), a well-established low-sulfur alternative to petroleum, is drawing
attention as a next-generation clean fuel because it significantly reduces the emission of air pollutants such as sulfur oxide, nitrogen
oxide, carbon dioxide, and soot [2]. The LNG-fueled ships require cryogenic fuel tanks suitable for the long-term storage of LNG at temperatures below its liquefaction
temperature of −162°C. One of the most widely used materials for the construction of such cryogenic storage tanks is 9% Ni steel,
owing to its good tensile strength and excellent impact toughness, which makes it highly suitable for cryogenic applications [3, 4]. Ni-
based superalloys such as Inconel® and Hastelloy® are used as welding materials for this steel, and mainly comprise an austenite
phase, resulting in good crack resistance and excellent low-temperature fracture toughness [4, 5]. These properties make them highly
suitable for welding 9% Ni steel. However, in the weld joint between dissimilar metals (Fe-based base material and Ni-based
superalloys), a dilution area is created where the material microstructure and properties change. This dilution phenomenon plays a
crucial role in determining the toughness of the cryogenic storage tanks made by 9% Ni steel [6, 7]. It is important to understand the weld-joint properties of 9% Ni steel with Ni-based superalloys, and their increasing use in
manufacturing cryogenic LNG storage tanks requires a more in-depth examination of the mechanical and structural properties of the
Ni-based weld joints to ensure an improved low-temperature toughness. The conventional welding methods such as shielded metal
arc welding (SMAW) and submerged arc welding (SAW) have been widely used for 9% Ni steel and Ni-based weld metal welding [4, 5,
8−10]. Moreover, flux-cored arc welding (FCAW) is expected to have a good efficiency and adaptability for welding 9% Ni steel, but the
microstructure and properties of the FCAW joint remain to be explored. In this study, the Ni-based superalloys, Inconel® and Hastelloy®, were used as welding metals for 9% Ni steel. The microstructure
and low-temperature toughness of the welding area were systematically examined depending on the type of Ni-based weld metal
used for the FCAW of the 9% Ni steel base metal. Research Article 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 The International Journal of Advanced Manufacturing Technology on
April 18th, 2022. See the published version at https://doi.org/10.1007/s00170-022-09173-5. Page 1/12 Page 1/12 Abstract The microstructural and mechanical evaluation of 9% Ni steel with Flux-Cored Arc Welding was performed with two different Ni-
based weld metals: Inconel 625 and Hastelloy 609. Weld metals showed the microstructural change depending on the temperature
gradient and crystal growth rate for each region during the cooling after welding. At the bottom of the weld metal, which is rapidly
cooled in contact with the cold base metal, a cellular/planar growth was exhibited due to a large temperature gradient and low crystal
growth rate. While, columnar dendrites were exhibited in the central region cooled relatively slowly and precipitates were observed in
the interdendritic region. In the low-temperature toughness test, the absorbed impact energies were 89 and 55 J for Inconel 625 and
Hastelloy 609, respectively. When Inconel 625 is used as the weld metal compared to Hastelloy 609, the high content of the γ
stabilizer and martensite start temperature decreasing elements leads to the formation of a thicker γ-phase layer and thinner
martensite layer in the transition region. In addition, high content of these elements suppresses the martensite transformation and
maintains the stability of the weld joint interface even at low temperatures, resulting in the higher absorbed impact energy. Results The SEM images of the Inconel 625 bead reveal columnar-dendritic growth from most areas of the center of the bead till the bottom
(Fig. 2e). Dendrite-to-cellular transition morphologies appear in some areas near the weld joint interface, as shown in Fig. 2f. The SEM images of the Inconel 625 bead reveal columnar-dendritic growth from most areas of the center of the bead till the bottom
(Fig. 2e). Dendrite-to-cellular transition morphologies appear in some areas near the weld joint interface, as shown in Fig. 2f. Moreover, similar to Hastelloy 609, precipitates are observed in the interdendritic regions. The compositional analysis reveals that the
interdendritic regions are relatively rich in Mo and Nb (Table 4). These results suggest the liquid segregation of Mo and Nb during the
weld joint solidification [8, 13]. The compositional analysis of the interdendritic precipitates reveals that the Nb-enriched phase is
NbC. It has been established that the Inconel 625 alloy solidifies to the γ phase, M6C, and Nb-rich phase (NbC, Laves) from its liquid
state through the following process: L→ L + γ→ L + γ + NbC→ L + γ + NbC +M6C→ L + γ + NbC +M6C + Laves→ γ + NbC +M6C +
Laves [13−15]. The secondary phase (NbC) has a high Nb content and can be distinguished from the Laves phase. However, it was
difficult to identify the Laves phase and M6C even through magnified observations in this study. It was envisaged that a more
detailed sample analysis in the future might be able to overcome this limitation. The SEM images of the joint interface welded with Hastelloy 609, together with the EDS line analysis and EBSD images of the
interface area, are presented in Fig. 3. In this case, elements with less than 1 wt. % content are excluded from the analysis since such
low values are considered to be below the accuracy limit of the EDS measurement. Compositional analysis was performed along a
straight line over the joint interface, as shown in Fig. 3a. The results from the corresponding compositional analysis, shown in
Fig. 3b, reveal a transition area in the Hastelloy 609 sample near the joint interface, where the elemental composition changes
linearly in the direction of the weld metal. This transition area has a thickness of approximately 20 µm, where the Fe content
decreases rapidly, while the Ni, Cr, Mo, and W contents increase. Experimental Methods Charpy impact tests were
performed for the weld metal area in a cryogenic environment at −196 ℃. Results Fig. 1 shows the XRD pattern of the base metal, weld metals, and weld-joint fabricated using FCAW of 9% Ni steel with Hastelloy 609
and Inconel 625. The base metal shows the diffraction peaks of martensite and austenite-γ phases (Fig. 1a). However, the weld metal
as well as the weld-joint of the base metal and weld metal mainly show diffraction peaks of the austenite-γ phases. The austenite
peaks for Hastelloy 609 and its joint interface (Fig. 1c and 1e) appear at a lower scattering angle compared to those of the sample
welded with Inconel 625 (Fig. 1b and 1d). The most likely reason for the peak shift is the difference in the solid-solution content of
the minor element. Fig. 2 shows the cross-sectional OM and SEM images of the joints welded with Hastelloy 609 and Inconel 625, respectively. With
Fig. 2a and Fig. 2d, it is noted that the two weld joints are composed with clearly distinguishable weld metal, joint interface, and the
heat-affected zone (HAZ) of the base metal. The center of the Hastelloy 609 bead comprises of columnar dendrites elongated
upward, while the bottom of the bead exhibits dendrite-to-cellular transition morphologies (Fig. 2b and Fig. 2c). Precipitates of
various sizes and shapes are located at the interdendritic regions. Similar precipitates were also observed at the bottom of the bead. In addition, the number of precipitates decreased toward the bottom of the bead. EDS was performed to investigate the
compositional distribution in the weld metal, and the results are presented in Table 4. The Mo content in the interdendritic regions is
higher than that in the dendritic regions, indicating that Mo tends to be rejected to an interdendritic residual liquid. Moreover, the
interdendritic precipitates have high contents of W, indicating a high W solubility. These results imply that the solidification of the
molten Hastelloy 609 starts in the γ phase, while the σ and ρ phases start to form at later stages. In this case, the low Cr content of
Hastelloy 609 stabilizes the ρ phase rather than the σ phase [11, 12]. The precipitates located at the interdendritic regions are inferred
to be in the ρ phase based on the compositional analysis of Cieslak [12] and Qiu [11]. The µ phase, which results from the long-term
phase transformation of the ρ phase, is not present as heat was not applied for a sufficiently long time during FCAW [12]. Experimental Methods The chemical composition of the base metal and weld metals was investigated using the inductively coupled plasma (ICP, Perkin
Elmer, Optima 7300DV, USA) analysis. The composition of the 9% Ni steel (Nippon steel & Sumitomo metal corporation) and Ni-
based weld metals are listed in Table 1 and Table 2, respectively. Flux-cored wire (FCW) with a diameter of 1.2 mm was the weld
metal. The welding was carried out in a single pass using FCAW for each of the weld metals. The welding conditions are summarized
in Table 3. The same welding conditions were used for all the specimens, whereas different weld metal samples were used each
time. The microstructure of the weld joint was analyzed using optical microscopy (OM, Hirox, MXB-5000REZ, Japan) and field
emission-scanning electron microscopy (FE-SEM, Hitachi, SU8230, Japan) equipped with an electron backscatter diffraction (EBSD, Page 2/12 Page 2/12 Oxford, Aztec HKL Nordlys Nano, United Kingdom) equipment. Compositional analysis of the weld joint was performed through point
and line measurements using energy-dispersive spectrometry (EDS, Oxford, Ultim Max100, United Kingdom). For the crystal structure
analysis, X-ray diffraction (XRD, Panalytical, EMPYREAN, United Kingdom) was performed within a 2θ scan range of 20–100° with
Cu-Kα radiation (λ = 1.54056 Å). The scan speed was 3 °/min scaling with a step size of 0.02°. The beam size was 7 mm 1/2 degree. A multi-layer FCAW process was performed to evaluate the low-temperature impact toughness of 9% Ni steel with different weld
metals. The multi-layer welded sample had a V-type notch with a 2 mm depth in the weld metal region. Charpy impact tests were
performed for the weld metal area in a cryogenic environment at −196 ℃. Oxford, Aztec HKL Nordlys Nano, United Kingdom) equipment. Compositional analysis of the weld joint was performed through point
and line measurements using energy-dispersive spectrometry (EDS, Oxford, Ultim Max100, United Kingdom). For the crystal structure
analysis, X-ray diffraction (XRD, Panalytical, EMPYREAN, United Kingdom) was performed within a 2θ scan range of 20–100° with
Cu-Kα radiation (λ = 1.54056 Å). The scan speed was 3 °/min scaling with a step size of 0.02°. The beam size was 7 mm 1/2 degree. A multi-layer FCAW process was performed to evaluate the low-temperature impact toughness of 9% Ni steel with different weld
metals. The multi-layer welded sample had a V-type notch with a 2 mm depth in the weld metal region. Discussion The microstructural analysis of the weld metal showed that the type of the dendritic growth varied depending on the position within
the weld metal. The columnar dendrite structures in the central part of the weld bead exhibited well-defined secondary arms. At the
bottom of the bead, the secondary dendritic arms nearly disappeared, and the morphology transitioned from dendritic to cellular. The
temperature gradient and the crystal growth rate are the two main parameters that govern the microstructure of the weld joint during
solidification. With the decrease of the temperature gradient and the increase of the growth rate, the microstructure transitions from
planar through cellular, cellular-dendritic, and columnar-dendritic, to equiaxed dendritic [16]. The bottom of the weld bead that is in
contact with the cold base metal cools more rapidly during the welding process, while the top of the bead cools slower. As a result,
the planar or cellular growth predominantly occurs at the bottom of the bead. In contrast, in the center of the bead, the cooling is
slower, which results in a lower temperature gradient and higher growth rate. Consequently, a columnar dendrite growth with
distinctively developed secondary arms takes place in this part of the weld bead. During welding, a dilution area is formed between the 9% Ni steel and Ni-based weld metal, where both metals are mixed. The
transition area of the joint interface where a compositional change occurs due to the dilution process consists of the martensite layer
(region 1) and the γ-phase-planar/cellular growth area (region 2). This microstructure of the transition area can be attributed to the
temperature gradient and the elemental composition of the area. Since Ni is a γ-phase stabilizing element, the Ni content in the base
metal (9% Ni steel) helps to stabilize the γ-phase at low temperatures. In the case of Hastelloy 609, the γ-phase in region 2 with a Ni
content of 20–45 wt. % was preserved despite the Fe dilution. Additionally, planar growth morphology appeared in the same region
due to the large temperature gradient and low growth rate induced by the rapid cooling from the cold base metal. In region 1, high Fe
dilution of 60–90 wt. % and low Ni content of 10–20 wt. % were observed. Thereby, the martensite in the corresponding area became
a relatively stable phase. Results An area farther away from the interface (region 3 in Fig. 3a) exhibits
20 wt. % Fe, indicating a considerably wide dilution area. The transition area consists of a uniform martensite layer (region 1 in
Fig. 3c and 3d) and a γ-phase-planar growth area (region 2 in Fig. 3c and 3d). Page 3/12 Page 3/12 Page 3/12 Page 3/12 The SEM image of the Inconel 625 joint interface (Fig. 4) reveals a similar structure to that of the Hastelloy 609 joint interface
(Fig. 4a). The transition area near the interface is 33 µm thick. Within the transition area, the Fe content decreases when scanning in
the direction from the base metal toward the weld metal, while the Ni, Cr, Mo, and Nb contents increase simultaneously (Fig. 4b). A
high Nb content is detected in the interdendritic precipitates, confirming the presence of an NbC phase. In Fig. 4c and 4d, two layers
can be distinguished within the transition: the martensite layer (region 1 in Fig. 4c and 4d) and the γ-phase-cellular growth area
(region 2 in Fig. 4c and 4d). To assess the toughness of the weld metals after welding, multilayer welding specimens, prepared from the same base metal and
weld metal pairs, were subjected to low-temperature impact tests. The absorbed impact energies were higher than or equal to 55 J
and 89 J for Hastelloy 609 and Inconel 625, respectively, as shown in Fig. 5. These values greatly exceed the impact absorption
energy standards stipulated by the classification regulation (standard of Charpy impact energy: at −196 ℃, 27 J in the transverse
direction and 41 J in the longitudinal direction). Discussion Moreover, rapid cooling accelerates the martensite formation; hence, the presence of fine martensites
indicate rapid cooling of the area. In the case of Inconel 625, the joint interface was also structured in two layers (region 1 and region
2). The factors that governed the growth morphology of the layers were the rapid cooling and the composition. The martensite layer
was formed in areas with a high Fe and low Ni content, while the γ-phase-cellular morphology appeared in areas with a high Ni and
low Fe content. In addition, as a result of the dilution phenomenon, the lattice parameter of the γ-phase increases due to the solid-solution of Mo and
Nb atoms (with larger atomic radii than Ni and Fe) in the γ-phase during welding (for reference, the atomic radii of Ni, Fe, Mo, and Nb
are 0.124, 0.126, 0.139, and 0.146 nm, respectively). This lattice expansion generates a low-angle shift of the γ peak in the XRD
spectrum. The EDS data, summarized in Table 4, indicate that the Hastelloy 609 sample has a Mo content of approximately 10–16
wt. %, while the Inconel 625 sample has Mo and Nb contents in the range of 7–10 wt. %. Therefore, the γ-phase peaks of Hastelloy
609 bead region and its joint interface appear at a lower diffraction angle because of the higher Mo solid-solution content. Moreover,
the intensity of the γ-phase peak is higher at the weld joint interface compared to the peak intensity at the weld metal area, indicating
an intense texture growth. The most likely reason for the strong γ-phase peak is the aligned dendrite-to-cellular transition morphology
that developed near the joint interface (or the bottom area of the weld metal bead) due to heat transfer. There is a deviation in the
preferential growth direction for the joint interface areas by Hastelloy 609 and Inconel 625. The low volume fraction of the NbC and ρ
phases prevents their reliable identification in the XRD pattern. In addition, as a result of the dilution phenomenon, the lattice parameter of the γ-phase increases due to the solid-solution of Mo and
Nb atoms (with larger atomic radii than Ni and Fe) in the γ-phase during welding (for reference, the atomic radii of Ni, Fe, Mo, and Nb
are 0.124, 0.126, 0.139, and 0.146 nm, respectively). This lattice expansion generates a low-angle shift of the γ peak in the XRD
spectrum. Discussion The EDS data, summarized in Table 4, indicate that the Hastelloy 609 sample has a Mo content of approximately 10–16
wt. %, while the Inconel 625 sample has Mo and Nb contents in the range of 7–10 wt. %. Therefore, the γ-phase peaks of Hastelloy
609 bead region and its joint interface appear at a lower diffraction angle because of the higher Mo solid-solution content. Moreover,
the intensity of the γ-phase peak is higher at the weld joint interface compared to the peak intensity at the weld metal area, indicating
an intense texture growth. The most likely reason for the strong γ-phase peak is the aligned dendrite-to-cellular transition morphology
that developed near the joint interface (or the bottom area of the weld metal bead) due to heat transfer. There is a deviation in the
preferential growth direction for the joint interface areas by Hastelloy 609 and Inconel 625. The low volume fraction of the NbC and ρ
phases prevents their reliable identification in the XRD pattern. Page 4/12 Page 4/12 There are microstructural changes that occur due to the cooling rate variations, by the position within the Hastelloy 609 and Inconel
625 beads, during the welding process. In both cases, aligned columnar dendrites were formed in the center of the bead, while the
morphology changed from dendrite-cellular to cellular/planar growth toward the bottom of the bead. Moreover, precipitates were
formed in the interdendritic regions of both the samples. The transition area of the weld joint was structured with two layers
(martensite layer and γ-phase cellular/planar layer) depending on the actual Fe and Ni content resulting from the dilution in the
transition area. However, the microstructure of the transition area differs for the two weld metals. The use of Inconel 625 resulted in a
transition area of 33 µm, compared to a 20 µm thick transition area for Hastelloy 609, with an 8 µm thick martensite layer. In contrast,
the martensite layer for Hastelloy 609 was 11 µm thick. Elements such as Cr, Mo, and Ni lower the onset temperature for martensite
transformation (martensite start temperature, Ms) in steel [6]. This implies an increase in the γ-phase volume fraction and the
inhibition of martensite formation. The high content of those elements in the transition area of Inconel 625 can explain the thick γ-
phase layer and thin martensite layer observed in the transition. Discussion This microstructure and the elemental composition of the transition area can influence the low-temperature fracture toughness. At
low-temperatures, the difference in the thermal expansion coefficients between the Ni-based weld metal and the Fe-based base metal
can cause cold shrinkage and mechanical stress at the joint interface. Such stresses can induce a transformation from austenite γ-
phase to martensite in the joint transition area. In turn, the stress from the martensite transformation can augment the deformation in
the adjacent HAZ, and the residual deformation can lead to crack propagation [6, 17, 18]. Furthermore, the two materials commonly
exhibited precipitation formation in the interdendritic regions upon microstructural analysis (the ρ phase for 609 weld metal and NbC
for 625 weld metal). Studies have reported that even a small number of interdendritic precipitations can induce intergranular crack
propagation at low-temperatures, thus promoting brittle failure of the sample [8]. Additionally, the bond between the dendrite and the
precipitate weakens due to the difference in the contraction rate between the γ-phase and the precipitate, which deteriorates the low-
temperature fracture toughness. This crack propagation mechanism diagram is illustrated in Fig. 6. However, the impact absorption
energies of Hastelloy 609 and Inconel 625 were higher than the value suggested in the classification regulations. Further, the high
content of the γ-stabilizing and Ms-decreasing elements present in Inconel 625 prevents martensite transformation. The γ-phase
stabilization process (martensite inhibition process) enhances the stability of the joint interface at low temperatures. In addition, in
Inconel 625, the initially thin martensite layer and thick γ-phase layer that help to maintain the residual deformation at a low level
increase its toughness. Consequently, a high low-temperature impact absorption energy of 89 J, and an outstanding low-temperature
toughness was observed when Inconel 625 was used. Depending on the type of weld metal utilized, the difference in the dilution area extent and microstructure can be generated even
under equal welding conditions. Furthermore, some microstructure types in the transition area along with the above-mentioned
precipitation can negatively affect the fracture toughness. Therefore, in the future, a detailed discussion is necessary on the
mechanical properties and microstructures dependent on weld metals and welding conditions. Conclusions The mechanical properties of the FCAW weld joints between 9% Ni steel and Ni-based weld metals (Hastelloy 609 and Inconel 625)
were investigated using the microstructure analysis. The findings of this study can be summarized in the following points: At the heterojunction between the 9% Ni steel (Fe-based base metal) and Ni-based weld metals, a compositional transition area
was created due to a dilution process at the joint interface. The transition areas for Hastelloy 609 and Inconel 625 were 20 μm
and 33 μm, respectively. Depending on the distribution of Fe and Ni in the transition area, two clearly distinguished layers were
identified: the martensite layer, where the Fe content is relatively high, and the γ-phase-cellular/planar layer, where the Ni content
is high. The microstructure and compositional distribution at the welded part play an important role in determining the low-
temperature fracture toughness of the material. First, the observed precipitates in the interdendritic regions facilitate the
intergranular crack propagation, thus adversely affecting the material toughness. In contrast, the high content of the γ-stabilizing
and Ms-decreasing elements impede martensite transformation, resulting in a relatively thick and stable γ-phase layer. Thus,
excellent toughness can be achieved by hindering additional martensite transformation and maintaining stability at the joint
interface. These microstructures were found in the transition area with the application of Inconel 625, which is considered to
have a higher toughness compared to Hastelloy 609. At the heterojunction between the 9% Ni steel (Fe-based base metal) and Ni-based weld metals, a compositional transition area
was created due to a dilution process at the joint interface. The transition areas for Hastelloy 609 and Inconel 625 were 20 μm
and 33 μm, respectively. Depending on the distribution of Fe and Ni in the transition area, two clearly distinguished layers were
identified: the martensite layer, where the Fe content is relatively high, and the γ-phase-cellular/planar layer, where the Ni content
is high. The microstructure and compositional distribution at the welded part play an important role in determining the low-
temperature fracture toughness of the material. First, the observed precipitates in the interdendritic regions facilitate the
intergranular crack propagation, thus adversely affecting the material toughness. In contrast, the high content of the γ-stabilizing
and Ms-decreasing elements impede martensite transformation, resulting in a relatively thick and stable γ-phase layer. Thus,
excellent toughness can be achieved by hindering additional martensite transformation and maintaining stability at the joint
interface. Authors’ contributions Hyo Yun Jung : supervision, review and editing Funding This study has been conducted with the support of the Korea Institute of Industrial Technology as "Research project (kitech JA-21-
0008)". Conclusions These microstructures were found in the transition area with the application of Inconel 625, which is considered to
have a higher toughness compared to Hastelloy 609. In the Hastelloy 609 weld joint, an 11 μm martensite layer was observed, amounting to nearly half the thickness of the transition
area. In contrast, despite having a thicker transition zone, the thickness of the martensite layer in the Inconel 625 weld joint was In the Hastelloy 609 weld joint, an 11 μm martensite layer was observed, amounting to nearly half the thickness of the transition
area. In contrast, despite having a thicker transition zone, the thickness of the martensite layer in the Inconel 625 weld joint was Page 5/12 Page 5/12 Page 5/12 approximately 1/4 of the total thickness of the transition zone. In the Inconel 625 transition area, the high content of elements
such as Cr, Mo, and Ni, which reduce the martensite transformation starting temperature of steel, inhibited the formation of
martensite and increased the austenite volume fraction (thin martensite layer and thick γ-phase layer). The variations in temperature gradients and crystal growth rates result from different cooling rates in various positions inside
the bead. In the bead center, the relatively low temperature gradient and high growth rate result in directional columnar dendrite
structures. Toward the bottom of the weld bead, where the temperature gradient is higher and the growth rate is lower, a
transition from dendrite-cellular structures to cellular/planar morphologies was observed. Additionally, precipitates of the ρ
phase and NbC were found in the interdendritic regions of the Hastelloy 609 and Inconel 625 bead, respectively. The variations in temperature gradients and crystal growth rates result from different cooling rates in various positions inside
the bead. In the bead center, the relatively low temperature gradient and high growth rate result in directional columnar dendrite
structures. Toward the bottom of the weld bead, where the temperature gradient is higher and the growth rate is lower, a
transition from dendrite-cellular structures to cellular/planar morphologies was observed. Additionally, precipitates of the ρ
phase and NbC were found in the interdendritic regions of the Hastelloy 609 and Inconel 625 bead, respectively. Conflict of interest/Competing interests The authors have no conflicts of interest to declare that are relevant to the content of this article. References J Mater Eng Perform 29:3004-3015. https://doi.org/10.1007/s11665-020-04861-3 [16] Sindo K (2002) Welding metallurgy, 2nd edn. John Wiley & Sons, Inc., New Jersey pp 155-166 [17] Fritz A, Jonathan DHP, Peter S, Michael O, Otmar K, Jozef P, Franz DF (2018) The effect of residual stresses and strain reversal on
the fracture toughness of TiAl alloys. Mater Sci Eng A 709:17-29. https://doi.org/10.1016/j.msea.2017.10.010 [17] Fritz A, Jonathan DHP, Peter S, Michael O, Otmar K, Jozef P, Franz DF (2018) The effect of residual stresses and strain reversal on
the fracture toughness of TiAl alloys. Mater Sci Eng A 709:17-29. https://doi.org/10.1016/j.msea.2017.10.010 References [1] IMO 2020 – cutting sulphur oxide emissions. International Maritime Organization. https://www.imo.org/en/MediaCentre/HotTopics/Pages/Sulphur-2020.aspx [2] LNG: an energy of the future. elengy. https://www.elengy.com/en/lng/lng-an-energy-of-the-future.html Page 6/12 [3] Du WS, Cao R, Yan YJ, Tian ZL, Peng Y, Chen JH (2008) Fracture behavior of 9% nickel high-strength steel at various temperatures:
part I. tensile tests. Mater Sci Eng A 486:611-625. https://doi.org/10.1016/j.msea.2007.09.057 [4] Kobelco (2011) Kobelco welding today. Kobe steel, LTD 14:1-12. https://www.kobelco.co.jp/ english/welding/files/kwt2011-02.pdf
[5] John ND, John CL, Samuel DK (2009) Welding metallurgy and weldability of nickel‐base alloys. 9% Ni steels, 1st edn. John Wiley [4] Kobelco (2011) Kobelco welding today. Kobe steel, LTD 14:1-12. https://www.kobelco.co.jp/ english/welding/files/kwt2011-02.pdf [5] John ND, John CL, Samuel DK (2009) Welding metallurgy and weldability of nickel‐base alloys. 9% Ni steels, 1st edn. John Wiley
& Sons, Inc., New Jersey, pp 357 [6] Weidong M, Yan C, Min W, Xueming H (2020) Microstructure characteristics and properties of fusion boundary in 9%Ni steel joint
filled with Ni-based alloy. Mater Charact 165:110390. https://doi.org/10.1016/j.matchar.2020.110390 [7] Dupont JN, Kusko CS (2007) Martensite formation in austenitic/ferritic dissimilar alloy welds. Weld J 86:51s-54s. [8] Weidong M, Yuzhang L, Yan C, Min W (2018) Cryogenic fracture toughness of 9%Ni steel flux cored arc welds. J Mater Process
Technol 252:804-812. https://doi.org/10.1016/j.jmatprotec. 2017.10.026 [9] Price AH (2013) Significant system parameters influencing HAZ properties in 9%Ni steel. Sci Technol Weld join 6:255-260. https://doi.org/10.1179/136217101101538758 [10] Yoon YK, Kim JH, Shim KT (2012) Mechanical characteristics of 9% Ni steel welded joint for LNG storage tank at cryogenic. Int J
Mod Phys Conf Ser 06:355-360. https://doi.org/ 10.1142/S2010194512003431 [11] Zhijun Q, Bintao W, Hanliang Z, Zhiyang W, Alan H, Yan M, Huijun L, Ondrej M, David W (2020) Microstructure and mechanical
properties of wire arc additively manufactured Hastelloy C276 alloy. Mater Des 195:109007. https://doi.org/10.1016/j.matdes.2020.109007 [12] Cieslak MJ, Headley TJ, Romig AD (1986) The welding metallurgy of Hastelloy alloys C-4, C-22, and C-276. Metall Mater Trans A
17:2035-2047. https://doi.org/10.1007/BF02645001 [13] Camila PA, Francisco CAM, Luís FGS, Jorge CFJ, Leonardo SA, Matheus CM (2019) Performance of the Inconel 625 alloy weld
overlay obtained by FCAW process. Rev Mater 24: https://doi.org/10.1590/S1517-707620190001.0627 [14] Cieslak MJ, Knorovsky GA, Headley TJ, Romig AD (1989) The solidification metallurgy of alloy 718 and other Nb-containing
superalloys. Superalloys 718:59-68. https://doi.org/10.7449/1989/ SUPERALLOYS_1989 _59_68 _59_68 [15] Camila PA, Soraia SS, Jorge CFJ, Luı´s FGS, Leonardo SA, Matheus CM, Jean D (2020) Microstructural characterization of
Inconel 625 nickel-based alloy weld cladding obtained by electroslag welding process. Tables Page 7/12
Tables
Table 1 Chemical composition of 9% Ni steel (wt. %)
Fe
Ni
C
Si
Mn
P
S
9% Ni steel
Bal. 8.860
0.060
0.250
0.600
0.007
0.001 Page 7/12
Tables
Table 1 Chemical composition of 9% Ni steel (wt. %)
Fe
Ni
C
Si
Mn
P
S
9% Ni steel
Bal. 8.860
0.060
0.250
0.600
0.007
0.001 Table 1 Chemical composition of 9% Ni steel (wt. %) Table 1 Chemical composition of 9% Ni steel (wt. %) Page 7/12 Table 2 Chemical composition of weld metals (wt. %)
Weld
metal
Ni
Cr
Mo
Nb+Ta
Fe
W
Si
Mn
Ti
C
Cu
P
S
Hastelloy
609
(609
weld
metal)
Bal. 15.700
16.200
6.600
3.300
0.270
0.270
0.010
0.012
0.002
Inconel
625
(625
weld
metal)
Bal. 21.600
8.600
3.570
1.800
0.370
0.280
0.110
0.030
0.030
0.010
0.003
Table 3 The welding conditions for 9% Ni Steel and Ni based weld metals Table 2 Chemical composition of weld metals (wt. %) Weld
metal
Ni
Cr
Mo
Nb+Ta
Fe
W
Si
Mn
Ti
C
Cu
P
S
Hastelloy
609
(609
weld
metal)
Bal. 15.700
16.200
6.600
3.300
0.270
0.270
0.010
0.012
0.002
Inconel
625
(625
weld
metal)
Bal. 21.600
8.600
3.570
1.800
0.370
0.280
0.110
0.030
0.030
0.010
0.003
Table 3 The welding conditions for 9% Ni Steel and Ni-based weld metals
Welding parameters
Welding machine
CMT/KUKA
Base metal
9% Ni steel
Weld metal (wire)
Ni-based alloy (Hastelloy 609, Inconel 625)
Shield gas
Ar-18% CO2
Welding current [A]
200
Welding speed [cm/min]
20
Table 4 The results of EDS point analysis (wt. %)
Analysis region
Phase
Ni
Cr
Fe
Mo
Nb
W
Hastelloy 609
Interdendrite
(Mo-rich) γ-phase
48.28
15.21
18.52
15.64
2.36
Dendrite
γ-phase
53.35
13.97
20.41
10.08
2.19
Precipitate
ρ-phase
26.45
13.49
11.72
43.01
5.33
Inconel 625
Interdendrite
(Mo, Nb-rich) γ-phase
59.99
21.70
8.75
7.12
2.44
Dendrite
γ-phase
61.46
21.42
9.51
6.22
1.39
precipitate
NbC
10.34
7.87
2.30
4.77
74.73 Table 3 The welding conditions for 9% Ni Steel and Ni-based weld metals
Welding parameters
Welding machine
CMT/KUKA
Base metal
9% Ni steel
Weld metal (wire)
Ni-based alloy (Hastelloy 609, Inconel 625)
Shield gas
Ar-18% CO2
Welding current [A]
200
Welding speed [cm/min]
20
Table 4 The results of EDS point analysis (wt. Figure 1 XRD results of the weld joints from the welding of 9% Ni steel with Hastelloy 609 and Inconel 625 weld metals; (a) 9% Ni steel, (b)
Inconel 625, (c) Hastelloy 609, (d) weld joint interface with Inconel 625 and base metal, (e) weld joint interface with Hastelloy 609
and base metal Figure 2
OM and SEM images of sample welded with (a−c) Hastelloy 609 and (d−f) Inconel 625; (a) cross section of weld joint by Hastelloy
609, SEM images of (b) center and (c) bottom part of Hastelloy 609 bead, (d) cross section of weld joint by Inconel 625, SEM images
of (e) center and (f) bottom part of Inconel 625 bead Tables %)
Analysis region
Phase
Ni
Cr
Fe
Mo
Nb
W
Hastelloy 609
Interdendrite
(Mo-rich) γ-phase
48.28
15.21
18.52
15.64
2.36
Dendrite
γ-phase
53.35
13.97
20.41
10.08
2.19
Precipitate
ρ-phase
26.45
13.49
11.72
43.01
5.33
Inconel 625
Interdendrite
(Mo, Nb-rich) γ-phase
59.99
21.70
8.75
7.12
2.44
Dendrite
γ-phase
61.46
21.42
9.51
6.22
1.39
precipitate
NbC
10.34
7.87
2.30
4.77
74.73 Table 3 The welding conditions for 9% Ni Steel and Ni-based weld metals Table 3 The welding conditions for 9% Ni Steel and Ni-based weld metals
Welding parameters
Welding machine
CMT/KUKA
Base metal
9% Ni steel
Weld metal (wire)
Ni-based alloy (Hastelloy 609, Inconel 625)
Shield gas
Ar-18% CO2
Welding current [A]
200
Welding speed [cm/min]
20 Table 4 The results of EDS point analysis (wt. %) Table 4 The results of EDS point analysis (wt. %)
Analysis region
Phase
Ni
Cr
Fe
Mo
Nb
W
Hastelloy 609
Interdendrite
(Mo-rich) γ-phase
48.28
15.21
18.52
15.64
2.36
Dendrite
γ-phase
53.35
13.97
20.41
10.08
2.19
Precipitate
ρ-phase
26.45
13.49
11.72
43.01
5.33
Inconel 625
Interdendrite
(Mo, Nb-rich) γ-phase
59.99
21.70
8.75
7.12
2.44
Dendrite
γ-phase
61.46
21.42
9.51
6.22
1.39
precipitate
NbC
10.34
7.87
2.30
4.77
74.73
Fi Figures Page 8/12 Figure 1
XRD results of the weld joints from the welding of 9% Ni steel with Hastelloy 609 and Inconel 625 weld metals; (a) 9% Ni steel, (b)
Inconel 625, (c) Hastelloy 609, (d) weld joint interface with Inconel 625 and base metal, (e) weld joint interface with Hastelloy 609
and base metal Figure 1 Figure 2 Figure 2 OM and SEM images of sample welded with (a−c) Hastelloy 609 and (d−f) Inconel 625; (a) cross section of weld joint by Hastelloy
609, SEM images of (b) center and (c) bottom part of Hastelloy 609 bead, (d) cross section of weld joint by Inconel 625, SEM images
of (e) center and (f) bottom part of Inconel 625 bead OM and SEM images of sample welded with (a−c) Hastelloy 609 and (d−f) Inconel 625; (a) cross section of weld joint by Hastelloy
609, SEM images of (b) center and (c) bottom part of Hastelloy 609 bead, (d) cross section of weld joint by Inconel 625, SEM images
of (e) center and (f) bottom part of Inconel 625 bead Page 9/12 Page 9/12 igure 3
oint interface welded with Hastelloy 609; (a) SEM image, (b) EDS line analysis, (c) magnified SEM image, (d) EBSD image Figure 3 Figure 3 Joint interface welded with Hastelloy 609; (a) SEM image, (b) EDS line analysis, (c) magnified Joint interface welded with Hastelloy 609; (a) SEM image, (b) EDS line analysis, (c) magnified SEM image, (d) EBSD image Page 10/12 ure 4
nt interface welded with Inconel 625; (a) SEM image, (b) EDS line analysis, (c) magnified SEM image, (d) EBSD image Figure 4 Joint interface welded with Inconel 625; (a) SEM image, (b) EDS line analysis, (c) magnified S oint interface welded with Inconel 625; (a) SEM image, (b) EDS line analysis, (c) magnified SEM Joint interface welded with Inconel 625; (a) SEM image, (b) EDS line analysis, (c) magnified SEM image, (d) EBSD image Joint interface welded with Inconel 625; (a) SEM image, (b) EDS line analysis, (c) magnified SEM image, (d) EBSD image Figure 5
Results of Charpy impact test performed for the weld metal areas in a cryogenic environment at −196 ℃ Figure 5 Results of Charpy impact test performed for the weld metal areas in a cryogenic environment at −196 ℃ Results of Charpy impact test performed for the weld metal areas in a cryogenic environment at −196 ℃ Page 11/12 Page 11/12 Figure 6
Schematic diagram of intergranular crack propagation in a cryogenic environment Figure 6 Figure 6 Figure 6 Schematic diagram of intergranular crack propagation in a cryogenic environment Page 12/12
|
https://openalex.org/W2146239126
|
https://www.spandidos-publications.com/10.3892/ijo.2015.3135/download
|
English
| null |
miRNA-99b-3p functions as a potential tumor suppressor by targeting glycogen synthase kinase-3β in oral squamous cell carcinoma Tca-8113 cells
|
International journal of oncology
| 2,015
|
cc-by
| 7,594
|
Introduction OSCC is the 6th most common malignancy and is a major
cause of cancer morbidity and mortality (1). OSCC accounts miRNA-99b is a member of the miR-125a~let-7e cluster
that is involved in a series of cellular activities such as cell
proliferation, differentiation and invasion (9-11). Recent
studies have shown that miR-125a expression decreased
in non-small cell lung cancer and breast cancer (12,13). Previously, it has been found that miR-99b-3p is expressed
in the Helicobacter pylori infection-dependent gastric
cancer (14) and that the miR-99b-3p is considered to be a
new tumor marker. This predicts the relapse-free survival
in patients with the follicular variant of papillary thyroid
carcinoma (15). However, the function of miR-99b-3p in
cancer, particularly in the pathogenesis of OSCC, has not
yet been reported. Correspondence to: Professor Ang Li, Department of
Periodontology, Stomatology Hospital, College of Medicine, Xi'an
Jiaotong University, Xi'an, Shaanxi 710004, P.R. China
E-mail: drliangyj@gmail.com Professor Chen Huang, Department of Genetics and Molecular
Biology, Xi'an Jiaotong, University School of Medicine, 76 Yan Ta
West Road, Xi'an, Shaanxi 710061, P.R. China DOI: 10.3892/ijo.2015.3135 Abstract. Dysregulation of microRNAs (miRNAs) has
been associated with carcinogenesis in oral squamous cell
carcinoma (OSCC). In the present study, we investigated the
expression and function of miR-99b-3p in human OSCC. We
found that the expression levels of miR-99b-3p decreased
in 21 clinical OSCC samples (84%). Furthermore, ectopic
expression of miR-99b-3p inhibited OSCC cell proliferation
by downregulating glycogen synthase kinase-3β (GSK3β), an
miR-99b-3p' target gene, at the mRNA and protein levels, both
in vitro and in vivo. Moreover, the silencing of GSK3β recapit-
ulated the cellular and molecular effects in a similar manner to
the overexpression of miR-99b-3p, which included inhibition
of OSCC cell proliferation and suppression of p65 (RelA) and
G1 regulators (cyclin D1, CDK4 and CDK6) in vitro. Our data
suggest that miR-99b-3p functions as a tumor suppressor in
OSCC via GSK3β downregulation. for ~90% of all oral cancers. Despite the advances in diagnosis
and treatment, only ~50% of the patients with OSCC survived
for 5 years in the past decade (2). Oral carcinogenesis arises as
a result of the activation of some oncogenes or the inactivation
of tumor suppressor genes (3). Growing evidence has shown
that non-coding small RNAs play an important role in OSCC
pathogenesis, which provides new insights into the treatment
of this cancer. MicroRNAs (miRNAs) are an abundant class of short
RNAs, which are 19-24 nucleotides in length; miRNAs
were shown to affect complementarity by binding at the 3'
untranslated region (UTR) of target genes, resulting in degra-
dation of target mRNAs and inhibition of translation (4). Many studies have shown that miRNA dysregulation occurs
in various human diseases, especially cancer. miRNAs are
involved in crucial cellular processes, including develop-
ment, differentiation, proliferation and apoptosis (5). Several
studies have suggested that dysregulation of miRNAs,
including miR-29b, miR-9, miR-29a, is related with OSCC
initiation and development (6-8). INTERNATIONAL JOURNAL OF ONCOLOGY 47: 1528-1536, 2015 INTERNATIONAL JOURNAL OF ONCOLOGY 47: 1528-1536, 2015 Kang He1,3*, Dongdong Tong3*, Si Zhang4, Donghui Cai3, Lumin Wang3, Yang Yang3,
Ling Gao2, Su'e Chang3, Bo Guo3, Tusheng Song3, Ang Li1 and Chen Huang3 Departments of 1Periodontology and 2Oral Maxillofacial Surgery, Stomatology Hospital of Xi'an Jiaotong University
College of Medicine, Xi'an, Shaanxi 710004; 3Department of Genetics and Molecular Biology, Key Laboratory of
Environment and Genes Related to Diseases, College of Medicine, Xi'an Jiaotong University, Xi'an, Shaanxi 710061;
4Department of Stomatology, First Affiliated Hospital of Xi'an Medical University, Xi'an, Shaanxi 710077, P.R. China Received June 23, 2015; Accepted August 17, 2015 Received June 23, 2015; Accepted August 17, 2015 Materials and methods Tissue samples. Formaldehyde-fixed, paraffin-embedded
(FFPE) tissue samples were obtained from surgical speci-
mens from 25 patients (15 male, 10 female, 59.07±10.69 and
53.62±15.29 years of age, respectively) diagnosed with OSCC,
between January 1987 and June 2011 at the Stomatological
Hospital, College of Medicine, Xi'an Jiaotong University
(Table I). Before the operation none of the patients had
received radiotherapy or chemotherapy. The non-tumor
tissues were taken more than 2 cm from the tumor to be
used as controls and were confirmed by an experienced
pathologist. The study was approved by our institutional
review board, and an informed consent was given by all the
patients. Stable transfection of miR-99b expression vector. The day
before transfection, Tca-8113 cells were plated in antibiotic-
free medium at ~25%. Pre-miR-99b and control vector were
transfected into Tca-8113 cells using LipoFiter™ (Hanbio,
Shanghai, China) in accordance with the manufacturer's
procedure, cultured by selection with 8 µg/ml blasticidin
(Invitrogen) containing medium for 2 weeks. Single cell
clones were picked and cultured in medium (RPIM-1640)
containing 4 µg/ml blasticidin for further study. The expres-
sion level of miR-99b-3p in transfected Tca-8113 cells was
identified by quantitative real-time PCR after further selec-
tion and expansion. Cell lines. Tca-8113, established in Ninth People's Hospital,
Shanghai Second Medical University in 1981, was purchased
from the Shanghai GeneChem Co., Ltd. (Shanghai, China)
and grown in RPMI‑1640 medium (PAA Laboratories GmbH)
supplement with 10% FBS (PAA Laboratories GmbH) at 37˚C
in a humidified atmosphere of 95% air and 5% CO2. Lentivirus infection. Lentivirus Luc was synthesized by
Shanghai Genechem. Stability enhanced pre-miR‑99b
Tca-8113 cells were infected by lentiviruses according to the
manufacturer's protocol. Quantitative real-time reverse transcription PCR. Total RNA
from Tca-8113 cells and prepared tissues sample was isolated
using the TRIzol reagent (Invitrogen) and RecoverAll™ Total
Nucleic Acid Isolation kit (Ambion, Austin, TX, USA)
according to the manufacturer's protocol. The cDNA was
synthesized according to the manufacturer's protocol (MBI
Fermentas). Quantitative real-time PCR (qRT-PCR) was
performed using a Maxima SYBR-Green qPCR Master Mixes
(MBI Fermentas) and PCR-specific amplification was Cell proliferation assay. The Tca-8113 cells (5,000 cells/
well) were seeded into 96-well plates with 200 µl of 1640
medium, and cultured 24-72 h after transfecting with vector
control, pre-miR-99b expression vector, inhibitor control,
miR‑99b-3p inhibitor, siRNA control and GSK3β siRNA. Cells washed with warm DMEM and MTT (Sigma) working
solution were incubated at 37˚C for 4 h. *contributed equally The aim of the present study was to determine the role
of miR-99b-3p in OSCC. The potential mechanisms under-
lying the regulation of the biological behavior of OSCC
by miR‑99b-3p were also investigated. Our findings will
contribute to understanding of the function of miR-99b-3p in
the progression of OSCC. Key words: oral squamous cell carcinoma, miR-99b-3p, GSK3β,
p65, proliferation he et al: miRNA-99b-3p TARGETS GSK3 IN Tca-8113 CELLs 1529 Table I. Relationship between clinicopathological factors and
miR-99b-3p expression levels. miR-99b-3p
expression
No. of
----------------------------
Characteristics
cases
High
Low
P-value
Age (years)
0.260
≥60
9
0
9
<60
16
4
12
Gender
0.656
Male
15
2
13
Female
10
2
8
Histology
0.357
Well
17
3
14
Moderate
5
2
3
poor
3
0
3
pTNM stage
0.552
I
9
1
8
II
5
1
4
III
2
1
1
IV
9
1
8 Table I. Relationship between clinicopathological factors and
miR-99b-3p expression levels. conducted in the Roche LightCycler® 480 II real-time PCR. The relative expression of genes (miR-99b-3p, U6, GSK3B
and β-actin) was calculated with the 2-∆∆Ct method (16). The
primers used are: qRT-PCR, miR-99b-3p‑F5'-ATCC
AGTGCGTGTCGTG-3', miR-99b-3p-R5'-TGCTCAAGCT
CGTGTCTGT-3'; GSK3β-F5'-CCTCTGGCTACCATCCT
TATTC-3', GSK3β-R5'-TTATTGGTCTGTCCACGGT
CTC-3'; U6-F5'-TGCGGGTGCTCGCTTCGGCAGC-3',
U6-R5'-CCAGTGCAGGGTCCGAGGT-3'; β-actin-F5'-CG
TGACATTAAGGAGAAGCTG-3', β-actin-R5'-CTAGAAG
CATTTGCGGTGGAC-3'. Construction of expression plasmids. The miR-99b-3p expres-
sion vector (pre-miR-99b) and control vector were constructed
with synthetic oligonucleotides and cloned between the EcoRI
and HindIII sites of the pcDNA6.2-GW/EmGFP vector
(Invitrogen): pre-miR-99b-F'-AATTCGGCACCCACCCG
TAGAACCGACCTTGCGGGGCCTTCGCCGCACACAAG
CTCGTGTCTGTGGGTCCGTGTCA-3'; R-5'-AGCTTGAC
ACGGACCCACAGACACGAGCTTGTGTGCGGCGAAGG
CCCCGCAAGGTCGGTTCTACGGGTGGGTGCCG-3'. Primers contained 5'EcoRI and 3'HindIII restriction sites to
facilitate cloning into the vector. The inhibitor of miR-99b-3p
and small interfering RNA (siRNA) targeting GSK3β were
purchased from GenePharma. The siRNA-GSK3B target
sequence GAUGAGGUCUAUCUUAAUC (nt:1353-1371) (17). Bioinformatic analysis. The information of human miR‑99b-3p
was registered, and obtained from miRBase (http://www. miRBase.org/). We used the publicly available programs:
RegRNA(http://regrna.mbc.nctu.edu.tw/), miRanda(http://
www.microrna.org/) to acquire the prediction of miRNA
targets. Results Aberrant miR-99b-3p expression in human OSCC. To validate
the expression of miR-99b-3p in human OSCC, we analyzed
the expression of miR-99b-3p in 25 paired human OSCC
tissue samples and adjacent non-cancerous oral mucosa using
real-time PCR. Compared with their peritumor counterparts,
we observed significant downregulation of miR-99b-3p in 84%
(21/25) of the OSCC samples (Fig. 1A). Moreover, miR‑99b-3p
was significantly downregulated in Tca-8113 cells compared
with normal human oral epithelial cells (Fig. 1B). This indi-
cated that the miR-99b-3p may play a role as a tumor suppressor
in the OSCC. Therefore, we analyzed the relationship between
miR-99b-3p levels and clinicopathological factors in OSCC
samples (Table I). There was no significant difference between
low expression of miR-99b-3p and clinicopathological factors
such as age (p=0.260), gender (p=0.656), histology (p=0.357)
and pT stage (p=0.552). Dual luciferase assay. HEK293 cells were seeded in a
96-well plate at a density of 1x104 cells/well one day before
transfection. miR-99b-3p expression vector was co-transfected
with wild or mutated 3'-UTR of GSK3β reporter constructs
and a blank pmirGLO Dual-luciferase as a positive control
group. After 24 h, the Dual-luciferase reporter assay system
(Promega) was used to measure the reporter activity according
to the manufacturer's protocol. Colony formation assay. The transfected Tca-8113 cells were
seeded into 6-well plates at a density of 1,000/well, incubated
for two weeks. Colonies were then stained with 0.1% crystal
violet for 30 min, counted and normalized to the control
group. Overexpression of miR-99b-3p suppresses Tca-8113 cell
growth and induces G1-S arrest in vitro. To explore the role of
miR-99b-3p in OSCC, Tca-8113 cells were transfected with an
miR-99b precursor overexpression vector or negative controls. The efficiency of vector transfection was monitored with a
GFP-label and an average of 70% efficiency was observed at
a concentration of 100 nmol/l without causing obvious cell
toxicity. qRT-PCR was performed to determine the expression
levels of miR-99b-3p after transfection of the miR-99b-3p
precursor construct vector. The results showed that the expres-
sion of miR-99b-3p in the pre-miR-99b-transfected cells was
~8-fold higher than that in the control vector-transfected cells
(Fig. 1C). The MTT assay and colony formation assay indicated
significant inhibition of cell growth and colony formation after
pre-miR-99b transfection, as compared to that seen for cells
transfected with the empty vector (Fig. 1D and H). The next
cell cycle experiment demonstrated that miR-99b-3p over-
expression caused cell cycle arrest at G1-S in Tca-8113 cells
(Fig. 1F). Materials and methods Using acidic isopro-
panol (0.04 M HCl in absolute isopropanol) solubilized the INTERNATIONAL JOURNAL OF ONCOLOGY 47: 1528-1536, 2015 1530 converted dye. Absorbance of the converted dye was measured
at a wavelength of 490 nm with FLUOstar Optima (BMG). mean ± SD. Differences between 2 groups were calculated
with the Student's t-test (two-tailed). The associations between
clinicopathological factors and miR-99b-3p levels were
analyzed using the chi-square test. P<0.05 was considered to
be significant. Cell cycle analysis. The Tca-8113 cells at 1x106 cells/well
were cultured in 12-well plates in triplicate and transfected
with DNA vectors or siRNAs for 24 h. Cells were harvested
by trypsinization, then washed in PBS, and fixed in ice-cold
ethanol at 4˚C overnight. Then cells were washed twice in PBS
and incubated in 1 ml of staining solution (20 mg/ml propidium
iodide and 10 U/ml RNaseA) for 30 min at room temperature. Cell cycle distribution was evaluated by fluorescence-activated
cell sorting by flow cytometry (FACSort; Becton-Dickinson). Results Overexpression and inhibition of miR-99b-3p affect OSCC progression in vitro: (A) expression
of miR-99b-3p in OSCC tissue samples (84% oscc tissue is upregulation), (B) expression of miR-99b-3p in Tca-8113 cell line. (*P<0.05, Student's t-test). (C) qRT-PCR analysis of miR-99b-3p in Tca-8113 cells transfected with pre-miR-99b overexpression construct and empty vector. (D and E) the effects of pre-
miR-99b vector or miR-99b-3p inhibitor on Tca-8113 cell proliferation were determined by MTT assay at 24, 48 and 72 h after transfection with pre-miR‑99b
or miR-99b-3p inhibitor, with empty vector or ASO-NC, respectively. (F and G) Histogram represented the percentage of cells in G0-G1, S, and G2-M cell cycle
phases after transfection for 48 h based on the flow cytometric analysis. (H) Representative micrographs of crystal violet-stained cell colonies were analyzed by
colony formation assay at day 14 after transfection. (I) Expression of the cell cycle regulators transfected by pre-miR-99b or miR-99b-3p inhibitor in Tca-8113
was analysis by western blot experiments. Figure 1. Low expression of miR-99b-3p in 25 oral squamous cell carcinoma (OSCC) tissue samples and OSCC cell line (tca-8113) compared with non-
cancerous samples and normal human oral epithelial cells. Overexpression and inhibition of miR-99b-3p affect OSCC progression in vitro: (A) expression
of miR-99b-3p in OSCC tissue samples (84% oscc tissue is upregulation), (B) expression of miR-99b-3p in Tca-8113 cell line. (*P<0.05, Student's t-test). (C) qRT-PCR analysis of miR-99b-3p in Tca-8113 cells transfected with pre-miR-99b overexpression construct and empty vector. (D and E) the effects of pre-
miR-99b vector or miR-99b-3p inhibitor on Tca-8113 cell proliferation were determined by MTT assay at 24, 48 and 72 h after transfection with pre-miR‑99b
or miR-99b-3p inhibitor, with empty vector or ASO-NC, respectively. (F and G) Histogram represented the percentage of cells in G0-G1, S, and G2-M cell cycle
phases after transfection for 48 h based on the flow cytometric analysis. (H) Representative micrographs of crystal violet-stained cell colonies were analyzed by
colony formation assay at day 14 after transfection. (I) Expression of the cell cycle regulators transfected by pre-miR-99b or miR-99b-3p inhibitor in Tca-8113
was analysis by western blot experiments. GSK3β expression was correlated with OSCC progress
(Table II) (19-24). The protein level of GSK3β was examined
in paired OSCC tissues from 3 cases by western blot analysis
and immunohistochemistry. As Fig. 2D and E show, GSK3β
protein levels were significantly higher in OSCC tissue than in
paired adjacent tissues. Results Compared to the findings for control
vector-transfected cells, significant reduction in GSK3β and
p65 expression was observed in Tca-8113 cells when trans-
fected with miR-99b-3p precursor (Fig. 2C, G and H). These
results indicated that miR-99b-3p might directly target the
3'UTR of GSK3β mRNA and inhibit GSK3β translation. Alteration in the p65 protein expression level occurred in both
the cell cytoplasm and the nucleus. GSK3β is a direct target gene of miR-99b-3p. We searched the
bioinformatic databases RegRNA and miRanda, and identi-
fied a large number of potential target genes of miR‑99b-3p. GSK3β was selected for further analysis among these
candidate genes. As shown in Fig. 2A, the binding site at the
GSK3β 3'-untranslated region (UTR) is displayed. To confirm
whether miR-99b-3p directly targets GSK3β, we constructed
3'UTR fragments of GSK3β (WT/MT) and a binding site for
miR-99b-3p, which was subcloned into the pmirGLO dual-
luciferase reporter vector. HEK293 cells were cotransfected
with pre-miR-99b, pmirGLO control vector, and a reporter
plasmid (GSK3β WT- or MT-3' UTR). Consequently, pre-
miR‑99b/GSK3β (WT)_UTR transfected cells showed
significant reduction (~40%) of luciferase activity (Fig. 2B),
which suggested that miR-99b-3p could suppress gene expres-
sion through its binding sequences at the 3'UTR of GSK3β. GSK3β is a direct target gene of miR-99b-3p. We searched the
bioinformatic databases RegRNA and miRanda, and identi-
fied a large number of potential target genes of miR‑99b-3p. GSK3β was selected for further analysis among these
candidate genes. As shown in Fig. 2A, the binding site at the
GSK3β 3'-untranslated region (UTR) is displayed. To confirm
whether miR-99b-3p directly targets GSK3β, we constructed
3'UTR fragments of GSK3β (WT/MT) and a binding site for
miR-99b-3p, which was subcloned into the pmirGLO dual-
luciferase reporter vector. HEK293 cells were cotransfected
with pre-miR-99b, pmirGLO control vector, and a reporter
plasmid (GSK3β WT- or MT-3' UTR). Consequently, pre-
miR‑99b/GSK3β (WT)_UTR transfected cells showed
significant reduction (~40%) of luciferase activity (Fig. 2B),
which suggested that miR-99b-3p could suppress gene expres-
sion through its binding sequences at the 3'UTR of GSK3β. Silencing of GSK3β suppresses Tca-8113 cell growth and
induces G1-S arrest, similarly to miR-99b-3p. Next, we
used RNA interference (RNAi) methods to silence GSK3β
expression to determine whether GSK3β was involved in the
antitumor effects of miR-99b-3p. GSK3β could be specifically
knocked down by siRNA (Fig. 3A and E). Results To further explore the potential molecular mecha-
nisms of miR-99b-3p-induced cell proliferation and cell cycle
arrest, we analyzed the protein levels of related G1 regulators
after miR-99b-3p overexpression in Tca-8113 by using western
blot analysis. Our results showed that miR-99b-3p expression
may reduce the expression of cyclin D1, CDK4, and CDK6,
which are the essential regulators of the G1-S phase transition
(Fig. 1I). Western blot analysis. All Tca-8113 cells or tissue were lysed
using RIPA buffer, supplemented with protease inhibitor
(Invitrogen). Protein was then separated with 10% SDS
polyacrylamide gels, and electrophoretically transferred
to polyvinylidene difluoride membrane (Millipore). The
membrane was incubated with primary antibodies to GSK3β
(abcam; antibody dilutions: 1:1,500), p65, cyclin D1, CDK4,
CDK6, Lamin B1 (Proteintech, antibody dilutions: 1:1,000),
β-actin antibody (cst, antibody dilutions: 1:2,000). The blots
were scanned and the band density was measured on Quantity
One imaging software. Immunohistochemistry. Immunohistochemistry (IHC) was
performed according to the methods previously described (18). The tissue sections were incubated in the primary antibodies
overnight. Staining intensity was assessed by Leica Q550
image analysis system. In vivo tumor xenograft model. Six-week-old male nude
mice (BALB/c-nude) were used to analyse tumorigenicity. Tca-8113 cells were stably tranfected with pre-miR-99b
and control vector which were infected with LV-Luc and
resuspended in PBS, then 1x107 cells were injected subcuta-
neously into both posterior flanks of nude mice. Tumor size
was measured every 3 days. At 18 days after injection, mice
from the pre-miR-99b group (n=3) and control group (n=3)
were subjected in vivo to endpoint experiments, the biolu-
minescence images in vivo were obtained by the system of
photobiology (Xenogen). Loss-of-function studies were also performed by using
anti-miR-99b-3p oligonucleotides to silence the expression
of miR-99b-3p (Fig. 1E, G and H). Unexpectedly, inhibition
of miR-99b-3p could only slightly increase the expression of
these proteins in Tca-8113 cells (Fig. 1I). The low expression
of endogenous miR-99b-3p in Tca-8113 cells may account
for this phenomenon. These results suggest that miR-99b-3p
inhibits the proliferation of Tca-8113 cells and arrests the cell
cycle by controlling cell cycle-related gene expression. Statistical analysis. We repeated each experiment at least
3 times independently. Numercial data are presented as he et al: miRNA-99b-3p TARGETS GSK3 IN Tca-8113 CELLs 1531 Figure 1. Low expression of miR-99b-3p in 25 oral squamous cell carcinoma (OSCC) tissue samples and OSCC cell line (tca-8113) compared with non-
cancerous samples and normal human oral epithelial cells. Results Moreover, silencing
of GSK3β suppressed the growth and proliferation of Tca-8113
cells and induced G1-S arrest, which is similar to the role of
miR-99b-3p in Tca-8113 cells (Fig. 3B-D). Next, we analyzed GSK3β is dysregulated in OSCC cancer cell lines and tissues. We used the Oncomine cancer microarray database, which
enabled multiple comparisons among different studies, to
analyze the expression profile of GSK3β mRNA in OSCC
tissue. In 6 studies, OSCC tissue samples had higher GSK3β
expression than normal tissue. These results showed that INTERNATIONAL JOURNAL OF ONCOLOGY 47: 1528-1536, 2015 1532 Figure 2. GSK3β is experimentally validated as a direct target of miR-99b-3p in Tca-8113 cell line. (A) Wild-type or mutant miR-99b target sequences of
the GSK3β 3'UTR. (B) Luciferase assay in HEK293 cells. Pre-miR-99b was cotransfected with target gene reporter construct (WT or MT version of pGLO
constructs) or NS-control. Relative repression of luciferase expression was standardized to β-gal signal. Luciferase activity in pGLO-GSK3β-WT group was
statistically decreased following ectopic expression of miR‑99b-3p. (C) GSK3β protein expression level in Tca-8113 was measured by western blot analysis
48 h after transfection with pre-miR-99b vector or miR-99b-3p inhibitor. (D and E) expression of GSK3β was determined by western blot analysis and
immunohistochemical in OSCC tissues and their corresponding non-tumorous oral mucosa. (F) qRT-PCR were performed to determine the expression level of
GSK3β after transfection of pre-miR-99b vector (*P<0.05, Student's t-test). (G and H) the protein expression level of p65 was detected by immunofluorescence
staining and western blot analysis after transfection with pre-miR-99b vector for 48 h. Figure 2. GSK3β is experimentally validated as a direct target of miR-99b-3p in Tca-8113 cell line. (A) Wild-type or mutant miR-99b target sequences of
the GSK3β 3'UTR. (B) Luciferase assay in HEK293 cells. Pre-miR-99b was cotransfected with target gene reporter construct (WT or MT version of pGLO
constructs) or NS-control. Relative repression of luciferase expression was standardized to β-gal signal. Luciferase activity in pGLO-GSK3β-WT group was
statistically decreased following ectopic expression of miR‑99b-3p. (C) GSK3β protein expression level in Tca-8113 was measured by western blot analysis
48 h after transfection with pre-miR-99b vector or miR-99b-3p inhibitor. (D and E) expression of GSK3β was determined by western blot analysis and
immunohistochemical in OSCC tissues and their corresponding non-tumorous oral mucosa. (F) qRT-PCR were performed to determine the expression level of
GSK3β after transfection of pre-miR-99b vector (*P<0.05, Student's t-test). Results (G and H) the protein expression level of p65 was detected by immunofluorescence
staining and western blot analysis after transfection with pre-miR-99b vector for 48 h. Figure 2. GSK3β is experimentally validated as a direct target of miR-99b-3p in Tca-8113 cell line. (A) Wild-type or mutant miR-99b target sequences of
the GSK3β 3'UTR. (B) Luciferase assay in HEK293 cells. Pre-miR-99b was cotransfected with target gene reporter construct (WT or MT version of pGLO
constructs) or NS-control. Relative repression of luciferase expression was standardized to β-gal signal. Luciferase activity in pGLO-GSK3β-WT group was
statistically decreased following ectopic expression of miR‑99b-3p. (C) GSK3β protein expression level in Tca-8113 was measured by western blot analysis
48 h after transfection with pre-miR-99b vector or miR-99b-3p inhibitor. (D and E) expression of GSK3β was determined by western blot analysis and
immunohistochemical in OSCC tissues and their corresponding non-tumorous oral mucosa. (F) qRT-PCR were performed to determine the expression level of
GSK3β after transfection of pre-miR-99b vector (*P<0.05, Student's t-test). (G and H) the protein expression level of p65 was detected by immunofluorescence
staining and western blot analysis after transfection with pre-miR-99b vector for 48 h. protein levels of related G1 regulators after silencing the
GSK3β in Tca-8113 cells in western blot analysis. As shown
in Fig. 3E, the expression of NF-κB (p65) was suppressed by
si-GSK3β (Fig. 3E and F). For cell cycle regulation, si-GSK3β
may reduce the expression of cyclin D1, CDK4, and CDK6,
which are the essential regulators of the G1-S phase transition. Thus, we speculate that GSK3β and p65 dysregulation in
OSCC may promote OSCC tumorigenesis. miR-99b-3p inhibits OSCC tumor growth in vivo. To further
confirm the growth-inhibitory function of miR-99b-3p in
OSCC, we tested the effects of miR-99b-3p on tumor growth
Table II. Expression of GSK3β in human normal mouth mucosa tissue and OSCC carcinoma. Cancer vs. normal (sample number)
Correlation (up/down)
Fold change
p-value
Ref. Tongue squamous cell carcinoma (31) vs. tongue (26)
m
1.727
6.19E-10
(19)
Tongue squamous cell carcinoma (31) vs. tongue (26)
m
1.947
6.47E-9
(20)
Tongue squamous cell carcinoma (26) vs. tongue (12)
m
1.980
3.76E-5
(21)
Tongue squamous cell carcinoma (3) vs. mucosa (22)
m
1.259
0.049
(22)
Oral cavity squamous cell carcinoma (4) vs. squamous cell (16)
m
1.341
0.031
(23)
Oral cavity squamous cell carcinoma (57) vs. Results oral cavity (22)
m
1.197
0.002
(24)
Data were provided by the cancer microarray database Oncomine (www.oncomine.org). Table II. Expression of GSK3β in human normal mouth mucosa tissue and OSCC carcinoma. Cancer vs. normal (sample number)
Correlation (up/down)
Fold change
p-value
Ref. Tongue squamous cell carcinoma (31) vs. tongue (26)
m
1.727
6.19E-10
(19)
Tongue squamous cell carcinoma (31) vs. tongue (26)
m
1.947
6.47E-9
(20)
Tongue squamous cell carcinoma (26) vs. tongue (12)
m
1.980
3.76E-5
(21)
Tongue squamous cell carcinoma (3) vs. mucosa (22)
m
1.259
0.049
(22)
Oral cavity squamous cell carcinoma (4) vs. squamous cell (16)
m
1.341
0.031
(23)
Oral cavity squamous cell carcinoma (57) vs. oral cavity (22)
m
1.197
0.002
(24)
Data were provided by the cancer microarray database Oncomine (www.oncomine.org). Table II. Expression of GSK3β in human normal mouth mucosa tissue and OSCC carcinoma. Thus, we speculate that GSK3β and p65 dysregulation in
OSCC may promote OSCC tumorigenesis. protein levels of related G1 regulators after silencing the
GSK3β in Tca-8113 cells in western blot analysis. As shown
in Fig. 3E, the expression of NF-κB (p65) was suppressed by
si-GSK3β (Fig. 3E and F). For cell cycle regulation, si-GSK3β
may reduce the expression of cyclin D1, CDK4, and CDK6,
which are the essential regulators of the G1-S phase transition. miR-99b-3p inhibits OSCC tumor growth in vivo. To further
confirm the growth-inhibitory function of miR-99b-3p in
OSCC, we tested the effects of miR-99b-3p on tumor growth he et al: miRNA-99b-3p TARGETS GSK3 IN Tca-8113 CELLs 1533 Figure 3. silencing of GSK3β suppresses Tca-8113 growth and induces G1-S arrest. (A) qRT-PCR was performed to determine the expression level of GSK3β
after transfection with GSK3β siRNA. (B-D) MTT assay/cell cycle/ clone formation were performed to determine the impact of Tca-8113 cells treated with
siRNA of GSK3β. (E and F) expression analysis for GSK3β and p65 proteins expression in Tca-8113 cells at 48 h after transfected with control siRNA or
GSK3β siRNA by western blot analysis and immunofluorescence staining. β-actin and lamin B1 was used as a housekeeping control. Statistical differences
were analyzed with the Wilcoxon signed-rank test (*P<0.05; Student's t-test). Figure 3. silencing of GSK3β suppresses Tca-8113 growth and induces G1-S arrest. (A) qRT-PCR was performed to determine the expression level of GSK3β
after transfection with GSK3β siRNA. Results (B-D) MTT assay/cell cycle/ clone formation were performed to determine the impact of Tca-8113 cells treated with
siRNA of GSK3β. (E and F) expression analysis for GSK3β and p65 proteins expression in Tca-8113 cells at 48 h after transfected with control siRNA or
GSK3β siRNA by western blot analysis and immunofluorescence staining. β-actin and lamin B1 was used as a housekeeping control. Statistical differences
were analyzed with the Wilcoxon signed-rank test (*P<0.05; Student's t-test). proliferation (25), cell cycle (26) and metastasis (27) in OSCC. The miR-99b gene, located on chromosome 19q13.41, produces
two mature forms (miR-99b-3p and miR-99b-5p). Some
recent reports suggested that miR-99b-5p could influence
the sensitivity of pancreatic cancer cells to radiotherapy and
suppress the growth rate of lung cancer (28,29). Very little
research has been focused on miR-99b-3p, except for the
study by Chang et al (14) and Dettmer et al (15) showing that
miRNA‑99b-3p is correlated with papillary thyroid carcinoma
and H. pylori-positive gastric cancer. In the present study,
we found that miR-99b-3p was commonly downregulated
in OSCC tissue samples and Tca-8113 cells, indicating that
miR‑99b-3p might be a novel tumor suppressor miRNA. In
a series of cell experiments, gain and loss of function studies
showed that miR-99b-3p was able to inhibit cell proliferation
by arresting cells in the G1-S transition in vitro. The luciferase
assay showed that GSK3β is a direct target of miR-99b-3p in
OSCC. in an in vivo xenograft model. Cells from stably transfected
cell lines developed from miR-99b-3p- and miR-control-
transfected Tca-8113 cells were injected subcutaneously into
the posterior flank of the same nude mice. After injection,
palpable tumors developed at 1 week and were measured every
3 days. As shown in Fig. 4A-C, tumor growth was significantly
suppressed by pre-miR-99b-treated cells as compared to that
seen for control vector-treated cells, during the experiments. This trend was confirmed by the size and weight of tumors
excised from the animals. On day 18, the average volume of
pre-miR-99b-treated tumors was lesser than that for the control
group. The average tumor weights for the control and the
miR‑99b-3p groups on day 27 were 1.35 g and 0.84 g, respec-
tively. Next, we assessed the expression levels of miR-99b-3p
and GSK3β in the tumor tissues by qRT-PCR and western
blot analyses. We found that the tumors transfected with pre-
miR‑99b had an ~4-fold increase in miR-99b-3p, as compared
to the control group (Fig. 4D). Results The in vivo data showed that the
expression level of NF-κB (p65), cyclin D1, CDK4 and CDK6
proteins all decreased in pre-miR-99b-treated tumors, which
was consistent with the in vitro data (Fig. 4F). These data
indicated that miR-99b-3p expression is capable of inhibiting
tumor growth by inhibiting GSK3β expression levels in vivo. Oncomine algorithms were utilized in a preliminary
study to confirm higher expression of GSK3β in OSCC as
compared to that in normal mucosa, which concurred with
our experimental result. GSK3β is an isoform splice variant of
GSK3, which has been reported to phosphorylate over a dozen
transcription factors (30). GSK3β is recognized as an impor-
tant component in a large number of cellular processes and
diseases. The exact physiological effect of GSK3β in cancer
is unclear, but accumulated data have shown that GSK3β
plays an important role in tumorigenesis. Dysregulation of Discussion During the past 10 years, dysregulation of miRNAs has
been reported to be a common event that controls cell INTERNATIONAL JOURNAL OF ONCOLOGY 47: 1528-1536, 2015 1534 Figure 4. miR-99b-3p inhibits Tca-8113 cells progression in vivo. (A) at day 18, small animal imaging analysis was used to assess the tumor volume in situ
during the tumor development. (A) the flanks of nude mice were injected with NC-transfected (left flank) and miR-99b-transfected cells (right flank) in
3 nude mice, respectively. (B) The gross morphology of tumors showed the morphology of mice injected with miR-99b and miR-ctrl. (C) at day 18 after
the first measure, the mice were anesthetized and sacrificed at the experimental endpoint and tumors infected with pre-miR-99b and control vector were
weighted. (D) the expression levels of miR-99b-3p were detected by qRT-PCR analysis in the tumor tissues from the nude mice. (E) tumor growth curves. (F) Overexpression of miR-99b-3p in the xenografts decreased the GSK3β protein level and subsequently reduced the expression of p65 and G1 regulators as
determined using western blot analysis. Figure 4. miR-99b-3p inhibits Tca-8113 cells progression in vivo. (A) at day 18, small animal imaging analysis was used to assess the tumor volume in situ
during the tumor development. (A) the flanks of nude mice were injected with NC-transfected (left flank) and miR-99b-transfected cells (right flank) in
3 nude mice, respectively. (B) The gross morphology of tumors showed the morphology of mice injected with miR-99b and miR-ctrl. (C) at day 18 after
the first measure, the mice were anesthetized and sacrificed at the experimental endpoint and tumors infected with pre-miR-99b and control vector were
weighted. (D) the expression levels of miR-99b-3p were detected by qRT-PCR analysis in the tumor tissues from the nude mice. (E) tumor growth curves. (F) Overexpression of miR-99b-3p in the xenografts decreased the GSK3β protein level and subsequently reduced the expression of p65 and G1 regulators as
determined using western blot analysis. Figure 5. Proposed model for the effects of miR-99b-3p-mediated GSK3β on the NF-κB pathways, and suppression of proliferation. GSK3β is important for
the activation of direct IκB phosphorylation and degradation. p65(relA) translocates into the nuclei to activate the transcription of cyclin D1, CDK4 and CDK6. As a result of the cyclin activation, the NF-κB pathway is involved in promoting proliferation. Figure 5. Proposed model for the effects of miR-99b-3p-mediated GSK3β on the NF-κB pathways, and suppression of proliferation. References 1. Parkin DM, Bray F, Ferlay J and Pisani P: Estimating the world
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Cancer 46: 1456-1463, 2010. Discussion GSK3β inhibition by siRNA had the same effect
on OSCC cell growth as overexpression of miR-99b-3p. Our
data are consistent with recent studies on the regulation of cell
proliferation by GSK3β though an NF-κB-dependent pathway,
as we observed that the manipulation of GSK3β expression
may result in suppression of p65 and decrease in the expres-
sion of cell cycle proteins (cyclin D1, CDK4 and CDK6).i GSK3β, that is, either overexpression (31) or inhibition (32,33)
by a pharmacological inhibitor, is a frequent oncogenic event
in human mechanisms. Several studies support that GSK3β
functions as a tumor suppressor. The tumor suppressor action
is exemplified by GSK3β-mediated phosphorylation and
subsequent degradation of β-catenin, which is a transcriptional
co-activator that often promotes cellular proliferation (34). However, some studies suggest that GSK3β may promote
tumorigenesis. The expression of GSK3β is upregulated in
multiple cancers, including ovarian, pancreatic and colorectal
cancers (31,33,35). GSK3β regulates cell proliferation via
activation of NF-κB-dependent gene transcription in ovarian
and pancreatic cancers (31). p65(RelA) is one of the subunits
of NF-κB, which is capable of mediating interactions with
basal transcription factors and cofactors (36). p65 is typically
sequestered in the cell cytoplasm by IκB proteins (37). It has
been reported that GSK3β is a key component involved in
inducing activation of the IKK complex and degradation of
IκB in pancreatic cancer cells (32). After degradation of IκB
proteins, the released p65 proteins are further activated, and
they translocate to the nucleus where they bind to specific DNA
sequences and promote transcription of target genes (38,39). p65 is necessary for the positive regulation of gene expression
through cyclin D1, CDK4 and CDK6 (40-42). These 3 proteins
could be considered as active cell cycle regulators. When
quiescent cells are stimulated to enter the cell cycle, expres-
sion of cyclin D1 promotes progression through the G1 phase
through association and activation of CDK4 and CDK6 (43). In
our research, GSK3β expression was inversely correlated with
miR-99b-3p. GSK3β inhibition by siRNA had the same effect
on OSCC cell growth as overexpression of miR-99b-3p. Our
data are consistent with recent studies on the regulation of cell
proliferation by GSK3β though an NF-κB-dependent pathway,
as we observed that the manipulation of GSK3β expression
may result in suppression of p65 and decrease in the expres-
sion of cell cycle proteins (cyclin D1, CDK4 and CDK6).i Discussion GSK3β is important for
the activation of direct IκB phosphorylation and degradation. p65(relA) translocates into the nuclei to activate the transcription of cyclin D1, CDK4 and CDK6. As a result of the cyclin activation, the NF-κB pathway is involved in promoting proliferation. Figure 5. Proposed model for the effects of miR-99b-3p-mediated GSK3β on the NF-κB pathways, and suppression of proliferation. GSK3β is important for
the activation of direct IκB phosphorylation and degradation. p65(relA) translocates into the nuclei to activate the transcription of cyclin D1, CDK4 and CDK6. As a result of the cyclin activation, the NF-κB pathway is involved in promoting proliferation. he et al: miRNA-99b-3p TARGETS GSK3 IN Tca-8113 CELLs 1535 (2010ZDKG-50), the National Natural Science Foundation of
China (81171398 and 31100921), and The Program for Chang
Jiang Scholars and Innovative Research Team in University
(PCSIRT; 1171). GSK3β, that is, either overexpression (31) or inhibition (32,33)
by a pharmacological inhibitor, is a frequent oncogenic event
in human mechanisms. Several studies support that GSK3β
functions as a tumor suppressor. The tumor suppressor action
is exemplified by GSK3β-mediated phosphorylation and
subsequent degradation of β-catenin, which is a transcriptional
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tumorigenesis. The expression of GSK3β is upregulated in
multiple cancers, including ovarian, pancreatic and colorectal
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and pancreatic cancers (31). p65(RelA) is one of the subunits
of NF-κB, which is capable of mediating interactions with
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sequestered in the cell cytoplasm by IκB proteins (37). It has
been reported that GSK3β is a key component involved in
inducing activation of the IKK complex and degradation of
IκB in pancreatic cancer cells (32). After degradation of IκB
proteins, the released p65 proteins are further activated, and
they translocate to the nucleus where they bind to specific DNA
sequences and promote transcription of target genes (38,39). p65 is necessary for the positive regulation of gene expression
through cyclin D1, CDK4 and CDK6 (40-42). These 3 proteins
could be considered as active cell cycle regulators. When
quiescent cells are stimulated to enter the cell cycle, expres-
sion of cyclin D1 promotes progression through the G1 phase
through association and activation of CDK4 and CDK6 (43). In
our research, GSK3β expression was inversely correlated with
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31. Acknowledgements 19. Talbot SG, Estilo C, Maghami E, Sarkaria IS, Pham DK,
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Effects of a lifestyle programme on objectively measured physical activity level and sedentary time on overweight and obese women of Somali background living in Norway Running
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Research Square (Research Square)
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cc-by
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Effects of a lifestyle programme on objectively
measured physical activity level and sedentary time
on overweight and obese women of Somali
background living in Norway Running Eivind Andersen
(
eivind.andersen@usn.no
)
University of South-Eastern Norway Keywords: Posted Date: January 22nd, 2024 DOI: https://doi.org/10.21203/rs.3.rs-3853254/v1 DOI: https://doi.org/10.21203/rs.3.rs-3853254/v1 License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Read Full License Additional Declarations: No competing interests reported. Page 1/20 Results The women took on average 6804 (SD = 3286) steps per day and were sedentary for 9.1 hours (SD = 3)
per day at baseline. There were no differences between groups on any objectively measured PA variable
at any timepoint. There were significant differences on vigorous intensity PA and total PA at the 12-month
measurement session between the two groups using the IPAQ. Background Given the elevated prevalence and impact of overweight and the potential risk of non-communicable
diseases among women of Somali background in high-income countries and recognising the potential
positive impact of physical activity (PA) on these health conditions, it becomes imperative to focus on
understanding the PA behaviour of this specific population. The objectives of this paper were twofold:
firstly, to provide a comprehensive description of both objectively and subjectively measured PA level and
sedentary time in a group of overweight women of Somali background in Norway, and secondly, to
assess the effectiveness of a tailored, culturally sensitive, and community-based intervention in
enhancing PA levels and reducing sedentary time. Methods 169 overweight women of Somali background in Norway were randomised by borough to either a lifestyle
programme or a comparison group. The programme consisted of two sessions per week for 12 weeks,
combining classroom discussion with graded group-based PA activity led by coaches, followed by
monthly sessions over nine months. PA was measured objectively using the ActivPAL monitor and
subjectively using the international PA questionnaire short form (IPAQ). Introduction The immigrant population constitutes 18.9% of the total Norwegian population of 5.4M, with people of
Somali descent representing 5.3% of immigrants (1). Most Somali immigrants came to Norway in the
early 2000s to escape conflict, civil unrest and instability in their home country (2). A high prevalence of
type 2 diabetes (T2D) has been found among women of Somali background in the U.S. (3); moreover,
studies from Norway, Sweden, Italy, and New Zealand all found a prevalence of overweight/obesity above
50% (range 50.9–87.2%) (4–8). In a study with 4194 immigrants from 12 different countries in Norway,
women of Somali background had the highest odds of being overweight/obese (9). The length of
residence in Norway, adjusted for age, is linked to a higher likelihood of developing T2D and overweight
(10). This association is likely a consequence of shifts in dietary patterns and physical activity (PA),
driven by the processes of adaptation to the new country and urbanisation. These changes can be
attributed to the influence of labour-saving technologies and the increased availability of high-calorie
foods (11). PA plays a crucial role in preventing and managing overweight and obesity (12), conditions strongly
associated with the risk of T2D (13), cardiovascular diseases (CVDs) (14, 15), and overall mortality (16). The impact of a lifestyle intervention on cardiovascular risk factors is tied to the extent of body weight
loss (17). Notably, the reduction of body fat is likely a key mediator in the relationship between changes
in PA and modifications in risk factors for cardiometabolic diseases (18). However, studies have
demonstrated that an increase in PA can influence biomarkers of cardiometabolic risk even when there is
no simultaneous reduction in body weight (19). Moreover, sedentary time - defined as any waking
behaviour characterised by an energy expenditure ≤ 1.5 metabolic equivalents (METs) while in a seated,
lying or reclining posture (20) - is found to be associated with higher mortality but may be attenuated to
some degree by moderate-to-vigorous-intensity PA (MVPA) (21). Research on the PA level of women of
Somali background is scarce, but consistent, in that all found the women to be little engaged in PA or
exercise (3, 7, 22–24). To our knowledge, data on sedentary time in this group has not been published. Conclusion Despite observing initially low PA levels and high sedentary time at baseline and thus a considerable
potential for intervention, the intervention failed in attaining an increase in objectively measured PA or
reduction in sedentary time compared to the control condition. However, self-reported measures indicated
success in these aspects. The potential threats to the programme's reliability and validity include
contamination, low attendance rates, and high drop-out rates possible due to the COVID-19 pandemic. These challenges underscore the complexity of interventions in this demographic, emphasising the need
for further exploration and refinement of study methodologies to effectively enhance PA levels and
reduce sedentary time in immigrant women living in high-income countries. Page 2/20 Page 2/20 Trial registration : clinicaltrials.gov NCT04578067. Keywords: Immigrant Somali women, Lifestyle programme, Overweight,
Physical activity, Sedentary time, ActivPAL Introduction Although studies consistently show a low level of PA among women of Somali background in Norway
and other high-income countries, the results may be flawed using self-reported methods when collecting
PA data. These methods carry certain weaknesses. Immigrants of Somali background, and immigrants in
general, may find it difficult to fill in questions that are not presented in their language, and validity
evidence is lacking for PA assessment for Somali populations. In addition, accurate information about
the duration and intensity of the activity is a problem. These are all methodological issues that can be
overcome by using objective measurements such as an accelerometer. To our knowledge, there are no
papers giving objectively measured PA levels and/or sedentary time in women of Somali background. Although accelerometers have been found to have good measurement properties to assess sedentary, Page 3/20 standing, and stepping time and postural transitions in adults (25, 26), they place a bigger burden on the
participants, are more expensive, time-consuming and require relatively higher technical expertise than
self-report methods. The Somali population appears to have shifted from a predominantly pastoral and nomadic way of life
to a more settled one when emigrating to a high-income country, leading to a significant alteration in their
daily PA routines (27). In addition to labour-saving technologies and other advancements that enable
reduced physical exertion, reasons for the low PA level may be lack of suitable places to exercise, cold
weather and short daylight periods during winter, large family size and limited childcare resources, safety
concerns, tradition and religious beliefs and norms (23, 24, 28, 29). PA promotion is particularly crucial for
groups at high risk of chronic diseases who experience disparities in PA participation. This includes
immigrant women who face a plethora of barriers to PA. Despite calls for interventions to address PA
among immigrant populations (30, 31), few interventions have been reported. To our knowledge, only
three randomised controlled studies have been conducted with women of Somali background, where two
were unsuccessful in increasing the participants’ PA level (32, 33) and one managed to increase the
participants’ PA level, but had a small sample size (N = 27) and used self-reported measure of PA (34). Introduction We
lack knowledge of how best to promote awareness of the importance of PA and engage women of
immigrant background in PA, but research suggests that community-based culturally safe approaches
that are tailormade to the participants and accommodate culture, gender and socioeconomic factors are
needed (35). The study’s primary objective was to foster enduring changes in lifestyle through the application of
motivational theories, specifically drawing from Self-Determination Theory and Achievement Goal Theory. The programme's focus was to empower women in cultivating intrinsic and personally meaningful
motivation to embrace a more active, less sedentary, and healthier dietary lifestyle. Notably, the
programme was customised with a gender- and culturally sensitive approach, encompassing
considerations in context, content, and delivery style and thus placed significant emphasis on adapting
the intervention to align with the cultural norms, beliefs, and traditions of its participants. Since the prevalence of obesity is especially high among women of Somali background in Norway and
other high-income countries, and PA can beneficially influence this condition, special attention to the PA
behaviour of this population group is needed. Based on the aforementioned limitations in existing
research, the aims of this paper were firstly, to describe objectively, and subjectively, measured PA
patterns in a group of overweight and obese women of Somali background living in Norway, and
secondly, to examine the long-term efficacy of the tailormade, culturally sensitive and community-based
intervention in increasing PA and reducing sedentary time. Methods Page 4/20 Page 4/20 This study is based on data from the Increased Physical Activity and a Healthier Lifestyle among
Immigrant Women study (ASLI) (clinicaltrials.gov NCT04578067) which was designed to investigate the
effect of a lifestyle programme (increased PA, reduced SED, and healthier eating) on weight loss. To
mitigate the potential risk of contamination between the intervention and comparison groups, treatment
conditions were randomly allocated from two distinct boroughs. The study was approved by the
Norwegian Centre for Research Data (ref: 724880). Recruitment and participants Participants were recruited through Somali radio, women’s groups, general practitioners, and female user
representatives from Somali community-based organisations who were included in the study team to
help with information and recruitment, from September 2020 through May 2023 from two boroughs in
Oslo, Norway. Women with a Somali background aged 20 and above with a body mass index (BMI) of ≥
27.0 kg.m− 2 were eligible for the study. Women who were pregnant, participating in a formal weight loss
programme, taking medications that may affect weight loss, have severe musculoskeletal problems or
difficulty walking, and had significant medical co-morbidities such as cancer, uncontrolled diabetes or
diagnosed eating disorders, were excluded from the study. Written consent was obtained from all eligible
participants who understood the nature of the research and were willing to participate. Intervention ASLI was designed, in collaboration with female representatives of Somali background, to support
overweight and obese women to lose weight through increased PA level (10 min per day), reduced
sedentary time (30 min per day) and healthier eating, and maintain these changes at least 12 months
after baseline. The programme was delivered at the Healthy Life Centres in two boroughs in the
municipality of Oslo to groups of 13 to 26 women over 12 weeks. The programme consisted of 90-min
sessions twice per week combining classroom discussion with graded group-based PA activity led by
coaches, followed by monthly sessions over nine months. ASLI aimed to promote sustained lifestyle change by drawing on motivational theories (Self-
Determination Theory and Achievement Goal Theory) to encourage the women to develop internalised
and self-relevant motivation for becoming more active, sitting less, and eating a healthier diet. The
programme was gender-sensitised concerning context, content, and style of delivery. In relation to
content, ASLI explicitly targeted theory-derived mechanisms of action (e.g., autonomous motivation, task-
oriented goals) and made use of the most evidence-based self-regulation techniques (e.g., self-
monitoring, goal setting, and implementation intentions). Special emphasis was placed on adapting the
intervention to the cultural norms, beliefs, and traditions of the participants. Study materials, including the
informed consent form, participant information, and food booklets were translated into Somali language
either in written form or verbally. Moreover, the intervention was provided by a multidisciplinary team
comprising nutritionists, nurses, and public health specialists. The research team developed nutrition and PA manuals which were based on the Norwegian Directorate
of Health’s recommendations. The PA manual contained modules intending to increase both awareness Page 5/20
The research team developed nutrition and PA manuals which were based on the Norwegian Directorate
of Health’s recommendations. The PA manual contained modules intending to increase both awareness Page 5/20 Page 5/20 of own PA level and opportunities and knowledge about PA in general (e.g. to be more active and increase
PA levels, how active and sedentary are we, short-term and long-term goals, the link between PA and
health, strategies to cope with PA barriers and relapse prevention). of own PA level and opportunities and knowledge about PA in general (e.g. to be more active and increase
PA levels, how active and sedentary are we, short-term and long-term goals, the link between PA and
health, strategies to cope with PA barriers and relapse prevention). Intervention Using the supporting manual developed by the study team, coaches (who received two days of
standardised training to deliver ASLI) provided participants with a toolbox of behaviour change
techniques, which were reinforced and practised through interaction and discussion between participants
during face-to-face group sessions. The materials were designed to help participants embed the new
behaviours into their everyday lives so that they were able to maintain these changes in the long term. Participants chose from the skills and strategies in the ASLI toolbox to change their PA, sedentary
behaviour, and diet. Simple, practical, relevant messages allowed participants to understand what they
can do to personally improve their PA, sedentary behaviour, and diet. The women were supported to
choose to engage in the behaviours that personally fit in their lives and to develop a clear rationale for
why they value these new behaviours. Moreover, interactions with other participants provided support to
collaboratively tackle challenges and encourage changes being made. Together these components
should foster the formation of new, self-endorsed, healthy lifestyle routines that sustain behaviour
change. When it comes to the style of delivery, ASLI coaches helped the women feel comfortable and
receptive to change from the outset by reinforcing the experience that they are with other ‘women-like-me’
and that their efforts each week, within and between programme sessions, were valued by the coaches. In
particular, the coaches were instructed on how to create a motivational, and autonomy- and mastery-
supportive climate, and on the importance of understanding and respecting participants’ perspectives
and preferences for lifestyle change. This delivery style aimed to promote intrinsic interest and foster
sustained engagement among participants. The coaches learned to provide a rationale for behavioural
change, to collaboratively develop change options for the women to choose from, and to facilitate the
development of participants’ personally relevant goals (rather than imposing goals on them). Engagement was promoted by ensuring the sessions were enjoyable, fun, non-dogmatic, experiential, and
interactive. It was also important to create a mutually supportive ‘team’ environment that helped women
learn from each other by sharing tips and advice, whilst facilitating interactional styles that women are
familiar with in other (predominantly) female contexts. Engagement was promoted by ensuring the sessions were enjoyable, fun, non-dogmatic, experiential, and
interactive. Intervention It was also important to create a mutually supportive ‘team’ environment that helped women
learn from each other by sharing tips and advice, whilst facilitating interactional styles that women are
familiar with in other (predominantly) female contexts. Positive feedback and celebration of individual progress (not just achievement) towards small, short-term
goals helped participants feel competent and confident that they can succeed in their long-term PA,
sedentary behaviour, healthy eating targets (as well as weight loss, if appropriate). Women were also
encouraged to recognise the personal value and benefits of the changes that they were making (e.g.,
feeling fitter, having more energy). Throughout the programme, long-term social support was promoted
within the group by encouraging positive interactions to build relationships and by encouraging the
women to use social media (i.e., Facebook, WhatsApp) to support each other outside the sessions and to
meet up between sessions to exercise or cooking food together, as well as by encouraging the women to
enlist the support of their wider social networks (e.g., family, friends). The monthly sessions following the
intensive weekly sessions provided women with an opportunity to share their experiences of maintaining
the changes they made during the first part of the programme. Page 6/20 Page 6/20 Objectively assessed PA The two primary outcomes in this paper were total PA (steps per day) and total sedentary time (min per
day), objectively measured at baseline and 12 months after randomisation using the ActivPAL monitor
(model ActivPAL micro; PAL Technologies, Glasgow, United Kingdom). The ActivPAL has been found to
have good measurement properties to assess sitting, standing, stepping, and postural transitions in
adults (25, 26, 36). The ActivPAL is a small activity monitor attached to the thigh and worn for seven consecutive full days. Researchers provided face-to-face instruction on how to use the ActivPAL at both visits and ensured the
women all succeeded in affixing the device to their thigh correctly, offering assistance where needed. This
allowed them to re-apply the device should it come off for any reason during the week. They were asked
to wear the device continuously, except during water-immersing activities (e.g., swimming, bathing). Participants returned approximately seven days later, during which the ActivPAL was removed, and the
data were downloaded on a computer. Analysis was restricted to participants who wore the accelerometer
for a minimum of 24 hours per day for at least three days (37). Measurements Data collection took place at the Healthy Life Centres in the two boroughs. All measurements were
conducted by a trained study team (nutritionist, nurse, epidemiologist, and a public health expert) who
followed and used standardised protocols and tools. Each assessment included a physical examination,
collection of blood samples, and questionnaires, as previously described in detail (4). Other measures Weight was measured without shoes in light clothing by an Omron medical scale to the nearest 0.1 kg. Height was measured without shoes with a manual height measuring instrument (SECA stadiometer) to
the nearest 0.1 cm. Waist circumference was measured midpoint between the lower margin of the last
palpable rib and the top of the iliac crest using a measuring tape (SECA 201) to the nearest 0.1 cm with
the participant standing and breathing normally. Overweight and obesity were defined as a BMI of 25–29
and ≥ 30, respectively. Data on education, employment, number of children in the household, years living
in Norway and marital status were obtained through a self-complete questionnaire. Statistical analysis All statistical analyses were performed using SPSS (Statistical Package for the Social Sciences for
Windows, version 29, IBM, Inc., Chicago, IL, USA). Descriptive data are presented as proportions, means,
and standard deviations (SDs) with 95% confidence intervals (CIs) where appropriate. Within- and
between-group differences in interval data were evaluated by t-tests (independent t-tests and paired t-
tests). For objective and subjective PA analysis a multiple imputation with predictive mean matching was
executed, assuming data is missing at random, and aiming to reduce bias and improve precision (41). With this approach, the distribution of the observed data is used to estimate multiple possible values for
the data points (41). For multiple imputation and predictive mean matching, 40 imputations were used,
reflecting the percentage of participants with a missing outcome, and ten was used as the number of
nearest neighbours for predictive mean matching. A multiple imputation plot with an explanation is
attached as supplement material (Figures A1 and A2). The multiple imputation analysis and linear
regression after imputation was performed using STATA (version 17, StataCorp. LLC, Texas, USA). Differences between the groups in terms of education level, employment and marital status were
assessed using chi-squared tests. All significance tests were two-sided. Subjectively assessed PA The International PA Questionnaire Short Form (IPAQ-SF) (38) was used to assess subjective PA levels. The IPAQ focuses on the amount of PA performed over the past seven-day period. The IPAQ includes
questions about the time spent engaging in vigorous PA, moderate PA and walking in 10-min bouts or
longer. Within these domains, participants are asked to consider all types of physical activities, including
activities performed during leisure time, domestic and gardening activities, work-related activities, and
transport-related activities. Relevant examples of activity performed at the different intensities were
mentioned in the items. The data obtained from the IPAQ were used to estimate the total amount of PA
completed in a seven-day period by weighting the reported min per week in each domain by a metabolic
equivalent (MET) energy expenditure estimate. The weighted MET minutes per week were then calculated
by multiplying the duration (minutes), frequency (days) and MET intensity and then summing the three
domains, namely, vigorous (8 METs), moderate (4 METs) and walking (3.3 METs), to produce a weighted
estimate of total PA per week (min∙week− 1) (39). The participants were categorised into low, moderate
and high PA levels according to the IPAQ scoring criteria (38). Those categorised as having moderate or
high activity were classified as being sufficiently active according to the WHO PA guidelines (40). Data Page 7/20 cleaning and processing were carried out following the guidelines published by the IPAQ Research
Committee, and the methods used to score the IPAQ are described in the IPAQ scoring protocol (38). Results Of 271 women of Somali background invited to participate in the study, 169 accepted to participate,
attended the first visit, and were found eligible. The reason for the exclusion of women who showed
interest in the study was BMI < 27.0 kg.m− 2. Figure 1 shows the flow of participants through the trial. There was a significant difference between those with valid accelerometer recordings at the 12-month
measurement session (N = 83) and those without valid accelerometer recordings (N = 86) on years of
education of 1.4 years (95% CI = 2.7,0.1; p = 0.03). There were no other differences between those with
valid accelerometer recordings at the 12-month measurement session and those without valid
accelerometer recordings on any other background variables or PA level. Page 8/20 Table 1 shows relevant sociodemographic information for both groups. The women had lived in Norway
for an average of 16 years, only one was born in Norway. Number of children in the household ranged
from zero to seven. On average the women had completed around seven years of education ranging from
zero to 16 years, where 7.8% had completed college or higher. Seventy-two per cent had a BMI above 30
kg.m− 2. The participants took on average 6804 (SD = 3286) steps per day and were sedentary for 9.1
hours (SD = 3) per day at baseline. Twenty-eight (17 per cent) women reached 10,000 steps per day at
baseline. There were no statistical differences between the groups at baseline. Table 1
Baseline sociodemographic characteristics of participants allocated to the lifestyle
programme immediately (intervention) or after 12 months (comparison). Results Data are
mean (SD) or N (%)
Sociodemographic characteristics
Intervention (n = 82)
Comparison (n = 87)
Age (years)
45.8 (10.1)
47.9 (10.9)
Height (cm)
161.1 (4.9)
161.6 (4.5)
Weight (kg)
87.0 (12.9)
89.4 (14.5)
BMI (kg.m− 2)
33.5 (5.0)
34.1 (5.2)
Waist (cm)
99.1 (10.5)
97.2 (10.8)
Married (%)
43 (52)
61 (70)
Number of children living at home
2.2 (1.8)
2.2 (1.9)
Years living in Norway
16.3 (6.3)
16.8 (6.5)
Years in school
6.8 (4.4)
7.5 (4.2)
Working or studying (%)
42 (51)
43 (49)
PA (ActivPAL)
Number of valid wear days
4.7 (1.8)
5.0 (1.9)
Steps per day
7175 (2975)
6455 (3536)
Sedentary time (min∙day− 1)
553 (174)
544 (187)
Stepping time (min∙day− 1)
96 (38)
85 (44)
Standing time (min∙day− 1)
258 (102)
252 (108)
Upright time (min∙day− 1)
354 (127)
337 (135)
Abbreviations: SD, standard deviation; BMI, body mass index; PA, physical activity. Except for the number of valid days at the 12-month measurement, there were no differences between
groups on any PA variable at any time point (Table 2). The number of steps per day and sedentary time
were only 106 min and 20 min higher in the intervention group compared to the control group with a very
wide 95% confidence interval. Furthermore, there were no within-group differences in any of the PA
variables between the baseline and post-programme time points (data not shown). Table 2
Objectively assessed physical activity and sedentary time outcome measures for participants in the
intervention compared to a comparison group after multiple imputations (n = 169)
PA variable
Mean treatment difference
(95%CI)a
Adjusted mean treatment difference
(95%CI)b
ΔSteps per day
243.8 (-719.1, 1206.8)
106.1 (-870.3, 1082.5)
ΔSedentary time
(min∙day− 1)
19.8 (-46.1, 85.7)
20.5 (-46.6, 87.5)
ΔStepping time
(min∙day− 1)
6.5 (-5.9, 19.0)
4.8 (-7.9, 17.5)
ΔStanding time
(min∙day− 1)
-9.5 (-54.2, 35.3)
-9.8 (-56.0, 36.5)
ΔUpright time (min∙day−
1)
-3.6 (-55.9, 48.7)
-5.3 (-59.5, 48.9)
Abbreviations: PA, physical activity; CI, confidence interval. aAdjusted for baseline value and number
of valid wear days at 12 months. bAdjusted for baseline value, number of valid wear days at 12
months, age, education, employment, marital status, number of children in the household, and length
of Norwegian residence. Table 2 Abbreviations: PA, physical activity; CI, confidence interval. aAdjusted for baseline value and number
of valid wear days at 12 months. Results bAdjusted for baseline value, number of valid wear days at 12
months, age, education, employment, marital status, number of children in the household, and length
of Norwegian residence. Abbreviations: PA, physical activity; CI, confidence interval. aAdjusted for baseline value and number
of valid wear days at 12 months. bAdjusted for baseline value, number of valid wear days at 12
months, age, education, employment, marital status, number of children in the household, and length
of Norwegian residence. From a total of 24 sessions, the women attended 9.6 (6.5) sessions. Thirty-nine percent of the women
attended more than half of the sessions and seven women did not attend any of the sessions. On
average the women gave the programme a rating of 9.7 (0.5) on a scale of 1 to 10, and similarly rated the
coaches at 9.7 (0.5). Furthermore, 95% would recommend the programme to others. There were no
correlations between change in steps or sedentary time with attendance recorded by the coach. There
was no evidence that change in steps or sedentary time varied by age, years of education, number of
children in the household, years living in Norway or baseline BMI. Women achieving 10,000 steps per day at baseline, post-programme and 12 months in the intervention
group was 15.8%, 11.9% and 15.8%, and for the comparison group, it was 20.3%, 12% and 12.2%. In the Page 10/20 Page 10/20 Page 10/20 intervention group, 42.1% increased the number of steps from baseline to 12 months, compared to 48.9%
in the comparison group, and 50% and 51.1% reduced sedentary time from baseline to 12 months, in the
intervention and comparison groups, respectively. intervention group, 42.1% increased the number of steps from baseline to 12 months, compared to 48.9%
in the comparison group, and 50% and 51.1% reduced sedentary time from baseline to 12 months, in the
intervention and comparison groups, respectively. There were no differences in self-reported PA levels between the two groups at baseline (data not shown). There were significant differences in vigorous intensity PA and total PA at the 12-month measurement
session between the two groups (Table 3). Results Table 3
Subjectively assessed physical activity outcome measures for participants in the intervention compared
to a comparison group after multiple imputations (n = 169)
PA variable
Mean treatment difference
(95%CI)a
Adjusted mean treatment difference
(95%CI)b
ΔWalking (min∙week−
1)
43.9 (-8.9, 96.6)
38.6 (-16.3, 93.5)
ΔModerate
(min∙week− 1)
12.4 (-0.2, 25.1)
12.2 (-0.5, 24.9)
ΔVigorous
(min∙week− 1)
26.6 (9.1, 44.1)1
25.9 (7.7, 44.1)2
ΔTotal PA (min∙week−
1)
82.5 (19.9, 145.1)3
77.6 (13.2, 142.1)4
Abbreviations: PA, physical activity; CI, confidence interval. aAdjusted for baseline value, bAdjusted for
baseline value, age, education, employment, marital status, number of children in the household, and
length of Norwegian residence. 1P-value = 0.004 2P-value = 0.006, 3P-value = 0.010, 4P-value = 0.019 Table 3 Abbreviations: PA, physical activity; CI, confidence interval. aAdjusted for baseline value, bAdjusted for
baseline value, age, education, employment, marital status, number of children in the household, and
length of Norwegian residence. 1P-value = 0.004 2P-value = 0.006, 3P-value = 0.010, 4P-value = 0.019 Abbreviations: PA, physical activity; CI, confidence interval. aAdjusted for baseline value, bAdjusted for
baseline value, age, education, employment, marital status, number of children in the household, and
length of Norwegian residence. 1P-value = 0.004 2P-value = 0.006, 3P-value = 0.010, 4P-value = 0.019 Most women in both groups were in the category of low PA level (not meeting the PA recommendations
of 150 min of MVPA per week) at baseline (Figure 2). A relatively large shift from the category low PA
level to the categories moderate and high was seen for the intervention group, but not for the comparison
group (Figure 2). The chi-square test of independence showed that women in the intervention group were
less likely to be in the low PA level category and more likely to be in the moderate and high PA level
category at the 12-month measurement session than women in the comparison group (X2 (2, N=99) =
23.9, P < 0.001). Discussion This study aimed to determine, both objectively and subjectively, PA level and sedentary time in
overweight and obese women of Somali background in Norway. In addition, to examine the efficacy of
the tailormade, culturally sensitive and community-based intervention in increasing PA and reducing
sedentary time. We found that the women had a very low PA level and a high amount of sedentary time at Page 11/20 Page 11/20 baseline and that despite this high intervention potential, the intervention did not manage to increase
objectively measured PA level or reduce sedentary time more than the control condition, although about
50% of the women in the intervention group did increase PA level and reduced sedentary time. However,
when measured subjectively, there was a highly significant difference in both vigorous intensity PA and
total PA level between the groups at the 12-month measurement session. The low objectively measured PA level and high amount of sedentary time are in line with the findings of
studies using self-reported measures in this group (3, 7, 22-24). This is of big concern, seeing that PA level
and/or sedentary behaviour are strongly linked to both morbidity and mortality (42, 43). The reasons for
these suboptimal behaviours are not known and are probably multiple and complex (23). Although the
study did take into consideration known barriers to PA in this group, and tailormade the intervention to
their culture, moral norms, and beliefs we did not succeed in increasing PA levels or reducing sedentary
time, even though they rated both the programme and the coaches very high and most of the women
reported that they would recommend the programme to others. There may be several reasons for the lack
of effect. First, the study was conducted during the coronavirus pandemic, and adherence to the
intervention was difficult due to quarantine, being infected with COVID-19, and occasional lockdowns of
schools and kindergartens. The programme’s lack of effect may thus be explained partly by the relatively
poor attendance rate and that following the whole programme may have yielded different results,
although we did not find a correlation between attendance rate and PA level. Second, another important
reason for the lack of effect may be that many women focused on losing weight through dietary changes
and not so much PA, and thus a stronger focus on PA and sedentary behaviour both in the programme
and among the women may have provided different results. Discussion Second, based on previously reported barriers to PA in this group a
major strength of the study is women-only groups, female trainers, information given in both Norwegian
and Somali and tools and advice tailormade to Somali culture, norms, and beliefs. Third, a strength of the
study is also the objective assessment of PA level and sedentary time, since self-reported measurements
are known to have serious flaws. However, although a minimum of three days of measurement data is
recommended, we cannot rule out the possibility that including only those with a full week of data would
have yielded more reliable and valid data (37). As discussed above, limitations to the study could be contamination, the relatively low attendance rate
and the high drop-out from the study. Seeing that the Somali community is relatively small and that the
women have several mutual meeting places we cannot rule out the possibility that contamination has
occurred. It is highly likely that the participants from the intervention group have interacted with those
from the comparison group and have shared details about the programme, which could have led to
unintended changes in behaviour within the comparison group. This can compromise the study's internal
validity by confounding the true impact of the intervention. Furthermore, the relatively low attendance
rates, mainly caused by the coronavirus pandemic, could have undermined the effectiveness of the
intervention. If a significant number of participants do not regularly attend the programme sessions, it
becomes challenging to assess the true impact of the intervention on PA levels. This limitation may also
raise questions about the programme's feasibility, acceptability, and relevance to the target population,
affecting the generalisability of the study findings. Lastly, participant drop-out is a common challenge in
these types of studies and can introduce bias, especially if the reasons for dropping out are related to the
intervention or outcome measures. In response to an open-ended question, the primary reasons for
dropping out were lack of interest, lack of time, and illness. When participants drop out due to
dissatisfaction with the programme or other unforeseen factors, the study's ability to draw accurate
conclusions about the programme's effectiveness diminishes. High drop-out rates may also affect the
representativeness of the sample and introduce selection bias. Discussion Third, seeing that around half of the women
in both groups increased PA levels and reduced sedentary time we cannot rule out the possibility of
contamination between the boroughs due to being part of the same community across the city of
Oslo (44). Fourth, this study had a high dropout rate which made the study underpowered, and we cannot
rule out a larger (or smaller) effect size if retention was higher. Fifth, the intervention season is a
limitation, as it mainly took place during the autumn and winter months, due to Ramadan (the fasting
month of Muslims), which is known to influence PA habits (45). Lastly, although we do not have data on
this, there could be that the women, despite voluntarily signing up for the programme, lacked motivation
and commitment to change their PA level and sedentary behaviour, or that important aspects of the
lessons and assignments were not fully grasped or understood. Page 12/20
An interesting observation emerged from our study, as there were no significant differences in the
objectively measured PA levels between the two groups at the 12-month measurement session, but a
noteworthy distinction did surface in the self-reported PA levels. This discrepancy could imply that the
women genuinely perceive an increase in their PA level at the 12-month assessment compared to
baseline. Alternatively, it raises the possibility of social-desirability bias, wherein participants may
respond in a manner they believe aligns with societal expectations rather than providing accurate
information. If the former holds true, with women feeling more physically active despite objective
measurements suggesting otherwise, it may influence their attitudes and behaviours toward maintaining
or further enhancing their PA levels. On the other hand, if social-desirability bias is at play, it prompts us to Page 12/20 Page 12/20 critically assess the reliability of self-reported data in capturing the true essence of participants'
experiences and behaviours in the study context. The ASLI study has several strengths. First, a major strength of this research lies in its inclusive approach,
wherein participants were not excluded based on their ability to speak or understand the Norwegian
language thus increasing the generalisability of the study. This inclusivity was facilitated by the presence
of a Somali-speaking researcher within the study team and the involvement of Somali user
representatives in both boroughs. Discussion In summary, our study aimed to comprehensively assess the PA and sedentary behaviour of overweight
and obese women with Somali background living in Norway, both objectively and subjectively, and
evaluate the efficacy of a tailormade, culturally sensitive, and community-based intervention. Despite
observing initially low PA levels and high sedentary time at baseline, the intervention did not yield a
statistically significant increase in objectively measured PA or reduction in sedentary time compared to
the control condition but managed to do so when using self-reported measures. Contamination, low
attendance rate and high drop-out rate are possible major threats to the reliability and validity of the Page 13/20 Page 13/20 Page 13/20 programme. These limitations underscore the complexity of conducting interventions in this demographic
and emphasise the need for further exploration and refinement of study methodologies to increase PA
levels and reduce sedentary time in immigrant women living in high-income countries. programme. These limitations underscore the complexity of conducting interventions in this demographic
and emphasise the need for further exploration and refinement of study methodologies to increase PA
levels and reduce sedentary time in immigrant women living in high-income countries. Abbreviations PA
physical activity
IPAQ
international PA questionnaire
T2D
type 2 diabetes
MET
metabolic equivalent
CI
confidence interval
ASLI
Increased Physical Activity and a Healthier Lifestyle among Immigrant Women study
BMI
body mass index
SED
sedentary
SD
standard deviation
CVDs
cardiovascular diseases
MVPA
moderate- to vigorous intensity physical activity Acknowledgments The authors would like to give thanks to all participants. The authors thank Inga Brenne for contributing
to the data collection and Hein Stigum for contributing to statistical analysis. The flow chart and
graphical abstract were created with BioRender.com. Authors' contributions AAM, HEM, MLW, MCW, and EA developed the original idea. AAM, and MCW acquired the funding for the
study. LB, MJL, DC, and AAM collected the data. AAM, LB, and EA had access to the data set. EA and LB
performed the statistical analyses and drafted the manuscript. HEM contributed with advice on the
statistical analyses. All authors revised the work for intellectual content and approved the final version to
be published and accept responsibility for the decision to submit for publication. Funding The project was supported financially by the Research Council of Norway under Grant number 296558,
the Norwegian Women’s Public Health Association, Grant number 46168 and Eckbo’s Legacy, Grant
number 87624. Consent for publication Page 14/20 After publication, anonymised data underlying the results and analysis can be made available to
researchers upon reasonable request to the corresponding author. A data access agreement needs to be
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mortality: systematic review and harmonised meta-analysis. BMJ. 2019;366:l4570. 42. Ekelund U, Tarp J, Steene-Johannessen J, Hansen BH, Jefferis B, Fagerland MW, et al. Dose-response
associations between accelerometry measured physical activity and sedentary time and all cause
mortality: systematic review and harmonised meta-analysis. BMJ. 2019;366:l4570. 43. Blodgett JM, Ahmadi MN, Atkin AJ, Chastin S, Chan HW, Suorsa K et al. Device-measured physical
activity and cardiometabolic health: the Prospective Physical Activity, Sitting, and Sleep (ProPASS)
consortium. Eur Heart J. 2023. 43. Blodgett JM, Ahmadi MN, Atkin AJ, Chastin S, Chan HW, Suorsa K et al. Device-measured physical
activity and cardiometabolic health: the Prospective Physical Activity, Sitting, and Sleep (ProPASS)
consortium. Eur Heart J. 2023. 44. Hunter RF, McAneney H, Davis M, Tully MA, Valente TW, Kee F. Hidden social networks in behavior
change interventions. Am J Public Health. 2015;105(3):513–6. 44. Hunter RF, McAneney H, Davis M, Tully MA, Valente TW, Kee F. Hidden social networks in behavior
change interventions. Am J Public Health. 2015;105(3):513–6. 45. Turrisi TB, Bittel KM, West AB, Hojjatinia S, Hojjatinia S, Mama SK, et al. Seasons, weather, and
device-measured movement behaviors: a scoping review from 2006 to 2020. Int J Behav Nutr Phys
Act. 2021;18(1):24. Figure 2 Distribution of PA level categories at baseline and 12 months, based on the IPAQ scoring protocol, for
participants allocated to the lifestyle programme immediately (intervention) or after 12 months
(comparison) Supplementfile.docx Figures Page 18/20 Figure 1
shows the flow of participants through the trial Figure 1 Figure 1 shows the flow of participants through the trial shows the flow of participants through the trial Page 19/20 Figure 2
Distribution of PA level categories at baseline and 12 months, based on the IPAQ scoring protocol, for
participants allocated to the lifestyle programme immediately (intervention) or after 12 months
(comparison)
S
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Hopkinson peak and superparamagnetic effects in BaFe<sub>12-x</sub>Ga<sub>x</sub>O<sub>19</sub>nanoparticles
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EPJ web of conferences
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a e-mail : Ibrahimbsoul@yahoo.com EPJ Web of Conferences
DOI: 10.1051/
C
⃝Owned by the authors, published by EDP Sciences, 2012
,
epjconf 20122
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29 00039 (2012)
900039 EPJ Web of Conferences
DOI: 10.1051/
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⃝Owned by the authors, published by EDP Sciences, 2012
,
epjconf 20122
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29 00039 (2012)
900039 Hopkinson
peak
and
superparamagnetic
effects
in
BaFe12-xGaxO19 nanoparticles S. H. Mahmood1, I. Bsoula2
1Physics Department, The University of Jordan, Amman, Jordan. Phone: (962) 796709673
2Physics Department, Al al-Bayt University, Mafraq 130040, Jordan. Phone: (962 2) 6297000 (ext. 3588), Fax: (962 2) 6297031 S. H. Mahmood1, I. Bsoula2
1Physics Department, The University of Jordan, Amman, Jordan. Phone: (962) 796709673
2Physics Department, Al al-Bayt University, Mafraq 130040, Jordan. Phone: (962 2) 6297000 (ext. 3588), Fax: (962 2) 6297031 S. H. Mahmood1, I. Bsoula2
1Physics Department, The University of Jordan, Amman, Jordan. Phone: (962) 796709673
2Physics Department, Al al-Bayt University, Mafraq 130040, Jordan. Phone: (962 2) 6297000 (ext. 3588), Fax: (962 2) 6297031 Abstract. In this article, the thermomagnetic properties of a system of Ga-substituted
barium hexaferrite nanoparticles (BaFe12-xGaxO19) prepared by ball milling were
investigated. The thermomagnetic curves for the samples with x ranging from 0.0 to 1.0
exhibited sharp peaks with high magnetization just below TC (Hopkinson peaks). The
height of the peak for our samples was similar or larger than previously observed or
calculated values. Theoretical treatment of the experimental data demonstrated that the
peaks are due to the effect of superparamagnetic relaxations of the magnetic particle. This effect was confirmed by hysteresis measurements at, and just below the
temperature at which the peak occurred. Consequently, the particle diameters were
calculated from the experimental data using a theoretical model based on the
superparamagnetic behavior of a system of uniaxial, randomly oriented, single domain,
non-interacting particles. The calculated diameters of 11 - 26 nm are less than the
physical diameters determined from TEM measurements. The factors responsible for the
low calculated values are discussed. 2 Experimental procedures BaFe12-xGaxO19 powders with x ranging from 0.0 to 1.0 were prepared using high energy ball
milling and appropriate heat treatment. The samples were characterized using XRD and transmission
electron microscopy (TEM), and the magnetic measurements were performed using a vibrating
sample magnetometer (VSM). For further details on the experimental procedures the reader is
referred to our earlier publication [24]. 1 Introduction Barium hexaferrite BaFe12O19 (BaM) possess interesting properties such as large saturation
magnetization, high coercivity, high Curie temperature, large uniaxial magnetic anisotropy and
chemical stability. These materials have been investigated due to their importance for both
fundamental research and technological applications in permanent magnets, high-density magnetic
recording, magneto-optics and microwave devices [1-6]. Different techniques have been used to
prepare and characterize hexaferrite particles [7-13]. The magnetic properties of these materials have
been tuned by substitution of Fe by different magnetic and nonmagnetic cations, and intensively
investigated by different techniques [14-20]. The magnetization of certain hexaferrites exhibits a peak (Hopkinson peak) near TC in the
thermomagnetic curve in a weak applied magnetic field. Popov and Mikhov explained this effect
using Stoner-Wohlfarth model for magnetically stable, single-domain (SD), randomly oriented
particles [21]. According to this model, the magnetization is given by: This i s an Open Acces s article distributed under the terms of the Creative Commons Attribution License 2.0, which permits
unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited. Article available at http://www.epj-conferences.org or http://dx.doi.org/10.1051/epjconf/20122900039 EPJ Web of Conferences H
T
H
T
M
p
M
a
s
SD
)
(
)
(
3
2
=
(1) (1) where p is the packing fraction of the powder, Ms(T) is the bulk saturation magnetization, and Ha(T)
is the anisotropy field at temperature T. The explanation was based on the argument that the
competition between the increase in magnetization due to the decrease in the anisotropy field, and
the decrease in magnetization due to the decrease in saturation magnetization as the temperature
increases may result in a peak in the magnetization. Later, the effect of the demagnetizing field was
taken into consideration in explaining the origin of Hopkinson peak [22]. Using a completely
different approach, the origin of the peak was explained by the superparamagnetic behavior of
magnetic particles which do not exhibit a peak as a result of the variations of the saturation
magnetization and anisotropy field of the material [23]. In the present work we prepared a system of BaM nanoparticles doped with Gallium, and
investigated its structural and magnetic properties. The Hopkinson peak height was analyzed in
terms of the superparamagnetic relaxation processes of the particles and compared with the
experimental data to arrive at a conclusion concerning the mechanism responsible for the peak, and
the particle size distribution of the synthesized powders. 3 Results and discussion Fig. 1. Standard JCPDS pattern for M-type hexagonal barium ferrite (file no: 043-0002) and XRD
patterns of BaFe12-xGaxO19 with different doping concentration. g. 1 shows the XRD patterns of samples of BaFe12-xGaxO19 along with the standard pattern
CPDS: 043-0002) for hexagonal barium ferrite (BaFe12O19) with space group P63/mmc. No Fig. 1. Standard JCPDS pattern for M-type hexagonal barium ferrite (file no: 043-0002) and XRD
patterns of BaFe12-xGaxO19 with different doping concentration. Fig. 1 shows the XRD patterns of samples of BaFe12-xGaxO19 along with the standard pattern
(JCPDS: 043-0002) for hexagonal barium ferrite (BaFe12O19) with space group P63/mmc. No Fig. 1 shows the XRD patterns of samples of BaFe12-xGaxO19 along with the standard pattern
(JCPDS: 043-0002) for hexagonal barium ferrite (BaFe12O19) with space group P63/mmc. No 00039-p.2 EMM-FM2011 secondary phases were detected in the diffraction patterns indicating the formation of a pure phase
with variations in the lattice parameters less than 0.1%. secondary phases were detected in the diffraction patterns indicating the formation of a pure phase
with variations in the lattice parameters less than 0.1%. p
The average crystallite size was determined using Scherrer formula [25], The average crystallite size was determined using Scherrer θ
β
λ
cos
k
D =
,
(2) θ
β
λ
cos
k
D =
, (2) where D is the crystallite size, k the Scherrer constant (= 0.94), λ the wavelength of radiation
(1.54056 Å), β the peak width at half maximum measured in radians, and θ the peak position. The
average crystallite size for the pure and doped samples ranges from 37 nm to 45 nm. Fig. 2. TEM images of BaFe12-xGaxO19, a) x = 0.0, b) x =1.0 Fig. 2. TEM images of BaFe12-xGaxO19, a) x = 0.0, b) x =1.0 TEM images of representative samples are shown in Fig. 2. The average particle size for the pure
sample is (42 ± 13) nm, and for the sample with x = 1.0 is (41 ± 13) nm. These values indicate that
the synthesized powders consist of single domain magnetic nanoparticles with a relatively narrow
particle size distribution. Fig. 3. Saturation magnetization and coercivity variations with x for BaFe12-xGaxO19 Fig. 3. 3 Results and discussion Saturation magnetization and coercivity variations with x for BaFe12-xGaxO19 Table 1: Coercivity, Saturation magnetization, remanence ratio (Mrs = Mr/Ms), and Curie temperature for Ga-
substituted hexaferrite samples rcivity, Saturation magnetization, remanence ratio (Mrs = Mr/Ms), and Curie temperature for Ga-
substituted hexaferrite samples x
Hc (kOe)
Ms (emu/g)
Mrs
Tc (°C)
0.0
4.02
70.9
0.523
460
0.2
3.95
69.3
0.519
430
0.4
4.00
68.3
0.518
420
0.6
4.26
68.0
0.519
400
0.8
4.49
65.3
0.517
385
1.0
4.55
60.3
0.514
370 00039-p.3 00039-p.3 EMM-FM2011 Fig.4 shows the thermomagnetic curves as a function of temperature for the samples at a constant
applied field of 100 Oe. All curves exhibit sharp pronounced Hopkinson peaks just below TC, and the
height of the peak relative to the minimum magnetization (RPH) for all samples is shown in Table 2. The sharpness of the peaks indicates a narrow particle size distribution. The relative peak heights for
our samples are similar or higher than the observed value of about 10 for Co-Ti substituted sample,
and the calculated value of about 8 based on superparamagnetic relaxation [23]. To investigate the
origin of the peak, a sample is prepared from bulk Barium hexaferrite (Aldrich made) powder of
grain size ~ 0.5 - 2 µm (with small fraction of smaller particles), and Ms for the sample is measured
against temperature. The anisotropy field Ha for the sample is determined from the switching field
distribution evaluated by differentiating the reduced DC demagnetization curve [24]. The
magnetization is then calculated from these values by adopting Stoner-Wohlfarth model for SD
blocked particles (eq. (1)) and the results are shown in Fig. 5. The figure shows only a very small
rise in the magnetization below TC which is insignificant compared with the peak heights observed
for our samples. Further, the magnetization is measured versus T for the bulk sample (Fig. 5). The
measured magnetization shows a relatively small sharp peak with relative height of 1.8, and a
behavior similar to that of the calculated magnetization in the temperature range below the peak. The
higher values of the measured magnetization in this temperature range could be associated with the
effects of interparticle interactions, or the presence of multidomain particles in the sample, which are
not accounted for in Stoner-Wohlfarth theory. Thus the observed sharp peaks in the thermomagnetic
measurements on our samples cannot be due to the temperature dependences of the saturation
magnetization and anisotropy field. The small peak observed for the bulk sample could be associated
with a small fraction of superparamagnetic particles in the bulk powder, or with the temperature
dependence of the saturation magnetization and the anisotropy field [21, 22]. However, the relative
height of this peak cannot account for the large observed peaks in our samples. Accordingly, we are
led to believe that the observed Hopkinson peaks in our synthesized samples are associated with the
superparamagnetic relaxations of the particles in the samples. Fig. EPJ Web of Conferences The initial magnetization of the pure sample was checked and found to increase slowly at low
fields followed by a rapid increase at higher fields [24]. This behavior is typical for randomly
oriented single domain magnetic particles. The variations of the saturation magnetization and
coercivity with x are shown in Fig. 3, and their values together with Curie temperatures for the
samples are listed in Table 1. The behavior of the saturation magnetization and coercivity indicates
that Ga ions replace Fe ions at both spin-up and spin-down sites. The small initial drop in coercivity
and slow decrease in saturation magnetization for x values up to 0.2 are consistent with the
substitution of Ga at spin-up 2a and spin-down 4f1 sites with preference for occupying 2a sites. This
is consistent with the substitution of small amounts of Ti-Ru at these sites as confirmed by
Mossbauer spectroscopy [26]. For higher x values, spin-up 12k sites which contribute negatively to
the anisotropy field start getting occupied by Ga ions, leading to the observed increase in coercivity
and decrease in saturation magnetization. The change in behavior of Ms at x = 0.6 suggests that
beyond this value, the fraction of Ga ions substituting Fe ions at spin-down sites remains constant at
a value of 0.2, where the remaining fraction substitute Fe ions at spin-up 12k sites. This substitution
would lead to the observed 5% drop in Ms at x = 0.6, and the 15% drop at x = 1.0. The remanence
ratio Mrs = Mr/Ms is ~ 0.5 (Table 1), which is consistent with the theoretical value for a system of
uniaxial, single domain, randomly oriented particles. Fig. 4. Thermomagnetic curves of BaFe12-xGaxO19. Fig. 5. Experimental and calculated thermomagnetic curves of bulk barium hexaferrite. Fig. 4. Thermomagnetic curves of BaFe12-xGaxO19. Fig. 4. Thermomagnetic curves of BaFe12-xGaxO19. Fig. 4. Thermomagnetic curves of BaFe12-xGaxO19. Fig. 5. Experimental and calculated thermomagnetic curves of bulk barium hexaferrite. Fig. 5. Experimental and calculated thermomagnetic curves of bulk barium hexaferrite. 00039-p.4 EMM-FM2011 6. Hysteresis loops for the sample with x = 0 at different temperatures. Fig. 6. Hysteresis loops for the sample with x = 0 at different temperatures. The magnetic relaxations of the particles are further confirmed by measuring the hysteresis loops
at the peak temperature and at lower temperatures. Fig. 6 shows the loops for the sample with x = 0,
which show superparamagnetic behavior with almost zero coercivity at the peak temperature, and the
appearance of coercivity at the temperature of the minimum magnetization just before the rise of the
peak (at 460 ºC). The increase in coercivity as the temperature is lowered is a consequence of the
gradual blocking of the particles as the temperature is lowered. All samples show similar behavior,
as illustrated in Fig. 7 for the sample with x = 0.4. 00039-p.5 00039-p.5 EPJ Web of Conferences
Fig. 7. Hysteresis loops for the sample with x = 0.4 at different temperatures. EPJ Web of Conferences Fig. 7. Hysteresis loops for the sample with x = 0.4 at different temperatures. Assuming that the volumes of the superparamagnetic particles have a flat top distribution
between V1 and V2, the upper and lower limits of the particle volumes can be calculated following
the model calculation in [27]. Following this model, the upper limit is calculated from the initial
susceptibility given by: kT
V
M
dH
dM
s
3
2
2
=
=
χ
(3) (3) where Ms is the saturation magnetization at temperature T. The lower limit is calculated from the
slope of the magnetization versus 1/H in the high field region, which is given by [27]:
k where Ms is the saturation magnetization at temperature T. The lower limit is calculated from the
slope of the magnetization versus 1/H in the high field region, which is given by [27]: H
V
kT
M
M
s
1
−
= H
V
kT
M
M
s
1
−
=
(4) H
V
kT
M
M
s
1
−
= (4) Fig. 8. Magnetization curve for the sample with x = 0.4 at peak temperature (414 oC). tial susceptibility, saturation magnetization, and the slope of M vs. 1/H are determined
magnetization curve (Fig. 8) for each sample at the peak temperature where the sample
perparamagnetically. Fig. 8. Magnetization curve for the sample with x = 0.4 at peak temperature (414 oC). EMM-FM2011 The initial susceptibility, saturation magnetization, and the slope of M vs. 1/H are determined
from the magnetization curve (Fig. 8) for each sample at the peak temperature where the sample
behaves superparamagnetically. The initial susceptibility, saturation magnetization, and the slope of M vs. 1/H are determined
from the magnetization curve (Fig. 8) for each sample at the peak temperature where the sample
behaves superparamagnetically. 00039-p.6 4 Conclusions Barium hexaferrite nanoparticle systems doped with different concentrations of Ga prepared by ball
milling exhibit single hexagonal phase with crystallite size ranging between 37 and 45 nm. The
magnetic measurements as a function of temperature exhibit sharp peaks with high relative
magnetization which cannot be explained on the basis of Stoner-Wohlfarth model for an assembly of
randomly oriented, non-interacting, single-domain particles. The magnetization curves at the peak
temperatures for all samples are consistent with the behavior of a system of superparamagnetic
particles, which are blocked at slightly lower temperature, indicating narrow superparamagnetic
particle size distribution. The calculated particle diameters for these samples are between 11 nm and
26 nm. These values suggest that the samples consist of single magnetic domain particles. Acknowledgement This work is supported by a generous grant from the Scientific Research Support Fund (SRF) in
Jordan under grant number (S/1/21/2009). The work is accomplished during a sabbatical leave
provided by Yarmouk University to one of the authors (S.H.M.) which was spent at Al al-Bayt
University. The Authors would like to thank Munir Khdour, Yarmouk University, for his technical
assistance in electron microscopy. 00039-p.6 EMM-FM2011 Table 2: Hopkinson peak temperature (Tp), saturation magnetization at the peak temperature, particle
diameters, and relative peak height (RPH) for Ga-substituted hexaferrite samples. x
Tp (°C)
Ms(Tp)(emu/g)
D1(nm)
D2(nm)
RPH
0.0
453
5.83
12
14
10.8
0.2
428
7.12
12
13
9.3
0.4
414
10.07
12
22
8.9
0.6
398
9.72
13
26
12
0.8
382
8.12
12
20
10.5
1.0
362
8.72
11
14
10.8 The calculated particle diameters, assuming spherical particles, are listed in Table 2. The particle
diameters range from 11 nm – 26 nm, which are lower than previously reported results [24]. Differences between particle diameters evaluated from the magnetic data and the previously reported
values could be due to deviation of the particle size distribution from the assumed flat top
distribution, and to interparticle interactions. However, these effects are possibly not enough to
account for the observed reduction of more than 50%. A number of reasons, in addition to the
assumptions of the theory, could be responsible for the low calculated values. Firstly, the calculated
volume is the volume of the magnetic core of the particle, and a nonmagnetic shell (dead layer)
could be surrounding the particles, which would give smaller particle sizes than the physical sizes. Secondly, the particles could be platelets in shape rather than spherical as suggested by the TEM
images. A rough estimate of the platelet thickness could be obtained by assuming that the observed
physical diameter is that of a cylindrical platelet of volume equals to that calculated from the
magnetic data. In this case, the thickness of the platelets is found to be between 1.5 nm and 4.0 nm. The lower limit of the thickness is smaller than the lattice parameter c, which is evidence that the
magnetic volume is an under estimate of the physical volume. 1. J. Fritzsche, R. B. Kramer, V. V. Moshchalkov, Phys. Rev. B 79, 132501 (2009) References 1. J. Fritzsche, R. B. Kramer, V. V. Moshchalkov, Phys. Rev. B 79, 132501 (2009). 1. J. Fritzsche, R. B. Kramer, V. V. Moshchalkov, Phys. Rev. B 79, 132501 (2009). 00039-p.7 EPJ Web of Conferences 2. P. Novak, J. Rusz, Phys. Rev. B 71, 184433 (2005). 3. J. Qiu, Q. Zhang, M. Gu, J. Appl. Phys. 98, 103905 (2005). 4. H. Pfeiffer, R. W. Chantrell, P. Gornert, W. Schuppel, E. Sinn, M. Rosler, J. Magn. Magn. Mater. 125, 373 (1993). 5. G. Li, G. Hu, H. Zhou, X. Fan, X. Li, Mater. Chem. Phys. 75, 101 (2002). 6. N. Dishovske, A. Petkov, IEEE Trans. Magn. 30, 969 (1994). 7. M. Han, Y. Ou, W. Chen, L. Deng, J. Alloys Compd. 474, 185 (2009). 8. A. Mali, A. Ataie, J. Alloys Compd. 399, 245 (2005). y
p
9. S. Chaudhury, S.K. Rakshit, S.C. Parida, Z. Singh, K.D. Singh Mudher, V. Venugopal, J. Alloys
Compd. 455, 25 (2008). 10. U. Topal, H. Ozkan, L. Dorosinskii, J. Alloys Compd. 428, 17 (2007). 11. A. Ataie, S. E. Zojaji, J. Alloys Compd. 431, 331 (2007). 12. K. S. Moghaddam, A. Ataie, J. Alloys Compd. 426, 415 (2006). 13. T. Yamauchi, Y. Tsukahara, T. Sakata, H. Mori, T. Chikata, S. Katoh, Y. Wada, J. Magn. Magn. Mater. 321, 8 (2009). 14. A. Ghasemi, A. Morisako, J. Alloys Compd. 456, 485 (2008). 15. X. Tang, Y. Yang, K. Hu, J. Alloys Compd. 477, 322 (2009). 16. G. Litsardakis, I. Manolakis, K. Efthimiadis, J. Alloys Compd. 427, 194 (2007). 17. F. Tabatabaie, M. H. Fathi, A. Saatchi, A. Ghasemi, J. Alloys Compd. 474, 206 (20 18. Ch. Venkateshwarlu, Ch. Ashok, B. A. Rao, D. Ravinder, B. S. Boyanov, J. Alloys Compd. 426,
1 (2006). 19. M. J. Iqbal, M. N. Ashiq, P. H. Gomez, J. Alloys Compd. 478, 736 (2009). 20. F. Tabatabaie, M. H. Fathi, A. Saatchi, A. Ghasemi, J. Alloys Compd. 470, 332 (2 21. O. Popov, M. Mikhov, J. Magn. Magn. Mater. 75, 135 (1988). 22. J. Geshev, O. Popov, M. Mikhov, J. L. Sanchez, N. Suarez, F. Leccabue, J. Magn. Magn. Mater. 117, 190 (1992). ,
(
)
23. H. Pfeiffer and W. Schüppel, J. Magn. Magn. Mater. 130, 92 (1994). 23. H. Pfeiffer and W. Schüppel, J. Magn. Magn. Mater. 130, 92 ( 24. I. Bsoul, S.H. Mahmood, J. Alloys Compd. 489, 110 (2010). 26. I. Bsoul, S.H. Mahmood, A-F. Lehlooh, J. Alloys Compnd. 498, 157 (2010) 27. S.H. Mahmood, J. Magn. Magn. Mater. 118, 359 (1993). 00039-p.8
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https://escholarship.org/content/qt35q9k8bv/qt35q9k8bv.pdf?t=p9wctq
|
English
| null |
In vitro evidence that platelet-rich plasma stimulates cellular processes involved in endometrial regeneration
|
Journal of assisted reproduction and genetics
| 2,018
|
public-domain
| 13,033
|
UCSF
UC San Francisco Previously Published Works
Title
In vitro evidence that platelet-rich plasma stimulates cellular processes involved in
endometrial regeneration
Permalink
https://escholarship.org/uc/item/35q9k8bv
Journal
Journal of Assisted Reproduction and Genetics, 35(5)
ISSN
1058-0468
Authors
Aghajanova, Lusine
Houshdaran, Sahar
Balayan, Shaina
et al.
Publication Date
2018-05-01
DOI
10.1007/s10815-018-1130-8
Peer reviewed UCSF
UC San Francisco Previously Published Works
Title
In vitro evidence that platelet-rich plasma stimulates cellular processes involved in
endometrial regeneration
Permalink
https://escholarship.org/uc/item/35q9k8bv
Journal
Journal of Assisted Reproduction and Genetics, 35(5)
ISSN
1058-0468
Authors
Aghajanova, Lusine
Houshdaran, Sahar
Balayan, Shaina
et al. Publication Date
2018-05-01
DOI
10.1007/s10815-018-1130-8
Peer reviewed UCSF
UC San Francisco Previously Published Works
Title
In vitro evidence that platelet-rich plasma stimulates cellular processes involved
endometrial regeneration
Permalink
https://escholarship.org/uc/item/35q9k8bv
Journal
Journal of Assisted Reproduction and Genetics, 35(5)
ISSN
1058-0468
Authors
Aghajanova, Lusine
Houshdaran, Sahar
Balayan, Shaina
et al. Publication Date
2018-05-01
DOI
10.1007/s10815-018-1130-8
Peer reviewed 1
Department of Obstetrics, Gynecology and Reproductive Sciences,
Center for Reproductive Sciences, and Center for Reproductive
Health, University of California San Francisco, 550 16th Street, 7th
Floor, Box 0132, San Francisco, CA 94158, USA * Lusine Aghajanova
lusine.aghajanova@ucsf.edu Abstract Transcripts for
inflammation markers/chemokines were upregulated by aPRP vs. aPPP (p < 0.05) in eMSC and eSF. No difference in estrogen or
progesterone receptor mRNAs was observed. p
g
p
Conclusions This is the first study evaluating the effect of PRP on different human endometrial cells involved in tissue regen-
eration. These data provide an initial ex vivo proof of principle for autologous PRP to promote endometrial regeneration in
clinical situations with compromised endometrial growth and scarring. Keywords Platelet-rich plasma . Endometrium . Regeneration . Stem cells . Proliferation Abstract Abstract
Purpose The study aims to test the hypothesis that platelet-rich plasma (PRP) stimulates cellular processes involved in endome-
trial regeneration relevant to clinical management of poor endometrial growth or intrauterine scarring. Purpose The study aims to test the hypothesis that platelet-rich plasma (PRP) stimulates cellular processes involved in endome-
trial regeneration relevant to clinical management of poor endometrial growth or intrauterine scarring. Methods Human endometrial stromal fibroblasts (eSF), endometrial mesenchymal stem cells (eMSC), bone marrow-derived
mesenchymal stem cells (BM-MSC), and Ishikawa endometrial adenocarcinoma cells (IC) were cultured with/without 5% activated
(a) PRP, non-activated (na) PRP, aPPP (platelet-poor-plasma), and naPPP. Treatment effects were evaluated with cell proliferation
(WST-1), wound healing, and chemotaxis Transwell migration assays. Mesenchymal-to-epithelial transition (MET) was evaluated
by cytokeratin and vimentin expression. Differential gene expression of various markers was analyzed by multiplex Q-PCR. Results Activated PRP enhanced migration of all cell types, compared to naPRP, aPPP, naPPP, and vehicle controls, in a time-
dependent manner (p < 0.05). The WST-1 assay showed increased stromal and mesenchymal cell proliferation by aPRP vs. naPRP, aPPP, and naPPP (p < 0.05), while IC proliferation was enhanced by aPRP and aPPP (p < 0.05). There was no evidence of
MET. Expressions of MMP1, MMP3, MMP7, and MMP26 were increased by aPRP (p < 0.05) in eMSC and eSF. Transcripts for
inflammation markers/chemokines were upregulated by aPRP vs. aPPP (p < 0.05) in eMSC and eSF. No difference in estrogen or
progesterone receptor mRNAs was observed. Methods Human endometrial stromal fibroblasts (eSF), endometrial mesenchymal stem cells (eMSC), bone marrow-derived
mesenchymal stem cells (BM-MSC), and Ishikawa endometrial adenocarcinoma cells (IC) were cultured with/without 5% activated
(a) PRP, non-activated (na) PRP, aPPP (platelet-poor-plasma), and naPPP. Treatment effects were evaluated with cell proliferation
(WST-1), wound healing, and chemotaxis Transwell migration assays. Mesenchymal-to-epithelial transition (MET) was evaluated
by cytokeratin and vimentin expression. Differential gene expression of various markers was analyzed by multiplex Q-PCR. Results Activated PRP enhanced migration of all cell types, compared to naPRP, aPPP, naPPP, and vehicle controls, in a time-
dependent manner (p < 0.05). The WST-1 assay showed increased stromal and mesenchymal cell proliferation by aPRP vs. naPRP, aPPP, and naPPP (p < 0.05), while IC proliferation was enhanced by aPRP and aPPP (p < 0.05). There was no evidence of
MET. Expressions of MMP1, MMP3, MMP7, and MMP26 were increased by aPRP (p < 0.05) in eMSC and eSF. Lusine Aghajanova1 & Sahar Houshdaran1 & Shaina Balayan1 & Evelina Manvelyan1 & Juan C. Irwin1 &
Heather G. Huddleston1 & Linda C. Giudice1 Lusine Aghajanova1 & Sahar Houshdaran1 & Shaina Balayan1 & Evelina Manvelyan1 & Juan C. Heather G. Huddleston1 & Linda C. Giudice1 Received: 30 November 2017 /Accepted: 23 January 2018 /Published online: 5 February 2018
# Springer Science+Business Media, LLC, part of Springer Nature 2018 Powered by the California Digital Library
University of California eScholarship.org Journal of Assisted Reproduction and Genetics (2018) 35:757–770
https://doi.org/10.1007/s10815-018-1130-8 REPRODUCTIVE PHYSIOLOGY AND DISEASE Study design Human primary eSF, eMSC, BM-MSCs, and Ishikawa endo-
metrial adenocarcinoma cells (IC) were cultured without (con-
trol group) and with 5% activated (a) PRP, non-activated
(na)PRP, activated platelet-poor-plasma (aPPP), and naPPP
(5 groups total). Effects of treatments were evaluated using
in vitro assays for cell proliferation (WST-1), wound-healing
migration, and chemotaxis Transwell migration. Mesenchymal-to-epithelial transition of eSF, eMSC, and
BM-MSC was evaluated with cytokeratin and vimentin im-
munofluorescence. Differential gene expression of growth
factor receptors, extracellular matrix markers, cell surface
markers, inflammation markers, and chemokines were ana-
lyzed by multiplex q-RT-PCR (Fluidigm, South San
Francisco, CA) (Fig. 1). PRP is an autologous concentration of platelets in plasma
that has been increasingly used to support tissue growth and
repair in orthopedics, dental and plastic surgery, diabetic
wound healing, and dermatology [21–24], but has been min-
imally investigated to date in gynecology. Platelets contain
granules that store growth factors and cytokines (e.g.,
VEGF, TGFβ, PDGF, IGF1, FGF, EFG, HGF, CXCL12,
CCL5) released upon platelet activation at the site of injury
or inflammation. These factors are critical in activation of
fibroblasts and recruitment of leukocytes to the injury site,
inducing and regulating proliferation and migration of other
cell types involved in tissue repair such as smooth muscle cells
and mesenchymal stem cells, and promoting angiogenesis
[25, 26]. Importantly, platelet-derived factors are essential
for endometrial progenitor cell activity [27], and PDGF iso-
forms significantly promote endometrial stromal cell prolifer-
ation, migration, and contractility [28]. Endometrium contains
epithelial, mesenchymal, and endothelial stem/progenitor
cells [29, 30]. BM-MSCs have been proposed as a potential
source of endometrial regeneration [31–34]. In mice, platelets
recruit circulating progenitors to exposed collagen in damaged
blood vessels and induce differentiation into mature endothe-
lial cells [25], which can be one of the regeneration mecha-
nisms initiated by platelets. Materials and methods Recently, the use of biologics has been pursued to boost the
regeneration process and/or minimize post-operative adhe-
sions, including intrauterine infusion of autologous bone
marrow-derived mesenchymal stem cells (BM-MSCs) [12],
human amnion graft placement [13], or autologous peripheral
blood CD133+ BM-MSC delivered into the uterine spiral ar-
terioles by catheterization [14]. These have not been widely
adopted likely due to the complexity of the techniques and
conflicting outcomes [12–14]. Human subjects Primary human eutopic eSF and eMSC were obtained through
the UCSF NIH Human Endometrial Tissue and DNA Bank. Written informed consent was given by all participants under
the active IRB protocol approved by the Institutional
Committee on Human Research. In the setting of persistent thin lining and normal uterine
cavity (without scarring), several strategies to improve thick-
ness have been investigated, including use of exogenous es-
trogens, vaginal sildenafil, low-dose aspirin, and granulocyte
colony stimulation factor [15–18]; however, a large propor-
tion of women with thin lining remain refractory to such ther-
apies. Two studies reported on successful use of an intrauter-
ine infusion of platelet-rich plasma (PRP) in infertile women
with thin endometrium [19, 20]. Introduction lactobacillus dominant endometrial microbiome [4], or struc-
turally due to uterine anomalies, scarring and intrauterine ad-
hesions (Asherman’s syndrome (AS)), or an unexplained thin
lining. Endometrial thickness is commonly used as a clinical
marker of endometrial receptivity and a prognostic factor for
pregnancy outcome after embryo transfer [5]. An endometrial
thickness <7–8 mm at the end of the follicular phase is asso-
ciated with reduced pregnancy rates [6, 7], poor pregnancy
outcomes [5], and may result in a cycle cancelation [8]. Unexplained thin endometrium and AS are among the most
challenging obstacles in fertility care, often resulting in pa-
tients pursuing gestational surrogacy [9]. About 13% of couples worldwide have infertility [1] due to
several factors, including impaired embryo quality and endo-
metrial receptivity. The latter can be affected by altered pro-
grammed responsiveness to steroid hormones [2, 3], a non- Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s10815-018-1130-8) contains supplementary
material, which is available to authorized users. Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s10815-018-1130-8) contains supplementary
material, which is available to authorized users. * Lusine Aghajanova
lusine.aghajanova@ucsf.edu The main complications of Asherman’s syndrome are in-
fertility (43%), impairment of menstrual flow (62%), and ab-
normal placentation if pregnancy is achieved [10], with sur-
gery (hysteroscopy) with or without oral estrogens being the
standard treatment [11]. 758 J Assist Reprod Genet (2018) 35:757–770 Endometrial stromal fibroblast isolation and culture All experiments were performed with the second
and third passages. treatments were performed in triplicate under identical culture
conditions. All experiments were performed with the second
and third passages. archived in our Tissue Bank, and we have extensively stud-
ied their stability and response to steroid hormones after
thawing, plating, and multiple passages in vitro [37]. Once
cultured, these cells retain their viability and steroid hor-
mone response uniformly over passages 1–4 [38]. IC This cell line (Sigma-Aldrich) was used in our study as a
model for primary endometrial epithelial cells because of
the limitations in obtaining sufficient numbers for primary
epithelial culture and expansion for all experiments
planned. Ishikawa cells were expanded in minimal essen-
tial Eagle’s medium with 10% charcoal-stripped FBS,
2 mM L-glutamine, 1% penicillin/streptomycin, and 1%
non-essential amino acids at 37 °C in a 95% air/5% CO2
humidified incubator [42]. The medium was changed every
second day. When cells became confluent, they were
trypsinized and plated at a specific density as required for
different experiments. All treatments were performed in
triplicate under identical culture conditions. IC This cell line (Sigma-Aldrich) was used in our study as a
model for primary endometrial epithelial cells because of
the limitations in obtaining sufficient numbers for primary
epithelial culture and expansion for all experiments
planned. Ishikawa cells were expanded in minimal essen-
tial Eagle’s medium with 10% charcoal-stripped FBS,
2 mM L-glutamine, 1% penicillin/streptomycin, and 1%
non-essential amino acids at 37 °C in a 95% air/5% CO2
humidified incubator [42]. The medium was changed every
second day. When cells became confluent, they were
trypsinized and plated at a specific density as required for
different experiments. All treatments were performed in
triplicate under identical culture conditions. Endometrial stromal fibroblast isolation and culture Isolated and cultured eSFs from women with either no
uterine pathology or non-cavity-distorting fibroids and
without endometriosis, (n = 3) before and after PRP/PPP
treatment, were used for the WST-1 proliferation assay,
migration scratch and Transwell assays, immunohisto-
chemistry, and real-time RT-PCR. Fresh endometrial sam-
ples were digested with collagenase as described previous-
ly [35]. Human eSF were separated from epithelium based
on size and plated with the use of Dulbecco modified Eagle
medium (DMEM; Life Technologies, Foster City, CA) and
25% MCDB-105 (Sigma-Aldrich, St Louis, MO), contain-
ing 10% charcoal-stripped fetal bovine serum (FBS;
HyClone, Thermo Scientific, Inc., Waltham, MA), 1 mM
sodium pyruvate (Sigma-Aldrich), 1% antibiotic-
antimycotic solution (Life Technologies), and 5 μg/ml in-
sulin (Gemini, Sacramento, CA), as described previously
[35, 36]. At passage 2, cells were cultured to near conflu-
ence in the same medium, followed by changing to low-
serum medium (2% FBS) and cultured for 24 h before the
onset of treatment. eSF culture purity (~99% stromal fibro-
blasts) was determined by cytokeratin, vimentin, and
CD45 by immunostaining, as described previously [35]. All cell culture experiments were conducted with the use
of second to fourth cell passages. Primary cells are Using an in vitro approach, our goal was to investigate the
potential pharmacologic use of PRP in promoting biological
processes involved in endometrial regeneration as relevant to
the management of Asherman’s syndrome or thin endometrial
lining in infertility patients. To this end, we studied the
effects of PRP and platelet-poor plasma (PPP) on the pro-
liferation and migration, as well as gene expression, of
endometrial stromal fibroblasts (eSF), endometrial mesen-
chymal stem cells (eMSC), BM-MSCs, and Ishikawa cells
(IC), as well as mesenchymal-to-epithelial transition
(MET) of eSF and eMSC. 759 J Assist Reprod Genet (2018) 35:757–770 Endometrial
stromal
fibroblasts
Endometrial
mesenchymal
stem cells
Bone marrow-
derived
mesenchymal
stem cells
Ishikawa
cells
aPRP
Vehicle
naPRP
aPPP
naPPP
aPRP
Vehicle
naPRP
aPPP
naPPP
aPRP
Vehicle
naPRP
aPPP
naPPP
aPRP
Vehicle
naPRP
aPPP
naPPP
Proliferation assay
Wound scratch assay
Transwell migration
assay
Immunofluorescence
QRT-PCR
Fig. 1 Flow diagram of
experimental design. aPRP
activated platelet-rich plasma,
naPRP non-activated platelet-rich
plasma, aPPP activated platelet-
poor plasma, naPPP non-
activated platelet-poor plasma aPRP
Vehicle
naPRP
aPPP
naPPP Endometrial
stromal
fibroblasts Endometrial
mesenchymal
stem cells Bone marrow-
derived
mesenchymal
stem cells mesenchymal
stem cells aPRP aPPP naPRP Ishikawa
cells treatments were performed in triplicate under identical culture
conditions. Preparation of PRP Leukocyte reduced apheresis platelets were purchased
from the Blood Centers of the Pacific (San Francisco,
CA), from a white 59-year-old male donor, blood group
A, Rh positive, with platelet concentration 2.2 × 1011 in
300 ml of apheresis platelet concentrate. We specifically
used a male donor to eliminate any potential influences of
hormonal variations in the growth factor/cytokine compo-
sition. The PRP and PPP were prepared as described [43,
44]. Briefly, the platelet-containing plasma was centri-
fuged for 15 min at 3200 rpm at room temperature (RT),
and PPP was separated out. The plasma supernatant was
used as PPP and the thrombocyte pellet in 1.0 ml of plasma
was used as PRP. Platelets were activated with thrombin
(1 U/ml) and 0.5 M of calcium chloride [45]. An activating Endometrial mesenchymal stem cell isolation
and culture CD146(+)/PDGFRB(+) eMSC that were isolated previously
from eutopic endometrial samples (healthy oocyte donors,
n = 3) using fluorescence-activated cell sorting (FACS) with
demonstration of their clonogenicity [39] were used in this
study. Primary cultures were established and used as described
[40], with modifications to the culture medium which was
composed of 75% high-glucose phenol red-free of DMEM/
MCDB-105 medium, supplemented with 10% charcoal-
stripped FBS, 1 mM sodium pyruvate, and 25 ng/ml basic
FGF (Sigma-Aldrich). Cells were expanded by serial passage
with subsequent cryopreservation of eMSC for further use. eMSC from [43] at second and third passages were used for
current experiments. As it was previously shown, eMSC cul-
tured in the presence of basic FGF will largely retain the
eMSC phenotype over serial passages with ~70% of
SUSD2+ cells and persistence of multi-lineage differentiation
capacity at passage 6 [41]. Fig. 1 Flow diagram of
experimental design. aPRP
activated platelet-rich plasma,
naPRP non-activated platelet-rich
plasma, aPPP activated platelet-
poor plasma, naPPP non-
activated platelet-poor plasma Transwell chemotaxis migration assay For the Transwell migration assay, 3 × 103 cells were seeded
on the upper surface of the polycarbonate Transwell filter
(8-μm pore size, Corning, New York, NY) with serum-free
DMEM (n = 3 for eSF, eMSC, and IC, and n = 2 for BM-
MSC, in duplicate or triplicate) [47]. DMEM with vehicle
and 5% aPRP, naPRP, aPPP, and naPPP were added to the
lower chambers. The incubation time was 14 and 24 h for
eSF and eMSC, 16 and 24 h for BM-MSC, and 24–96 h for
Ishikawa cells. At the termination of the experiments, the up-
per membrane was carefully cleaned with a q-tip to remove all
adherent cells, so that the cells on the upper surface of the
membrane were not mistaken for the migrated cells on the
bottom membrane of the insert and counted as such. Thereafter, membranes were washed twice in phosphate-
buffered saline (PBS) and fixed with methanol for 5 min at
−20 °C. Then, the chambers were stained with 0.5% crystal
violet solution for 15 min, air-dried, and photographed at
×200 magnification. A total of three fields were counted for
each Transwell filter, and the average number of cells was
used for statistical analysis. PRP dose-finding experiments in eSF proliferation
assay eSF were seeded at a density of 5 × 104 cells/well in 96-well
plates. After overnight serum starvation, the cells were cul-
tured in serum-free DMEM supplemented with 0 (control), 1,
5, 10, and 20% aPRP and aPPP for 3 and 5 days. WST-1
proliferation assay was performed as described above, with
results demonstrating no significant differences between pro-
liferation potential in the 1, 5, and 10% groups, and decrease
in proliferation at 20% PRP concentration (data not shown). Since 5% PRP use was also reported previously in studies on
dermal fibroblasts [34, 44], we used that concentration in our
experiments. Cell lines BM-MSCs were purchased from Cambrex Biosciences, East
Rutherford, NJ and expanded in high-glucose DMEM con-
taining 10% FBS, 1% penicillin, and 1% streptomycin [32]. At confluency, BM-MSCs were trypsinized and plated at a
specific density as required for different experiments. All J Assist Reprod Genet (2018) 35:757–770 760 24 h for eSF and eMSC, 48 h for BM-MSC, and 72 h for
Ishikawa cells. The total incubation time was identified by
preliminary experiments to evaluate the time needed for
complete closure of the wound line. Migration was monitored
by time-lapsed microscopy using a Leica DFC360FX 1.4-
megapixel monochrome digital camera mounted to a Leica
DMI6000B inverted microscope fitted with a motorized
stage and digital camera (DFC360FX) powered by a
Leica CTR6500 HS electronics box. Sequential images
for three randomly selected areas were acquired per well
per experimental group every 6–24 h as programmed for
different cell types, at ×100 magnification, and the coordinates
were saved for time-lapse imaging. 1:1 (v/v) mixture of calcium chloride and thrombin was
prepared in advance. A 10:1 (v/v) mixture of PRP or PPP
and activator was incubated for 10 min at RT. Activated
samples are designated as activated (a) PRP or PPP and
samples not activated are designated as non-activated
(na)PRP or PPP. As platelets will not survive extended
storage, aPRP and aPPP were centrifuged again at
3200 rpm for 15 min and the resulting supernatant was
stored at −20 °C until further use. WST-1 proliferation assay WST-1 assay was used to assess metabolic activity of all
primary cells and cell lines investigated herein. Cells were
seeded at a density of 5 × 104 cells/well in 96-well plates in
triplicate and cultured overnight (n = 3 in each group and
each cell type). After media change, the cells were incu-
bated for total of three (eSF and eMSC) or seven (BM-
MSC and Ishikawa) days, depending on the growth rate. Ten microliters of WST-1 reagent (Roche Diagnostics,
Laval, Quebec, Canada) was added to each well and incu-
bated for another 4 h at 37 °C (incubation time verified by
preliminary experiments; data not shown). The absorbance
was determined using a microplate reader at a test wave-
length of 450 nm and reference wavelength of 650 nm
(Bio-Rad Laboratories, Hercules, CA). The wound repair was assessed by calculating the area in
square micrometers between the lesion edges (the wound area)
using the public domain ImageJ program developed at the
National Institutes of Health (Bethesda, MD), and the
average of data from duplicate/triplicate cultures were used
for statistical analysis. Immunofluorescence Cells were then fixed in 2%
paraformaldehyde and 100% methanol and stored until
use. Cells were permeabilized with 0.1% Triton X-100,
blocked with 10% normal goat serum, and incubated over-
night at 4 °C with the following primary antibodies: rabbit
anti-human cytokeratin 18 (1:100; ab32118, Abcam,
Cambridge, MA), rabbit anti-human cytokeratin 5 (1:100;
ab24647, Abcam), mouse anti-human vimentin (1:100;
180,052, Life Technologies), and mouse anti-human E
cadherin (1:100; ab1416, Abcam). Cells were then washed
three times with PBS/0.1% Tween 20 buffer and incubated
for 1 h at room temperature with the corresponding Alexa
Fluor 488 conjugated goat anti-mouse, or 488 or 594 con-
jugated goat anti-rabbit secondary antibodies (1:250; A-
11001 and A-11008 or A-11012, respectively, Invitrogen)
and washed thereafter. Negative control wells were treated
with the corresponding mouse or rabbit non-immune IgG. Results were viewed on a Leica DM 5000 microscope
equipped with epifluorescence optics (Leica
Microsystems, Inc.). naPRP, aPPP, and naPPP. Cells were then fixed in 2%
paraformaldehyde and 100% methanol and stored until
use. Cells were permeabilized with 0.1% Triton X-100,
blocked with 10% normal goat serum, and incubated over-
night at 4 °C with the following primary antibodies: rabbit
anti-human cytokeratin 18 (1:100; ab32118, Abcam,
Cambridge, MA), rabbit anti-human cytokeratin 5 (1:100;
ab24647, Abcam), mouse anti-human vimentin (1:100;
180,052, Life Technologies), and mouse anti-human E
cadherin (1:100; ab1416, Abcam). Cells were then washed
three times with PBS/0.1% Tween 20 buffer and incubated
for 1 h at room temperature with the corresponding Alexa
Fluor 488 conjugated goat anti-mouse, or 488 or 594 con-
jugated goat anti-rabbit secondary antibodies (1:250; A-
11001 and A-11008 or A-11012, respectively, Invitrogen)
and washed thereafter. Negative control wells were treated
with the corresponding mouse or rabbit non-immune IgG. Results were viewed on a Leica DM 5000 microscope
equipped with epifluorescence optics (Leica
Microsystems, Inc.). All cDNA samples from cultured eSF, eMSC, BM-MSC, and
Ishikawa cells in control, aPRP, naPRP, aPPP, and naPPP groups
(n = 3 in each group, in duplicate) were assayed by q-RT-PCR
using the Fluidigm Dynamic Array Integrated Fluidic Circuits
and the BioMark HD system (www.fluidigm.com/biomark-
system.html) as previously described [40, 49]. Briefly, cDNA
was pre-amplified to generate a pool of target genes using Taq-
Man Pre-Amp master mix (Applied Biosystems), 100 ng cDNA,
and 500 nM for each primer pair. Immunofluorescence Samples were then treated with
exonuclease (Exonuclease I; New England BioLabs) per proto-
col and diluted 1:5 in a Tris-ethylenediaminetetraacetic acid di-
lution buffer (TEKnova) using previously generated optimal di-
lution curves. q-RT-PCR was performed using SsoFast
Evagreen supermix with low ROX binding dye (Biotium Inc.)
at a primer concentration of 5 μM (primers were designed by
Fluidigm). Data were processed by user-detected threshold set-
tings and linear baseline correction using Biomark real-time
PCR Analysis software (version 3.0.4). The YWHAZ housekeep-
ing gene was used as a normalizer. The comparative (ΔΔ) Ct
method was used to calculate relative fold changes (docs. appliedbiosystems.com/pebiodocs/04303859.pdf). RNA isolation Total RNA was purified using Qiagen RNeasy Plus Mini Kit
(Qiagen) according to the manufacturer’s instructions, and
quantified by spectroscopy as previously described [36, 40]. Purity was analyzed by the 260/280 absorbance ratio and
RNA integrity was assessed using an Agilent Bioanalyzer
2100 (Agilent Technologies, Santa Clara, CA). For quantita-
tive RT-PCR analysis, 1 μg of RNA was converted to com-
plementary DNA (cDNA) using the iScript cDNA Synthesis
Kit (Bio-Rad Laboratories, Hercules, USA). Immunofluorescence Cells were seeded in 6- or 12-well plates with 1 × 105 cells
per well and cultured to confluency (n = 3 for eSF, eMSC,
and IC and n = 2 for BM-MSC, in triplicate or duplicate). Monolayers of confluent cells were scratched with a
100-μl pipette tip without damaging the plastic and then
washed with PBS to remove non-adherent cells, as de-
scribed [46]. The wells were divided according to the study
protocol into control, aPRP, naPRP, aPPP, and naPPP
groups and treated accordingly, with incubation times of Immunocytochemistry analyses with cytokeratin and
vimentin antibodies were performed to analyze possible
mesenchymal-to-epithelial transition under the experimen-
tal conditions. Indirect immunofluorescence was conduct-
ed following previously reported methods [48]. Immunofluorescence Briefly,
cells were cultured in 96-well plates until confluency and
were divided into five groups and treated for 72 h accord-
ing to the study protocol as above with vehicle, 5% aPRP, J Assist Reprod Genet (2018) 35:757–770 761 Cells were then fixed in 2%
0%
h
l
d
d
il
Quantitative RT-PCR with multiplex Fluidigm array
*
*
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
day 0
day 1
day 3
Endometrial stromal fibroblasts, n=3
ctrl
aPRP
naPRP
aPPP
naPPP
**
*
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
day 0
day 1
day 3
Absorbance 450 nm
Endometrial mesenchymal stem cells, n=3
ctrl
aPRP
naPRP
aPPP
naPPP
*
*
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
day 0
day 3
day 7
Bone marrow-derived mesenchymal
stem cells, n=3
ctrl
aPRP
naPRP
aPPP
naPPP
*
**
*
*
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
day 0
day 3
day 7
Absorbance 450 nm
Ishikawa cells, n=3
ctrl
aPRP
naPRP
aPPP
naPPP
*
*
*
*
Absorbance 450 nm
Absorbance 450 nm Endometrial mesenchymal stem cells, n=3 *
*
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
day 0
day 1
day 3
Endometrial stromal fibroblasts, n=3
ctrl
aPRP
naPRP
aPPP
naPPP
**
*
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
day 0
day 1
day 3
Absorbance 450 nm
Endometrial mesenchymal stem cells, n=3
ctrl
aPRP
naPRP
aPPP
naPPP
*
*
Absorbance 450 nm Endometrial mesenchymal stem cells, n=3 mal
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
day 0
day 3
day 7
Absorbance 450 nm
Ishikawa cells, n=3
ctrl
aPRP
naPRP
aPPP
naPPP
*
*
*
* 0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
day 0
day 3
day 7
Bone marrow-derived mesenchymal
stem cells, n=3
ctrl
aPRP
naPRP
aPPP
naPPP
*
**
*
*
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
day 0
day 3
day 7
Absorbance 450 nm
Ishikawa cells, n=3
ctrl
aPRP
naPRP
aPPP
naPPP
*
*
*
*
Absorbance 450 nm 0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
day 0
day 3
day 7
Bone marrow-derived mesenchymal
stem cells, n=3
ctrl
aPRP
naPRP
aPPP
naPPP
*
**
*
*
0
0
1
1
2
2
3
3
4
4
Absorbance 450 nm
Absorbance 450 nm Bone marrow-derived mesenchymal Ishikawa cells, n=3
* Bone marrow-derived mesenchy
stem cells, n=3 Quantitative RT-PCR with multiplex Fluidigm array naPRP, aPPP, and naPPP. Results Activated PRP promoted the migration of human eSF,
eMSC, BM-MSC, and IC compared to non-activated
PRP, PPP, and vehicle controls, in both wound healing
(Fig. 3 and images in Supplemental Fig. 1) and chemo-
taxis assays (Fig. 4 and images in Supplemental Fig. 2),
at various time points studied (p < 0.05). As shown in
both figures, Ishikawa cells required substantially longer
time to close the wound or to migrate through the pores
of a Transwell membrane. The difference in migration
was largely not significant after short-time exposure,
but reached significance upon longer exposure in all cell
types in both migration assays. It is worth noting that
there were significant differences in the migration poten-
tial of different cell types studied herein between the
control (untreated) group and naPRP, aPPP, and naPPP
groups for the most part as well (p < 0.01; Figs. 3 and 4),
signifying the overall stimulatory effect of human
platelet-rich or platelet-poor plasma on endometrial cell
migration. Statistical analysis Statistical analysis for the WST-1 proliferation assay, scratch
migration, and Transwell migration assays and for the quanti-
tative RT-PCR was performed using two-way repeated mea-
sures ANOVA with post hoc Tukey-Kramer test for multiple
pairwise comparison corrections. Statistical significance was
determined at p ≤0.05. 762 J Assist Reprod Genet (2018) 35:757–770 a
0
10
20
30
40
50
60
70
80
90
100
0% control
aPRP
naPRP
aPPP
naPPP
eSF, n=3, in triplicate
8hrs
16hrs
24hrs
a
a
a
b
b
c
c
d
d
e
e
b
c
d
e
*
*
*
*
*
*
*
*
*
0
10
20
30
40
50
60
70
80
90
100
0% control
aPRP
naPRP
aPPP
naPPP
% covered surface
Ishikawa cells, n=3, in duplicate
24hrs
48hrs
72hrs
b
b
c
d
e
e
b
c
e
c
d
d
a
a
a
*
*
*
*
0
10
20
30
40
50
60
70
80
90
100
0% control
aPRP
naPRP
aPPP
naPPP
% covered surface
eMSC, n=3, in duplicate
8hrs
16hrs
24hrs
a
a
a
b
b
b
c
c
c
*
*
*
*
*
*
*
0
10
20
30
40
50
60
70
80
90
100
0% control
aPRP
naPRP
aPPP
naPPP
BM-MSC, n=2, in triplicate
8hrs
16hrs
24hrs
*
*
*
*
*
*
*
*
e
e
e
d
d
d
b
b
a a
a
c
c
c
*
*
*
% covered surface
% of covered surface
Fig. 3 a Wound-healing assays for endometrial stromal fibroblasts (eSF),
endometrial mesenchymal stem cells (eMSC), bone marrow-derived
mesenchymal stem cells (BM-MSCs), and Ishikawa cells. Graphs show
the percentage of covered surface (n = 3 images per well, in duplicate or
triplicate, n = 3 for each cell type) for the control and treatment groups. Data represent the mean ± SD. Statistical significance accepted at p ≤
0.05. Significant difference between different time points within the same
treatment groups indicated by the same letter. Asterisk indicates signifi-
cant difference compared to the respective time point in the aPRP group. aPRP activated platelet-rich plasma, naPRP non-activated platelet-rich
plasma, aPPP activated platelet-poor plasma, naPPP non-activated plate-
let-poor plasma. b Transwell migration assays for endometrial stromal
fibroblasts (eSF), endometrial mesenchymal stem cells (eMSC), bone
marrow-derived mesenchymal stem cells (BM-MSCs), and Ishikawa
cells at different time points. Statistical analysis b Transwell migration assays for endometrial stromal
fibroblasts (eSF), endometrial mesenchymal stem cells (eMSC), bone marrow-derived mesenchymal stem cells (BM-MSCs), and Ishikawa
cells at different time points. Graphs show the average number of migrat-
ed cells (n = 3 images per insert, in duplicate or triplicate, n = 3 for each
cell type) for the control and treatment groups. Data represent the mean ±
SD. Statistical significance accepted at p ≤0.05. Significant difference
between different time points within the same treatment group is indicated
by the same letter. Asterisk indicates significant difference compared to
the respective time point in the aPRP group. aPRP activated platelet-rich
plasma, naPRP non-activated platelet-rich plasma, aPPP activated
platelet-poor plasma, naPPP non-activated platelet-poor plasma. The y
axis range is not unified so that the smaller-scale changes remain apparent
to the reader Fig. 3 a Wound-healing assays for endometrial stromal fibroblasts (eSF),
endometrial mesenchymal stem cells (eMSC), bone marrow-derived
mesenchymal stem cells (BM-MSCs), and Ishikawa cells. Graphs show
the percentage of covered surface (n = 3 images per well, in duplicate or
triplicate, n = 3 for each cell type) for the control and treatment groups. Data represent the mean ± SD. Statistical significance accepted at p ≤
0.05. Significant difference between different time points within the same
treatment groups indicated by the same letter. Asterisk indicates signifi-
cant difference compared to the respective time point in the aPRP group. aPRP activated platelet-rich plasma, naPRP non-activated platelet-rich
plasma, aPPP activated platelet-poor plasma, naPPP non-activated plate-
let-poor plasma. b Transwell migration assays for endometrial stromal
fibroblasts (eSF), endometrial mesenchymal stem cells (eMSC), bone Statistical analysis Graphs show the average number of migrat-
ed cells (n = 3 images per insert, in duplicate or triplicate, n = 3 for each
cell type) for the control and treatment groups. Data represent the mean ±
SD. Statistical significance accepted at p ≤0.05. Significant difference
between different time points within the same treatment group is indicated
by the same letter. Asterisk indicates significant difference compared to
the respective time point in the aPRP group. aPRP activated platelet-rich
plasma, naPRP non-activated platelet-rich plasma, aPPP activated
platelet-poor plasma, naPPP non-activated platelet-poor plasma. The y
axis range is not unified so that the smaller-scale changes remain apparent
to the reader a
0
10
20
30
40
50
60
70
80
90
100
0% control
aPRP
naPRP
aPPP
naPPP
eSF, n=3, in triplicate
8hrs
16hrs
24hrs
a
a
a
b
b
c
c
d
d
e
e
b
c
d
e
*
*
*
*
*
*
*
*
*
Ishika a cells n 3 in d plicate
0
10
20
30
40
50
60
70
80
90
100
0% control
aPRP
naPRP
aPPP
naPPP
% covered surface
eMSC, n=3, in duplicate
8hrs
16hrs
24hrs
a
a
a
b
b
b
c
c
c
*
*
*
*
*
*
*
BM MSC n 2 in triplicate
% covered surface a 0
10
20
30
40
50
60
70
80
90
100
0% control
aPRP
naPRP
aPPP
naPPP
BM-MSC, n=2, in triplicate
8hrs
16hrs
24hrs
*
*
*
*
*
*
*
*
e
e
e
d
d
d
b
b
a a
a
c
c
c
*
*
*
% of covered surface Fig. 3 a Wound-healing assays for endometrial stromal fibroblasts (eSF), Fig. 3 a Wound-healing assays for endometrial stromal fibroblasts (eSF),
endometrial mesenchymal stem cells (eMSC), bone marrow-derived
mesenchymal stem cells (BM-MSCs), and Ishikawa cells. Graphs show
the percentage of covered surface (n = 3 images per well, in duplicate or
triplicate, n = 3 for each cell type) for the control and treatment groups. Data represent the mean ± SD. Statistical significance accepted at p ≤
0.05. Significant difference between different time points within the same
treatment groups indicated by the same letter. Asterisk indicates signifi-
cant difference compared to the respective time point in the aPRP group. aPRP activated platelet-rich plasma, naPRP non-activated platelet-rich
plasma, aPPP activated platelet-poor plasma, naPPP non-activated plate-
let-poor plasma. Platelet involvement in endometrial regeneration
may be mediated by inflammation
and chemoattraction demonstrated significant upregulation of CCL5 and CCL7 in
aPRP compared to other groups (p < 0.05; Fig. 4c; non-
significant data for BM-MSC and Ishikawa cells not shown). MMP7, TAGL, IL1B, and CXCL13 transcripts were not signif-
icantly regulated in eMSC (data not shown). We analyzed gene expression differences in all cell types and
groups studies herein. Interestingly, there was no significant
difference in estrogen or progesterone receptor gene expres-
sion between cells treated with or without PRP or PPP in all
cell types studied (data not shown). In contrast, expression of
matrix metalloproteinases MMP3, MMP7, and MMP26 were
increased in the PRP and PPP groups in eSF with significant
differences with control and between aPRP and other groups
(p < 0.05), while in eMSC MMP1, MMP3 and MMP26 were
significantly upregulated compared to controls and between
aPRP and other groups (Fig. 4a). There was no difference in
MMP2 and MMP7 transcript expression in any cell type stud-
ied, and no difference in messenger RNA (mRNA) expression
of MMPs in BM-MSC or Ishikawa cells (data not shown). The cell transformation marker transgelin (TAGL) mRNA
was upregulated in eSF in aPRP versus naPRP or aPPP
(p < 0.05) (Fig. 4b), with no difference in E-cadherin
(CDH1) expression (data not shown). Inflammation markers
IL1A, IL1B, and IL1R2 were significantly upregulated in the
aPRP group in eSF compared to all other groups; while in
eMSC IL1A, IL15, and IL1R2 followed the same pattern of
expression (Fig. 4c; non-significant data for BM-MSC and
Ishikawa cells (latter not shown)). Several chemokines
(CCL5, CCL7, and CXCL13) but not CCL2, CCL3, CCL4,
or IL8 (data not shown) were upregulated in aPRP versus
control, aPPP, naPRP, or naPPP-treated eSF, while eMSC Transcripts for some growth factor receptors such as
EGFR, PDGFRB, and FGFR2 were analyzed in eSF, eMSC,
BM-MSC, and IC, and the results demonstrated various de-
grees of upregulation (Fig. 4d), with EGFR showing most
consistent upregulation in response to treatment with aPRP
in all cell types (Fig. 4d). Activated platelet-rich plasma promotes cell
proliferation 3 (continued)
J Assist Reprod Genet (2018) 35:757–770 b
0
5
10
15
20
25
30
35
40
0% control
aPRP
naPRP
aPPP
naPPP
BM-MSC, n=2, in triplicate
16hrs
24hrs
*
*
*
*
*
*
*
0
20
40
60
80
100
120
140
0% control
aPRP
naPRP
aPPP
naPPP
Number of migrated cells
eMSC, n=3, in triplicate
14hrs
24hrs
a
a
b
b
e
e
*
0
50
100
150
200
250
0% control
aPRP
naPRP
aPPP
naPPP
eSF, n=3, in triplicate
14hrs
24hrs
*
*
*
0
20
40
60
80
100
120
0% control
aPRP
naPRP
aPPP
naPPP
Number of migrated cells
Ishikawa cells, n=3, in duplicate
24h
48h
72h
96h
a
a
a
b
b
*
c
c
*
*
*
* * *
a
a
b
b
c
c
d
d
e
e
*
*
*
a
a
b
b
c
c
d
d
*
* *
*
*
* *
Number of migrated cells
Number of migrated cells
Fig. 3 (continued) b
0
5
10
15
20
25
30
35
40
0% control
aPRP
naPRP
aPPP
naPPP
BM-MSC, n=2, in triplicate
16hrs
24hrs
*
*
*
*
*
*
*
0
20
40
60
80
100
120
140
0% control
aPRP
naPRP
aPPP
naPPP
Number of migrated cells
eMSC, n=3, in triplicate
14hrs
24hrs
a
a
b
b
e
e
*
0
50
100
150
200
250
0% control
aPRP
naPRP
aPPP
naPPP
eSF, n=3, in triplicate
14hrs
24hrs
*
*
*
0
20
40
60
80
100
120
0% control
aPRP
naPRP
aPPP
naPPP
Number of migrated cells
Ishikawa cells, n=3, in duplicate
24h
48h
72h
96h
a
a
a
b
b
*
c
c
*
*
*
* * *
a
a
b
b
c
c
d
d
e
e
*
*
*
a
a
b
b
c
c
d
d
*
* *
*
*
* *
Number of migrated cells
Number of migrated cells
Fig. 3 (continued) b Platelet involvement in endometrial regeneration
may be mediated by inflammation
and chemoattraction Mesenchymal-to-epithelial transition in eMSC
and BM-MSC at mRNA or protein level Under the experimental conditions tested, PRP did not induce
changes in major terminal METendpoints in eSF or eMSC, as
shown by sustained expression of vimentin mRNA and pro-
tein, with no induction of KRT7 mRNA expression and absent
cytokeratin 18 immunoreactivity (Fig. 5). Activated platelet-rich plasma promotes cell
proliferation Several chemokines
(CCL5, CCL7, and CXCL13) but not CCL2, CCL3, CCL4,
or IL8 (data not shown) were upregulated in aPRP versus
control, aPPP, naPRP, or naPPP-treated eSF, while eMSC
demonstrated significant upregulation of CCL5 and
aPRP compared to other groups (p < 0.05; Fig. significant data for BM-MSC and Ishikawa cells no
MMP7, TAGL, IL1B, and CXCL13 transcripts were n
icantly regulated in eMSC (data not shown). Transcripts for some growth factor receptors
EGFR, PDGFRB, and FGFR2 were analyzed in eS
BM-MSC, and IC, and the results demonstrated va
grees of upregulation (Fig. 4d), with EGFR show
consistent upregulation in response to treatment w
in all cell types (Fig. 4d). Mesenchymal-to-epithelial transition in eMSC
and BM-MSC at mRNA or protein level
Under the experimental conditions tested, PRP did n
changes in major terminal METendpoints in eSF or
shown by sustained expression of vimentin mRNA
tein, with no induction of KRT7 mRNA expression a
cytokeratin 18 immunoreactivity (Fig. 5). Discussion
General comments
The uterine endometrium is unique among adult h
sues in that it undergoes physiologic cyclic shedding
sequent regeneration without scarring at roughly m
tervals throughout women’s reproductive ye
b
0
5
10
15
20
25
30
35
40
0% control
aPRP
naPRP
aPPP
naPPP
BM-MSC, n=2, in triplicate
16hrs
24hrs
*
*
*
*
*
*
*
0
20
40
60
80
100
120
140
0% control
aPRP
naPRP
aPPP
naPPP
Number of migrated cells
eMSC, n=3, in triplicate
14hrs
24hrs
a
a
b
b
e
e
*
0
50
100
150
200
250
0% control
aPRP
naPRP
aPPP
naPPP
eSF, n=3, in triplicate
14hrs
24hrs
*
*
*
0
20
40
60
80
100
120
0% control
aPRP
naPRP
aPPP
naPPP
Number of migrated cells
Ishikawa cells, n=3, in duplicate
24h
48h
72h
96h
a
a
a
b
b
*
c
c
*
*
*
* * *
a
a
b
b
c
c
d
d
e
e
*
*
*
a
a
b
b
c
c
d
d
*
* *
*
*
* *
Number of migrated cells
Number of migrated cells
Fig. Activated platelet-rich plasma promotes cell
proliferation In the WST-1 assay, the absorbance values indicative of relative
cell proliferation showed an increase in stromal and mesenchy-
mal cell proliferation by activated PRP versus non-activated PRP
and PPP (p < 0.05), while Ishikawa epithelial cell proliferation
was significantly affected by both activated, but not non-activat-
ed, PRP and PPP (p < 0.05) (Fig. 2). It is important to note that
while aPRP had the most significant effect on proliferation of all
cell types studied, naPRP as well as aPPP and naPPP also
showed various degrees of increased proliferation when com-
pared to control cells after 3 days (Fig. 2). eSF and BM-MSC
demonstrated significantly higher proliferation in all groups com-
pared to control after 3 (eSF) and 7 (BM-MSC) days, respective-
ly, while such significant effect was seen in eMSC in aPPP and
naPRP groups, and in aPPP and naPPP in Ishikawa cells after
3 days. The decreased proliferation of Ishikawa cells on the
seventh day may be explained by confluent culture. 763 J Assist Reprod Genet (2018) 35:757–770 Platelet involvement in endometrial regeneration
may be mediated by inflammation
and chemoattraction
We analyzed gene expression differences in all cell types and
groups studies herein. Interestingly, there was no significant
difference in estrogen or progesterone receptor gene expres-
sion between cells treated with or without PRP or PPP in all
cell types studied (data not shown). In contrast, expression of
matrix metalloproteinases MMP3, MMP7, and MMP26 were
increased in the PRP and PPP groups in eSF with significant
differences with control and between aPRP and other groups
(p < 0.05), while in eMSC MMP1, MMP3 and MMP26 were
significantly upregulated compared to controls and between
aPRP and other groups (Fig. 4a). There was no difference in
MMP2 and MMP7 transcript expression in any cell type stud-
ied, and no difference in messenger RNA (mRNA) expression
of MMPs in BM-MSC or Ishikawa cells (data not shown). The cell transformation marker transgelin (TAGL) mRNA
was upregulated in eSF in aPRP versus naPRP or aPPP
(p < 0.05) (Fig. 4b), with no difference in E-cadherin
(CDH1) expression (data not shown). Inflammation markers
IL1A, IL1B, and IL1R2 were significantly upregulated in the
aPRP group in eSF compared to all other groups; while in
eMSC IL1A, IL15, and IL1R2 followed the same pattern of
expression (Fig. 4c; non-significant data for BM-MSC and
Ishikawa cells (latter not shown)). General comments The uterine endometrium is unique among adult human tis-
sues in that it undergoes physiologic cyclic shedding and sub-
sequent regeneration without scarring at roughly monthly in-
tervals throughout women’s reproductive years. The 764 J Assist Reprod Genet (2018) 35:757–770 0
5
10
15
20
25
30
35
CCL5
CCL7
CXCL13
Chemokine mRNA in eSF
aPRP
naPRP
aPPP
naPPP
0
1
2
3
4
5
CCL5
CCL7
Fold change to control
Chemokine mRNA in eMSC
aPRP
naPRP
aPPP
naPPP
0
1
2
3
4
5
6
7
8
IL1A
IL1B
IL1R2
Interleukins mRNA in eSF
aPRP
naPRP
aPPP
naPPP
0
2
4
6
8
10
12
14
IL1A
IL15
IL1R2
Fold change to control
Interleukin mRNA in eMSC
aPRP
naPRP
aPPP
naPPP
0
2
4
6
8
10
MMP3
MMP7
MMP26
MMPs mRNA in eSF
aPRP
naPRP
aPPP
naPPP
0
2
4
6
8
10
12
MMP1
MMP3
MMP26
Fold change to control
MMPs mRNA in eMSC
aPRP
naPRP
aPPP
naPPP
0
1
2
3
4
5
TAGLN
Transgelin mRNA in eSF
aPRP
naPRP
aPPP
naPPP
*
*
*
*
*
*
*
* * *
* *
*
*
*
*
*
*
* * *
* * *
* * *
*
*
*
* *
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
Fold change to control
Fold change to control
Fold change to control
Fold change to control
a
b
c
Fig. 4 mRNA expression of a MMPs (MMP 1, 3, 7, 26); b transgelin; c
interleukins (IL1A, IL1B) and receptor IL1R2, and chemokines (CCL5,
CCL7, and CXCL13); and d EGFR, FGFR2, PDGFRB, in endometrial
stromal fibroblasts (eSF), endometrial mesenchymal stem cells (eMSC),
bone marrow-derived mesenchymal stem cells (BM-MSCs) upon treat-
ment with PRP or PPP, normalized to vehicle controls. Statistical
significance accepted at p ≤0.05. Data represent the mean ± SD. Asterisk indicates significant difference compared to the aPRP group. aPRP activated platelet-rich plasma, naPRP non-activated platelet-rich
plasma, aPPP activated platelet-poor plasma, naPPP non-activated plate-
let-poor plasma. General comments The y axis range is not unified so that the smaller-scale
changes remain apparent to the reader 0
2
4
6
8
10
MMP3
MMP7
MMP26
MMPs mRNA in eSF
aPRP
naPRP
aPPP
naPPP
0
2
4
6
8
10
12
MMP1
MMP3
MMP26
Fold change to control
MMPs mRNA in eMSC
aPRP
naPRP
aPPP
naPPP
0
1
2
3
4
5
TAGLN
Transgelin mRNA in eSF
aPRP
naPRP
aPPP
naPPP
*
*
*
*
*
*
*
* * *
* *
*
*
*
*
*
*
Fold change to control
Fold change to control
a
b
c a b 0
5
10
15
20
25
30
35
CCL5
CCL7
CXCL13
Chemokine mRNA in eSF
aPRP
naPRP
aPPP
naPPP
0
1
2
3
4
5
CCL5
CCL7
Fold change to control
Chemokine mRNA in eMSC
aPRP
naPRP
aPPP
naPPP
0
1
2
3
4
5
6
7
8
IL1A
IL1B
IL1R2
Interleukins mRNA in eSF
aPRP
naPRP
aPPP
naPPP
0
2
4
6
8
10
12
14
IL1A
IL15
IL1R2
Fold change to control
Interleukin mRNA in eMSC
aPRP
naPRP
aPPP
naPPP
TAGLN
* * *
* * *
* * *
*
*
*
* *
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
Fold change to control
Fold change to control
c c Chemokine mRNA in eSF
Chemokine mRNA in eMSC
0
1
2
3
4
5
6
7
8
IL1A
IL1B
IL1R2
Interleukins mRNA in eSF
aPRP
naPRP
aPPP
naPPP
0
2
4
6
8
10
12
14
IL1A
IL15
IL1R2
Fold change to control
Interleukin mRNA in eMSC
aPRP
naPRP
aPPP
naPPP
* * *
* * *
* * *
*
*
*
*
*
*
*
*
Fold change to control
c 0
1
2
3
4
5
CCL5
CCL7
Fold change to control
aPRP
naPRP
aPPP
naPPP
*
*
*
*
*
* Fig. 4 mRNA expression of a MMPs (MMP 1, 3, 7, 26); b transgelin; c
interleukins (IL1A, IL1B) and receptor IL1R2, and chemokines (CCL5,
CCL7, and CXCL13); and d EGFR, FGFR2, PDGFRB, in endometrial
stromal fibroblasts (eSF), endometrial mesenchymal stem cells (eMSC),
bone marrow-derived mesenchymal stem cells (BM-MSCs) upon treat-
ment with PRP or PPP, normalized to vehicle controls. Statistical significance accepted at p ≤0.05. Data represent the mean ± SD. General comments 0
20
40
60
EGFR
FGFR2
PDGFRB
Fold change to control
Growth Factor receptors mRNA in
BM-MSC
aPRP
naPRP
aPPP
naPPP
*
* * * 0
50
100
150
EGFR
FGFR2
PDGFRB
Growth factor receptors mRNA in
eSF
aPRP
naPRP
aPPP
naPPP
*
* * *
*
Fold change to control
d 0
5
10
15
EGFR
FGFR2
PDGFRB
Fold change to control
Growth factor receptors mRNA in
IC
aPRP
naPRP
aPPP
naPPP
* *
*
*
*
* IC Fig. 4 continued. confirmed herein by upregulation of some of them in treated
cells by aPRP. interesting finding, not entirely unexpected, was that not only
aPRP but also naPRP and PPP did stimulate endometrial cell
proliferation and migration. This demonstrated that even plas-
ma that was relatively poor in its growth factor and cytokine
content was able to stimulate key cellular processes, which
could be an important asset in clinical situation. Interestingly, different cells types tested here demonstrated
more or less similar proliferative response to PRP, while
migratory responses were more pronounced in eSFs,
followed very closely by eMSC and then BM-MSC,
whereas IC had longer doubling time and required much
longer time for migration in our experimental settings. Whether this represents the order of first responders in
the process of tissue repair is unclear and warrants further
investigation, as well as potential effect that unique PRP
contents from different patients could have on the rate of
endometrial repair and regeneration. On the same note,
further experiments can address if different doses of PRP
may result in different proliferation rate of various endometrial
residual and circulating cell types. General comments Asterisk indicates significant difference compared to the aPRP group. aPRP activated platelet-rich plasma, naPRP non-activated platelet-rich
plasma, aPPP activated platelet-poor plasma, naPPP non-activated plate-
let-poor plasma. The y axis range is not unified so that the smaller-scale
changes remain apparent to the reader intracavitary scarring. Accordingly, our study evaluated for
the first time the effects of PRP on biological responses of
different human endometrial cells, and confirmed previously
published data on BM-MSC. The main finding of the study is
the demonstrated stimulatory effect of PRP on endometrial
cell proliferation and migration, as well as expression of sev-
eral factors potentially involved in endometrial regeneration
and repair. Because stimulation of proliferation and migration
is more robust when using a combination of growth factors
rather than single agents [53], our approach in this in vitro
study was to evaluate the effect of PRP, a complex mixture
of key agents involved in these processes, on different cell
types involved in endometrial regeneration. Another successful execution of this remarkable recurrent tissue repair
process requires the coordinated involvement of fibroblasts,
epithelial, endothelial, and adult stem/progenitor cells, and
their responses to local cues from the tissue microenviron-
ment, including cell proliferation, migration, lineage differen-
tiation, and transdifferentiation through MET [50, 51]. Disturbances of this tightly regulated homeostatic balance
have been implicated in endometrial pathologies, infertility,
and poor pregnancy outcomes [52]. In this context, the current study addressed the fundamental
questions of whether and how a therapeutic intervention may
affect these specific cellular processes towards alleviating re-
lated endometrial pathologies of inadequate growth or 765 J Assist Reprod Genet (2018) 35:757–770 0
50
100
150
EGFR
FGFR2
PDGFRB
Growth factor receptors mRNA in
eSF
aPRP
naPRP
aPPP
naPPP
*
0
20
40
60
EGFR
FGFR2
PDGFRB
Fold change to control
Growth Factor receptors mRNA in
BM-MSC
aPRP
naPRP
aPPP
naPPP
0
1
2
3
EGFR
FGFR2
PDGFRB
Growth factor receptors mRNA in
eMSC
aPRP
naPRP
aPPP
naPPP
* *
*
* *
*
* * *
* * *
*
0
5
10
15
EGFR
FGFR2
PDGFRB
Fold change to control
Growth factor receptors mRNA in
IC
aPRP
naPRP
aPPP
naPPP
* *
*
*
*
*
Fold change to control
Fold change to control
d
Fig. 4 continued. PRP stimulates endometrial cell proliferation
and migration 5 a Immunofluorescent analysis of vimentin (Vim) and cytokeratin
18 (KRT18) protein in human endometrial mesenchymal stem cells
(eMSC) in control cultures and cultures exposed to 5% PRP or PPP. Magnification, ×200. Negative controls are presented as inserts for
vimentin and cytokeratin 18, respectively. No difference was observed
between the groups. Data on endometrial stromal fibroblasts (eSF) or
bone marrow-derived mesenchymal stem cells (BM-MSCs) not shown. b mRNA expression of cytokeratin 7 (KRT7) in eMSC, eSF, and BM-
MSC and vimentin (VIM) in eSF. Significance accepted at p ≤0.05. Data
represent the mean ± SD. Vimentin mRNA data for eMSC or BM-MSC
not shown (no significant difference). aPRP activated platelet-rich plas-
ma, naPRP non-activated platelet-rich plasma, aPPP activated platelet-
poor plasma, naPPP non-activated platelet-poor plasma VIM-Control
VIM-aPRP
VIM-naPRP
VIM-aPPP
VIM-naPPP
KRT18-Control
KRT18-aPRP
KRT18-naPRP
KRT18-aPPP
KRT18-naPPP
a a naPRP
VIM-aPPP
VIM-naPPP
-naPRP
KRT18-aPPP
KRT18-naPPP
P
RP
P
P
0
0.5
1
1.5
2
2.5
3
VIM
Fold change to control
VIM mRNA in eSF
aPRP
naPRP
aPPP
naPPP
b mRNA expression of cytokeratin 7 (KRT7) in eMSC, eSF, and BM-
MSC and vimentin (VIM) in eSF. Significance accepted at p ≤0.05. Data
represent the mean ± SD. Vimentin mRNA data for eMSC or BM-MSC
not shown (no significant difference). aPRP activated platelet-rich plas-
ma, naPRP non-activated platelet-rich plasma, aPPP activated platelet-
poor plasma, naPPP non-activated platelet-poor plasma VIM-aPPP b
0
0.5
1
1.5
2
2.5
eSF
eMSC
BM-MSC
Fold change to control
KRT7 mRNA
aPRP
naPRP
aPPP
naPPP b 0
0.5
1
1.5
2
2.5
3
VIM
Fold change to control
VIM mRNA in eSF
aPRP
naPRP
aPPP
naPPP b mRNA expression of cytokeratin 7 (KRT7) in eMSC, eSF, and BM-
MSC and vimentin (VIM) in eSF. Significance accepted at p ≤0.05. Data
represent the mean ± SD. Vimentin mRNA data for eMSC or BM-MSC
not shown (no significant difference). aPRP activated platelet-rich plas-
ma, naPRP non-activated platelet-rich plasma, aPPP activated platelet-
poor plasma, naPPP non-activated platelet-poor plasma Fig. 5 a Immunofluorescent analysis of vimentin (Vim) and cytokeratin
18 (KRT18) protein in human endometrial mesenchymal stem cells
(eMSC) in control cultures and cultures exposed to 5% PRP or PPP. Magnification, ×200. Negative controls are presented as inserts for
vimentin and cytokeratin 18, respectively. No difference was observed
between the groups. Data on endometrial stromal fibroblasts (eSF) or
bone marrow-derived mesenchymal stem cells (BM-MSCs) not shown. PRP stimulates endometrial cell proliferation
and migration PRP has been shown to promote proliferation of human
adipose-derived stem cells, human dermal fibroblasts, human
synovial cells, human gingival fibroblasts, osteoblast-like
cells, and stromal cells [34, 45, 54–56] among others. We
were interested in evaluating if PRP (activated, non-
activated, or both) exerts a similar effect on human endome-
trial stromal fibroblasts and other cell types in the endometri-
um. The WST-1 assay demonstrated that PRP stimulates the
growth of all endometrial cell types studied (eSF, eMSC, and
Ishikawa cells), as well as BM-MSC. Moreover, the scratch
assay, which reflects the ability of cells to engage in wound
healing, demonstrated the stimulating effect of aPRP (and
even PPP) in this regard in all cell types studied. The
Transwell assay demonstrated that particularly aPRP can mo-
bilize endometrial cells by chemoattraction. Thus, our data
demonstrate that PRP promotes endometrial cell proliferation
and migration—characteristics that are fundamental for endo-
metrial regeneration. Of note is that while all treatment groups
(aPRP, naPRP, aPPP, and naPPP) affected proliferation and
migration processes to various degrees, aPRP had the greatest
and most consistent effect. Similar to data on other cell types,
these effects are likely mediated via growth factor receptors, Transgelin is a cytoplasmic protein that is highly expressed
in fibroblasts and participates in processes associated with a
remodeling of the actin cytoskeleton, including cell migration,
proliferation, differentiation, apoptosis, and matrix remodel-
ing [57]. It is expressed in human endometrium and is
overexpressed in ectopic endometriotic lesions [58]. Significant upregulation of its transcript in eSF upon aPRP
and even naPRP treatment suggests its potential involvement
in the fibroblast activation morphological transformation of
cells with migration and proliferation under these conditions. In contrast to our findings with eSF from women without any
gynecologic disorder, prolonged exposure of endometriotic
and adenomyosis lesions to activated platelets results in fibro-
sis due to epithelial-to-mesenchymal transition, fibroblast-to-
myofibroblast transdifferentiation, and repeated injury and
healing [59, 60]. These processes are mediated by TGF-β1 J Assist Reprod Genet (2018) 35:757–770 766 VIM-Control
VIM-aPRP
VIM-naPRP
VIM-aPPP
VIM-naPPP
KRT18-Control
KRT18-aPRP
KRT18-naPRP
KRT18-aPPP
KRT18-naPPP
a
b
0
0.5
1
1.5
2
2.5
eSF
eMSC
BM-MSC
Fold change to control
KRT7 mRNA
aPRP
naPRP
aPPP
naPPP
0
0.5
1
1.5
2
2.5
3
VIM
Fold change to control
VIM mRNA in eSF
aPRP
naPRP
aPPP
naPPP
Fig. PRP stimulates endometrial cell proliferation
and migration It was also proposed as a potential
treatment for endometritis in cattle based on its abilities to
downregulate the expression of pro-inflammatory genes in
primary cultures of bovine endometrial cells after 48 h of
incubation [65], and decreased intrauterine inflammatory
response in mares [66]. In the present study, we observed
a stimulatory effect of PRP on the expression of several
pro-inflammatory cytokines, chemokines, and MMPs (Fig. 4),
while others (IL8, IL1RL1; data not shown) did not sig-
nificantly change. Inflammation is a hallmark of the first
phase of wound healing, which is marked by platelet ac-
cumulation, coagulation, and leukocyte migration and me-
diated by inflammatory cytokines and growth factors [67]. Interestingly, post-menstrual repair is characterized by re-
epithelialization and angiogenesis, processes that play a
vital role in endometrial regeneration. While AS and thin
lining patients do not have a wound within the uterine
cavity per se, similar processes for wound healing and
tissue regeneration prevail, and our results of activation
of cytokine/chemokine gene expression support the effects
of PRP in promoting these processes. PRP stimulates endometrial cell proliferation
and migration and activation of TGF-β/SMAD signaling pathway, and anti-
platelet therapy in a mouse model was shown to decrease the
burden of endometriosis and adenomyosis [61, 62]. These
data underscore baseline molecular differences between
endometriotic/adenomyotic ectopic endometrial cells com-
pared to eutopic endometrial cells from subjects without such
pathology [36, 63], resulting in different molecular and func-
tional responses when exposed to activated platelets. Whether
eutopic endometrium from subjects with endometriosis/
adenomyosis will respond similarly to ectopic cells or normal
eutopic endometrial cells is currently unknown. Of note, the
focus of the current study—i.e., processes resulting in endo-
metrial regeneration in the setting of thin endometrium and
endometrial scarring—is fundamentally different from endo-
metriosis/adenomyosis, wherein ectopic endometrial cells are
exposed repeatedly to bleeding and activated platelets. This
clinical feature can explain the differences in the in vitro and
anticipated in vivo results. MMPs in human meniscocytes and articular chondrocytes,
thus supporting use of PRP in the management of cartilage
and meniscal injuries [64]. It was also proposed as a potential
treatment for endometritis in cattle based on its abilities to
downregulate the expression of pro-inflammatory genes in
primary cultures of bovine endometrial cells after 48 h of
incubation [65], and decreased intrauterine inflammatory
response in mares [66]. In the present study, we observed
a stimulatory effect of PRP on the expression of several
pro-inflammatory cytokines, chemokines, and MMPs (Fig. 4),
while others (IL8, IL1RL1; data not shown) did not sig-
nificantly change. Inflammation is a hallmark of the first
phase of wound healing, which is marked by platelet ac-
cumulation, coagulation, and leukocyte migration and me-
diated by inflammatory cytokines and growth factors [67]. Interestingly, post-menstrual repair is characterized by re-
epithelialization and angiogenesis, processes that play a
vital role in endometrial regeneration. While AS and thin
lining patients do not have a wound within the uterine
cavity per se, similar processes for wound healing and
tissue regeneration prevail, and our results of activation
of cytokine/chemokine gene expression support the effects
of PRP in promoting these processes. MMPs in human meniscocytes and articular chondrocytes,
thus supporting use of PRP in the management of cartilage
and meniscal injuries [64]. Strengths and limitations On the other hand, in the postpartum endometrial repair,
which is characterized by massive endometrial regeneration,
MET of endometrial progenitor cells has been described as an
important mechanism [68]. However, in the present in vitro
study on normal endometrial or progenitor cells, we did not
observe any evidence of MET, which could probably be ex-
plained by a Bsmaller scale^ or repair needed in our in vitro
conditions. While providing major novel information, our study has
both strengths and limitations. Strengths include the first
comprehensive evaluation of the effect of PRP on different
human endometrial cell types. The major limitation is the
inability to obtain sufficient cultures of primary endome-
trial epithelial cells for the experiments, thus necessitating
substitution of these with the Ishikawa endometrial adeno-
carcinoma cell line. While Ishikawa cells are used widely
in endometrial research and do express estrogen and pro-
gesterone receptors, the data obtained should be interpreted
with caution as this is an epithelial cancer cell line. Another limitation of the study is that we did not evaluate
the concentrations of the reportedly main growth factors
such as PDGF and TGFβ in PRP, which is going to be
addressed in follow-up study. In addition, while we chose
to analyze here PCR primers relevant to endometrial biology
and previously validated in our and others studies evaluating
endometrial gene expression in the mid-secretory phase and in
endometrial stromal and mesenchymal stem cells, microarray
analysis would be an important next step in the future to get a
more comprehensive and unbiased assessment of genes and
pathways affected. Matrix metalloproteinases (MMPs) are involved in tissue
regeneration and wound healing via degradation of extracel-
lular matrix (ECM) and wound remodeling [69]. Awide range
of cytokines and growth factors known to activate MMPs [70]
are present in PRP. In our study, PRP and, to some extent, PPP
increased expression of the matrix-degrading enzymes
MMP1, MMP3, MMP7, and MMP26 (Fig. 4). This correlates
well with previous data on human dermal fibroblasts and
tenocytes [71, 72]. MMP7 in particular is required for re-
epithelization of mucosal wounds [69], which adds to the
significance of its upregulation in endometrial stromal cells
upon PRP treatment in our study. PRP effect of sex hormone receptor expression In bovine endometrium, PRP upregulates progesterone recep-
tor protein in glandular epithelial cells when administered to
dairy cows compared to untreated controls [65]. In vitro, PRP
upregulated the gene expression of ERalpha, ERbeta, and the
progesterone receptor in cultured bovine endometrial cells
[65], leading the authors to suggest that intrauterine infusion
of PRP in cattle may improve their fertility [65, 73]. Activated
platelets also induced ERbeta (the predominant ER in medi-
ating estrogen action in endometriosis), mRNA, and protein
expression in endometriotic stromal cells from ovarian
endometrioma [74]. In our study, we did not observe differen-
tial expression of estrogen or progesterone receptor transcripts
in response to PRP or PPP treatment likely due to species
differences and different source of human cells used compared
to above studies. An additional limitation of the study is that we isolated
PRP from a single white 59-year-old male blood donor. While in clinical applications autologous PRP would be
anticipated for testing in women with AS and/or thin lin-
ing, we chose to use PRP from a male donor in order to
avoid possible effect of circulating hormones, which can
fluctuate based on menstrual cycle phase (latter informa-
tion is not provided to and by the blood bank). In addition,
we did not expect that the age of the blood donor would
have any significant effect on study results; however, this
can be evaluated in follow-up studies using pooled blood
from several donors. Despite these limitations, our study is
the first, to our knowledge, to evaluate the effect of PRP on
functional characteristics of endometrial cells, providing the
fundamental mechanistic foundation and proof-of-principle
for future controlled clinical studies. Inflammation, wound healing, and regeneration PRP has been shown to attenuate fibronectin-induced
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Riccaboni P, et al. Effects of platelet-rich plasma in a model of
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Incidência e características endoscópicas de lesões das vias aéreas associadas à intubação traqueal em crianças
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Revista da Associação Médica Brasileira
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INTRODUÇÃO (pressão = força/área), altas pressões podem
resultar de forças relativamente pequenas5. A
pressão exercida pelas paredes firmes do tubo
superou largamente a pressão de perfusão da
mucosa, estimada em 30mmHg e alcançou
400mmHg em modelo animal7, dando início a
um processo inflamatório local6. A resolução
desse processo pode acarretar graves seqüelas
na via aérea tais como estenose, sinéquias,
formação de granulomas, entre outras. exposição) após 48 horas de permanência do
tubo na via aérea em adultos3. Estudos em
crianças mostraram resultados similares. Striker
et al. reportaram os achados de autópsia em
32 crianças submetidas à intubação traqueal e
descreveram edema em laringe (50,0% das
autópsias) e traquéia (53,1%), ulceração na
laringe (28,1%) e traquéia (34,4%) e erosão em
laringe (12,5%) e traquéia (25%)4. Lesões de via aérea secundárias a trauma
decorrente da intubação começaram a ser
descritas à medida que o procedimento passou
a ser realizado com maior freqüência, particu-
larmente com a popularização desse método
como alternativa ou predecessor da traqueo-
tomia, e conseqüentemente para suporte
prolongado da via aérea, no início dos anos 501. Os relatos iniciais se limitavam a estudos de
autópsia que descreveram diferentes tipos de
lesões como ulceração e necrose de cartilagem
da traquéia, edema na laringe e traquéia em
adultos2. Perda completa ou focal do epitélio foi
detectada na mucosa que recobre os processos
vocais e região posterior da cricóide após
intubação por apenas 1 a 3 horas; ulceração em
processos vocais e subglote após 12 a 48 horas
de intubação e úlceras profundas e acometi-
mento de cartilagem (pericondrite, necrose e O mecanismo fisiopatológico primário da
lesão decorrente da permanência do tubo na
via aérea envolve a pressão exercida na
mucosa pelo tubo5,6. O mesmo se apóia e
exerce uma pressão na porção posterior da
laringe afetando três sítios principais: a super-
fície medial das cartilagens aritenóides, aspec-
tos mediais das junções cricoaritenóides e pro-
cesso vocal; a comissura posterior na região
interaritenóide e a subglote envolvendo a
superfície interna da cartilagem cricóide geral-
mente a lâmina posterior. Devido à natureza
tangencial do contato entre o tubo e a laringe
a área de contato é pequena. Artigo
Original Artigo
Original INCIDÊNCIA E CARACTERÍSTICAS ENDOSCÓPICAS DE LESÕES DAS VIAS
AÉREAS ASSOCIAD AS À INTUBAÇÃO TRAQUEAL EM CRIANÇAS ANDRÉA MARIA GOMES CORDEIRO*, SHIEH HUEI SHIN, IRACEMA DE CÁSSIA OLIVEIRA FERREIRA FERNANDES,
ALBERT BOUSSO, EDUARDO JUAN TROSTER ANDRÉA MARIA GOMES CORDEIRO*, SHIEH HUEI SHIN, IRACEMA DE CÁSSIA OLIVEIRA FERREIRA FERNANDES,
ALBERT BOUSSO, EDUARDO JUAN TROSTER
Trabalho realizado na Unidade de Terapia Intensiva Pediátrica do Hospital Universitário da USP, São Paulo, SP. Trabalho realizado na Unidade de Terapia Intensiva Pediátrica do Hospital Universitário da USP, São Paulo, SP. (IC95%: 6,6–14,8). Locais principalmente acometidos foram
glote (48,1% das lesões) e subglote (34,9% das lesões). Erosões
foram as mais incidentes em ambos os grupos etários ( p=0,88). Edema de prega vocal foi a principal lesão moderada em ambos
os grupos (p=0,96), seguida por ulcerações (p=0,92). Nódulos
fibrosos em pregas vocais e sinéqüias foram as principais lesões
graves em ambos os grupos etários ( p=0,12). Estenose
subglótica foi detectada em 2,8% da população sem diferença
entre as faixas etárias (p=0,35). RESUMO – OBJETIVO. Descrever a incidência e características
endoscópicas de lesões das vias aéreas em crianças submetidas à
intubação. RESUMO – OBJETIVO. Descrever a incidência e características
endoscópicas de lesões das vias aéreas em crianças submetidas à
intubação. MÉTODOS. Durante o período de dois anos (outubro/99 a outu-
bro/01) foi conduzido estudo prospectivo no qual todo paciente
intubado, excetuando-se aqueles que evoluíram para óbito e re-
cém-nascidos (RN) com peso inferior a 1.250g, foi submetido à
endoscopia respiratória na extubação. Achados endoscópicos fo-
ram classificados em leves, moderados ou graves. Descrições foram
realizadas por meio de proporções e medianas, comparações feitas
por teste qui-quadrado para proporções. CONCLUSÕES. Verificou-se elevada incidência de lesões em vias
aéreas, sem diferença significante entre os grupos etários com
relação à incidência e características das lesões. Houve predomínio
de lesões leves, lesões na glote e caracterizadas por erosões, edema
e ulcerações. RESULTADOS. Foram estudados 61 RN e 154 crianças. Em
89,8% dos pacientes, sendo 55 RN e 138 crianças ( p=0,89), foi
detectada pelo menos uma lesão somando 507. Pacientes com
lesões leves corresponderam a 54,8% (IC95%: 48,1–61,5),
aqueles com lesões moderadas foram 24,2% (IC95%: 18,5–
30,0) enquanto as graves ocorreram em 10,7% dos pacientes Unitermos: Intubação intratraqueal. Criança. Recém-nascido. Lesão das
vias aéreas. Estenose subglótica MÉTODOS Durante o período de outubro de 1999 a
outubro de 2001 todo paciente admitido na
Unidade de Terapia Intensiva Pediátrica e
Neonatal (UTIP/UTIN) do Hospital Univer-
sitário da Universidade de São Paulo (HU-
USP) que tenha necessitado de intubação
traqueal foi submetido à endoscopia respira-
tória no momento da extubação. Foram
excluídos os recém-nascidos de peso inferior
a 1.250g e aqueles que evoluíram ao óbito
previamente à extubação. Na população de estudo foram descritas e
analisadas as seguintes características: idade
(até 28 dias de vida ou idade superior a
28 dias), sexo, peso, escore de risco de óbito
(PRISM- Pediatric Risk of Mortality para aque-
les com idade superior a 28 dias21), duração da
intubação, principais diagnósticos de admissão
e indicações de intubação de acordo com a
falência ou insuficiência orgânica principal. INTRODUÇÃO Uma vez que
quanto menor a área sobre a qual uma força é
aplicada maior será a pressão resultante Estudos baseados em avaliação endos-
cópica das lesões de via aérea em crianças
submetidas à intubação endotraqueal têm
enfocado a incidência de estenose subglótica
provavelmente pela elevada morbidade asso-
ciada (necessidade de traqueotomia, dilatação
endoscópica ou cirurgia de reconstrução da
laringe)8-13. No entanto, lesões graves como a
estenose subglótica se desenvolvem a partir de
lesões fundamentais como ulcerações e expo-
sição de cartilagem. Existe uma seqüência de
eventos que culminaria com a seqüela em via
aérea5,6. Portanto, a prevenção de lesões gra-
ves requer o conhecimento da incidência de
outras formas de lesão que têm o potencial de *Correspondência:
Rua Jacques Felix, 96 – apt. 94
Cep: 04509-000 – São Paulo – SP *Correspondência:
Rua Jacques Felix, 96 – apt. 94
Cep: 04509-000 – São Paulo – SP 87 Rev Assoc Med Bras 2004; 50(1): 87-92 CORDEIRO AMG ET AL. evolução para formas graves. Estudos de inci-
dência das diferentes formas de lesão da via
aérea associadas à intubação traqueal se limi-
taram à faixa etária neonatal14,15 ou envolveram
pequeno número de pacientes pediátricos16-20. O presente estudo teve como objetivo des-
crever numa coorte de crianças submetidas à
intubação traqueal a incidência e caracte-
rísticas endoscópicas de lesões de via aérea. Quadro I – Alterações endoscópicas pós-extubação e prognóstico (Adaptado de Benjamin6)
Tipo de alteração
Aspecto endoscópico
Possível evolução
Precoce e
Hiperemia
Resolução
inespecífica
Edema
Erosão
Edema
Protusão da mucosa ventricular
Resolução
Edema prega vocal
Edema crônico de Reinke
Edema subglótico
Obstrução subglótica
Tecido de granulação
“Línguas” a partir do processo vocal
Resolução
das aritenóides
Granuloma
Nódulo fibroso
Sinéquia interaritenóide
Ulceração
Depressões ulceradas
Sulcos cicatriciais
Ulceração anular em glote posterior
Sinéquia da glote posterior
Ulceração subglótica com acometimento
Estenose subglótica
cricóide
Laceração
Cicatriz
Miscelânea
Sangramento
Hematoma
Deslocamento de aritenóide
Fixação da junção
cricoaritenóide
Perfurações
Infecção ou formação
de abscesso
Ulceração da cricóide
Fístula Quadro I – Alterações endoscópicas pós-extubação e prognóstico (Adaptado de Benjamin6) RESULTADOS •
Lesões com probabilidade de evolução
para seqüelas foram denominadas lesões
moderadas e incluíram edema sub-
glótico, edema de pregas vocais, ulcera-
ção, línguas de tecido de granulação,
laceração, hematoma, deslocamento de
aritenóide, exposição de cartilagem e
depressões ulceradas. •
Lesões com probabilidade de evolução
para seqüelas foram denominadas lesões
moderadas e incluíram edema sub-
glótico, edema de pregas vocais, ulcera-
ção, línguas de tecido de granulação,
laceração, hematoma, deslocamento de
aritenóide, exposição de cartilagem e
depressões ulceradas. Durante o período de estudo foram admi-
tidos 1.014 pacientes na UTIP e 421 na UTIN. Um total de 1.435 pacientes compôs a po-
pulação da unidade nesse período. Desses,
313 pacientes (21,8%) foram submetidos à
intubação traqueal. Foram excluídos 58 pa-
cientes (35 que evoluíram a óbito, 12 por não
consentimento e 11 que apresentavam peso
inferior a 1.250g). Em 40 pacientes não foi
possível a realização do exame (oito por im-
possibilidade de obtenção do consentimento,
14 por dificuldades operacionais, 10 por
extubação acidental e oito pacientes que foram
transferidos ainda intubados). g
p
p
O exame endoscópico foi realizado por
um dos pesquisadores (AMGC). Uma amostra
não-aleatória de 50 exames foi registrada em
vídeo e os resultados confrontados com outro
observador que desconhecia os diagnósticos
estabelecidos pelo primeiro, visando aferir a
concordância entre eles. Para realização do
exame endoscópico foi adotado um protocolo
de anestesia e todos os pacientes tiveram a
saturação de oxigênio, freqüência cardíaca e
pressão arterial monitorizados durante todo o
procedimento. •
Seqüelas de intubação foram denominadas
lesões graves e incluíram estenoses, siné-
quias, paresia ou paralisia de prega vocal,
granuloma, nódulos fibrosos, sulcos cica-
triciais e fixação da junção cricoaritenóide. Lesões situadas abaixo da subglote não
foram classificadas quanto à gravidade uma vez
que não estavam inclusas na classificação
proposta por Benjamin6 e adotada no estudo. •
Seqüelas de intubação foram denominadas
lesões graves e incluíram estenoses, siné-
quias, paresia ou paralisia de prega vocal,
granuloma, nódulos fibrosos, sulcos cica-
triciais e fixação da junção cricoaritenóide. Lesões situadas abaixo da subglote não
foram classificadas quanto à gravidade uma vez
que não estavam inclusas na classificação
proposta por Benjamin6 e adotada no estudo. •
Seqüelas de intubação foram denominadas
lesões graves e incluíram estenoses, siné-
quias, paresia ou paralisia de prega vocal,
granuloma, nódulos fibrosos, sulcos cica-
triciais e fixação da junção cricoaritenóide. RESULTADOS Tabela I – Características da população de estudo (215 pacientes)
Variável
Mediana (Variação)
≤ 28 d (61)
> 28 d (154)
Idade
5,4 d (1 - 26)
4 meses (1 mês - 14 anos)
Peso*
2.700 (1.250 - 4.600)
6,2 (2,1 - 54,0)
PRISM u
NA
12 (3 - 29)
Risco de morte (%)
u
NA
9,0 (2,2 - 64,5)
Duração da intubação em dias
4,4 (1 - 63 d)
5,0 (10h - 53 d) Tabela I – Características da população de estudo (215 pacientes) ç
Foram detectadas 507 lesões, sendo 358 em
138 crianças maiores que 28 dias de vida e 149
lesões em 55 recém-nascidos. Portanto foram
identificadas em média 2,6 e 2,7 lesões por
criança maior que 28 dias de vida e recém-
nascido, respectivamente. A distribuição topo-
gráfica e por faixa etária das lesões mostrou que a
glote (48,1% das lesões; IC 95%: 43,9 – 52,3) e
subglote (34,9% das lesões; IC 95%: 30,8 – 39,0)
foram os locais principalmente acometidos em
ambos os grupos. Lesões em via aérea inferior
foram incomuns (11,0% das lesões; IC 95%: 8,3
– 13,7) assim como em supraglote (5,9% das
lesões; IC 95%: 3,8 – 8,0). Não houve diferença
estatisticamente significante entre os grupos
etários com relação às lesões em glote (p= 0,11)
e subglote (p= 0,21) (Gráfico I). RESULTADOS *Peso em gramas para ≤ 28 dias, peso em Kg para > 28 dias; uAvaliados em 102 pacientes (excluídos 61 recém-nascidos, 10 pacientes
c/ shunt intracardíaco, 15 c/ doença pulmonar crônica e 27 pacientes com dados incompletos); h- horas, d- dias; NA- não se aplica Gráfico I – Distribuição percentual das 507 lesões por faixa etária e topografia
SUPGL - supraglote, GL - glote, SUBGL - subglote, VAI - via aérea inferior
0
25
50
75
100
SUPGL
GL
SUBGL
VAI
Tipo
%
> 28 dias
<= 28 dias
SUPGL-supraglote, GL-glote, SUBGL-suglote, VAI-via aérea inferior
Topografia Gráfico I – Distribuição percentual das 507 lesões por faixa etária e topografia
SUPGL - supraglote, GL - glote, SUBGL - subglote, VAI - via aérea inferior Gráfico I – Distribuição percentual das 507 lesões por faixa etária e topografia
SUPGL - supraglote, GL - glote, SUBGL - subglote, VAI - via aérea inferior ç
p
p
p g
SUPGL - supraglote, GL - glote, SUBGL - subglote, VAI - via aérea inferior Como houve com freqüência a associação
de lesões em um mesmo paciente apresenta-
remos o percentual de pacientes com lesões
de gravidades distintas. Para tal levamos em
consideração a presença de pelo menos uma
lesão grave, pelo menos uma lesão moderada
ou apenas lesões leves. Observamos que
54,8% dos pacientes (IC 95%: 48,1 – 61,5)
apresentaram apenas lesões leves, aqueles
com pelo menos uma lesão moderada
corresponderam a 24,2% da população estu-
dada (IC 95%: 18,5 – 30,0), enquanto os que
apresentaram pelo menos uma lesão grave
foram 10,7% (IC 95%: 6,6 – 14,8). Os diagnósticos principais à admissão foram
classificados, de acordo com o sistema acome-
tido, em problemas respiratórios, cardiovas-
culares, neurológicos e outros. Foram detecta-
dos 504 diagnósticos principais, sendo 377 na
população com idade acima de 28 dias e 127
nos recém-nascidos. Doenças de origem
respiratória prevaleceram em crianças maio-
res (56,5% dos diagnósticos) e também entre
recém-nascidos (54,3% dos diagnósticos)
(p= 0,67). Ao contrário, a doença neurológica
foi a condição menos comum em ambos os
grupos: 6,6% em crianças maiores e 7,9% em
recém-nascidos (p = 0,63). Broncopneu-
monia/pneumonia foram os principais diag-
nósticos de origem respiratória nos 154 pa-
cientes maiores (53,9%). Por outro lado, en-
tre os recém-nascidos, responderam por ape-
nas 22,9% dos diagnósticos de admissão
(p < 0,001). RESULTADOS Nesses a doença de membrana
hialina foi a doença respiratória mais prevalente
tendo ocorrido em 37,7% dos recém-nascidos. A intubação foi indicada por insuficiência
respiratória na maior parte dos pacientes
(57,8% dos pacientes acima de 28 dias de vida
e 73,8% dos recém-nascidos). Instabilidade
neurológica foi raramente indicação de
intubação nessa população, tendo ocorrido
em cinco pacientes maiores que 28 dias
(3,3%) e dois recém-nascidos (3,3%). Com-
parando os dois grupos etários com relação às
indicações de intubação, notamos diferenças
estatisticamente significantes apenas para a in-
suficiência respiratória que prevaleceu nos re-
cém-nascidos (p= 0,04). (
)
Lesões em via aérea inferior (56/507) fo-
ram representadas por erosões (23,2% das
lesões nessa região), ulcerações (17,9%),
traqueíte e/ou bronquite (55,4%), granuloma
em brônquio principal direito (1,8%) e
estenose do brônquio principal direito (1,8%). Na Tabela 2 descrevemos os tipos de le-
sões encontradas por faixa etária e gravidade. Erosões foram as lesões que apresentaram
maior incidência em ambos os grupos etários,
tendo representado 40,5% das lesões em crian-
ças fora do período neonatal e 37,6% daquelas
em recém-nascidos (p=0,61) e ocorreram
com maior incidência na subglote para as
crianças maiores de 28 dias de vida e de forma
similar na glote e subglote nos recém-nascidos. Lesões em via aérea inferior (56/507) fo-
ram representadas por erosões (23,2% das
lesões nessa região), ulcerações (17,9%),
traqueíte e/ou bronquite (55,4%), granuloma
em brônquio principal direito (1,8%) e
estenose do brônquio principal direito (1,8%). Na Tabela 2 descrevemos os tipos de le-
sões encontradas por faixa etária e gravidade. Erosões foram as lesões que apresentaram
maior incidência em ambos os grupos etários,
tendo representado 40,5% das lesões em crian-
ças fora do período neonatal e 37,6% daquelas
em recém-nascidos (p=0,61) e ocorreram
com maior incidência na subglote para as
crianças maiores de 28 dias de vida e de forma
similar na glote e subglote nos recém-nascidos. RESULTADOS Lesões situadas abaixo da subglote não
foram classificadas quanto à gravidade uma vez
que não estavam inclusas na classificação
proposta por Benjamin6 e adotada no estudo. Na Tabela I são descritas as principais
características dos 215 pacientes estudados. A
maior parte foi representada por pacientes
fora do período neonatal (71,6%). Nesse gru-
po houve predomínio de lactentes (60,5% da
população). Pacientes do sexo masculino pre-
dominaram em ambos os grupos, 57,4% para
os abaixo de 28 dias e 55,2% para os maiores
de 28 dias. Os valores do escore PRISM21
mostraram que a população acima de 28 dias,
à qual se aplica, apresentava baixo risco de
morte à admissão. Não houve diferença entre
as faixas etárias com relação à duração da
intubação (p=0,19). Reavaliação endoscópica foi realizada
apenas para os pacientes que necessitaram de
reintubação. Para esses foram registrados
apenas os novos achados endoscópicos. Os achados endoscópicos foram classifica-
dos de acordo com proposta de Benjamin
(1993) que definiu o aspecto endoscópico, a
topografia e possibilidade evolutiva das lesões
supraglóticas, glóticas e subglóticas6 (Quadro I). Consentimento para participação na pes-
quisa foi obtido para todos os pacientes junta-
mente aos pais ou representantes legais. A partir dessa classificação adotamos a
seguinte terminologia: A descrição das características populacionais
e das lesões foi realizada por meio de proporções
e medianas. Comparações, quando pertinentes,
foram realizadas por meio de teste qui-quadrado
para proporções. Foram consideradas diferenças
significantes quando o valor de p foi inferior a 0,05. •
Lesões com potencial de evolução para
resolução foram denominadas leves e
incluíram edema (exceto os citados abai-
xo), hiperemia e erosão. Rev Assoc Med Bras 2004; 50(1): 87-92 88 INCIDÊNCIA E CARACTERÍSTICAS ENDOSCÓPICAS DE LESÕES aérea. Um grupo de 55 pacientes necessitou
de reintubação sendo 39 dentre as 154 crian-
ças maiores que 28 dias de vida (25,3%), e 16
dentre 61 recém-nascidos (26,2%) (P= 0,97). Em alguns foram necessárias até três
reintubações e 83 procedimentos de rein-
tubação foram conduzidos. Estudo endoscópico Exame endoscópico normal foi observado
em apenas 10,2% (22/215) dos pacientes den-
tre os quais seis eram recém-nascidos e 16
eram crianças maiores que 28 dias (p= 0,89). Nos demais 89,8% da população estudada
(IC 95%: 85,8 – 93,8) sendo 55 recém-nasci-
dos e 138 crianças maiores que 28 dias de vida,
foi detectada pelo menos uma lesão na via 89 Rev Assoc Med Bras 2004; 50(1): 87-92 CORDEIRO AMG ET AL. Tabela 2 – Achados endoscópicos em 215 pacientes de acordo com a localização
anatômica e gravidade da lesão e por faixa etária
Prognóstico
Supraglote
Glote
Subglote
TOTAL
(n= 30)
(n= 244)
(n= 177)
(n= 451)
> 28d
≤≤ 28d
> 28d
≤≤ 28d
> 28d
≤≤ 28d
> 28d
≤≤ 28d
LEVES
Edema
1
0
9
7
4
2
14
9
Edema + Hiperemia
19
8
2
2
27
7
48
17
Erosão
0
0
65
30
80
26
145
56
Hiperemia
0
2
1
3
4
0
5
5
Subtotal
20
10
77
42
115
35
212
87
MODERADAS
Edema de PV
-
-
48
19
-
-
48
19
Ulceração
0
0
16
8
9
4
25
12
Tecido de granulação
0
0
7
3
0
0
7
3
Exposição cartilagem. 0
0
1
0
3
2
4
2
Lesão de PV
-
-
1
2
-
-
1
2
Subtotal
0
0
73
32
12
6
85
38
GRAVES
Granuloma
0
0
0
0
1
0
1
0
Sinéquia
0
0
5
2
1
1
6
3
Estenose
0
0
0
0
3
3
3
3
Nódulos Fibrosos PV
-
-
6
3
-
-
6
3
Paresia PV
-
-
0
1
-
-
0
1
Sulcos cicatriciais PrVcAr
3
0
3
0
Subtotal
0
0
14
6
5
4
19
10
TOTAL DE LESÕES
20
10
164
80
132
45
316
135
EM VAS e SUBGLOTE
VAS- via aérea superior; PV- prega vocal; PrVcAr- processos vocais das aritenóides de maior gravidade sofre possivelmente a
influência de variáveis relativas ao paciente
e ao procedimento de intubação. Aquelas re-
lativas ao procedimento são de grande inte-
resse, pois são passíveis de intervenção. Para
tal é essencial o conhecimento da incidência
desses eventos. Estudo endoscópico Tabela 2 – Achados endoscópicos em 215 pacientes de acordo com a localização
anatômica e gravidade da lesão e por faixa etária Observamos uma elevada incidência
(89,9% da população estudada) de lesões em
vias aéreas secundárias à intubação traqueal,
de gravidades variadas. A taxa geral de concor-
dância entre os dois observadores sugere
que os resultados demonstrados devem estar
próximos à realidade com mínima ocorrência
de um viés de observação. Uma vez que
reavaliação endoscópica foi realizada apenas
para os pacientes que necessitaram reintu-
bação, é possível que a incidência de lesões
graves tenha sido subestimada. O processo de
reparação que se inicia após o trauma se com-
pleta após a retirada do tubo podendo durar
até semanas. Dessa maneira é possível que
dentre aqueles não reavaliados, alguns tenham
evoluído com lesões graves em via aérea. Essa
possibilidade teria então interferido no cálculo
de incidência dessas lesões que poderiam estar
subestimadas. Para as lesões classificadas
como moderadas essa eventualidade é mais
remota, uma vez que lesões leves evoluem
para formas moderadas apenas com a persis-
tência da agressão. Já as moderadas podem
evoluir para resolução, ou para lesões graves
como formação de sinéquia ou estenose após
a retirada do tubo. VAS- via aérea superior; PV- prega vocal; PrVcAr- processos vocais das aritenóides VAS- via aérea superior; PV- prega vocal; PrVcAr- processos vocais das aritenóides Foram registrados em vídeo 50 exames,
sendo 44 em crianças maiores que 28 dias de
vida e seis em recém-nascidos (p=0,86). A
amostra analisada foi composta por cinco
exames normais e 45 exames alterados onde
foram diagnosticadas 90 lesões. O prognósti-
co das lesões em questão apresentou um
perfil similar ao do conjunto das lesões. Lesões leves representaram 60% da amos-
tra, moderadas representaram 21,1%, gra-
ves foram 5,6% da amostra e outras lesões
foram 13,3%. Com relação aos achados po-
sitivos houve 100% de concordância entre
os observadores. Houve discordância em
relação a cinco exames (10%) com relação a
achados não relacionados à intubação e às
lesões moderadas. A taxa geral de concor-
dância foi de 84% (IC 95%: 74 – 94%). Dentre as lesões classificadas como
moderadas observamos maior incidência de
edema em pregas vocais em ambos os grupos
(13,4% das lesões em crianças maiores de
28 dias de vida e 12,8% das lesões em
recém-nascidos) (p=0,96). Estudo endoscópico Fan et al.14, em estudo de 95 recém-
nascidos submetidos a intubação traqueal, ob-
servaram exame normal em apenas 19%. Com relação aos pacientes com exames alte-
rados, os autores adotaram classificação
segundo a qual lesões menores, tais como
ulceração na região interaritenóide, pregas
vocais e subglote, granulomas em pregas
vocais e paresia transitória das pregas, não
eram associadas à obstrução da via aérea. As
lesões moderadas cursavam com algum grau
de obstrução, e incluíam edema, pseudomem-
branas e granulomas maiores, associados ou
não a lesões menores. Lesões maiores
corresponderam à obstrução fixa e grave, ge-
ralmente decorrente de estenose subglótica,
membrana subglótica, defeitos em pregas
vocais e estenose traqueal. Os resultados des-
se estudo mostraram que 40% dos pacientes
apresentaram lesões menores, 34% lesões Lesões graves ocorreram principal-
mente ao nível da glote tendo sido 73,7%
das lesões dessa natureza e nessa topografia
no grupo etário fora do período neonatal e
60% em recém-nascidos (p=0,67). Siné-
quia foi a lesão grave de maior incidência
em crianças maiores de 28 dias de vida
(1,7% do total de lesões) assim como nódu-
los fibrosos em pregas vocais (1,7% do total
de lesões). Para os recém-nascidos siné-
qüias, estenoses e nódulos fibrosos em
pregas vocais apresentaram incidência simi-
lar (2% do total de lesões para cada). Estenose subglótica apresentou uma inci-
dência de 2,8% na população estudada
(6/215 sendo três pacientes recém-nasci-
dos e três pertencentes ao grupo fora do
período neonatal). INCIDÊNCIA E CARACTERÍSTICAS ENDOSCÓPICAS DE LESÕES podem ter contribuído para a elevada incidên-
cia de alguma lesão observada nessa popula-
ção. O diâmetro do tubo traqueal é freqüen-
temente citado como fator de risco para
lesões6,9,12. Identificamos uma variedade de
propostas para a escolha do diâmetro do tubo
traqueal em crianças, tais como o diâmetro do
dedo mínimo, diâmetro do leito ungueal do
dedo mínimo, fórmulas baseadas na idade ou
estimativas de peso a partir da estatura34-38. No
presente estudo a escolha do diâmetro do
tubo traqueal foi baseada em fórmulas
estruturadas no peso e idade com possibilida-
de de uso de tubos com diâmetros 0,5mm
maior ou menor do que os definidos pelas
propostas31. A precisão dessas recomen-
dações foi contestada anteriormente37. Dessa
maneira, uma postura prudente é aquela de se
basear em alguma recomendação e procurar
utilizar o tubo de menor diâmetro possível que
permita um escape e a adequada ventilação. De fato, o escape é recomendável e deve estar
presente com a aplicação de uma pressão
positiva de 20-30 cm H2O38. Apesar de termos
baseado a escolha do tubo traqueal em
fórmulas propostas na literatura, não é possí-
vel descartar que tenha ocorrido inade-
quação do diâmetro, pois não foi aferida a
presença de escape. Apesar dessa possibili-
dade a incidência de lesões graves, em
particular da estenose subglótica, foi compa-
rável aos estudos publicados. recém-nascidos intubados, exceto aqueles
com peso à extubação inferior a 1.250g. Pode-
mos perceber que as populações estudadas
são distintas, o que permite apenas inferir
similaridades ou diferenças entre os resultados
apresentados relativos à estenose subglótica. moderadas e 9% lesões maiores. Esses acha-
dos são muito similares aos por nós obser-
vados tanto em termos de incidência, quanto
na distribuição da gravidade das lesões. No
entanto, as classificações adotadas foram
distintas, e no estudo de Fan et al.14 os autores
estudaram apenas recém-nascidos, enquanto
no presente estudo incluímos também crian-
ças fora do período neonatal. É Encontramos uma maior incidência de
lesões na região da glote (48,1% das lesões) e
subglote (35,1% das lesões) sem diferença
estatisticamente significante entre os grupos
etários com relação aos achados em glote
e subglote, mas com diferença significante
(p< 0,001) quando comparamos as duas re-
giões, tendo sido mais freqüente o acome-
timento da glote independente da faixa etária. Esses resultados mostraram maior acome-
timento da via aérea superior, em particular
nas regiões posteriores da glote. INCIDÊNCIA E CARACTERÍSTICAS ENDOSCÓPICAS DE LESÕES ç
p
É possível estabelecer um paralelo com a
incidência de estenose subglótica que foi por
nós classificada como lesão grave e diag-
nosticada em seis pacientes (2,8%). A incidên-
cia reportada de estenose subglótica experi-
mentou uma redução nos últimos 30 anos. Estudos conduzidos nos anos 70 mostravam
uma incidência de 3,8% a 8,3%8,15,22-25. Aque-
les realizados posteriormente mostraram au-
sência de estenose subglótica em algumas sé-
ries26,27 ou baixa incidência em outros estudos
(0,4%-3,2%)10,11,13,14,28,29. Relatos com eleva-
das incidências dessa forma de lesão (12,8%-
24,5%) são provavelmente decorrentes das
características da população estudada (recém-
nascidos de muito baixo peso ao nascimento)
e pela maior sobrevida desses pacientes9,12. Os
estudos identificados se referem a uma popu-
lação específica de recém-nascidos prematu-
ros. A menor incidência de estenose subglótica
nos recém-nascidos incluídos no presente
estudo pode ter sido decorrente do fato de
a nossa população não ter sido fortemente
representada por neonatos prematuros de
muito baixo peso (média do peso a extubação
= 2.539,4 ± 833,3; variação= 1.250 -
4.600g). Nos demais estudos o peso de nasci-
mento médio foi de 1.229,7 ± 137,3 gramas
com variação de 496 a 4.850g9-14. A
prematuridade extrema pressupõe uma maior
duração da intubação com maior risco de
exposição às demais variáveis de risco, além da
imaturidade do sistema respiratório. Acometimento da via aérea abaixo da
região subglótica é descrito principalmente em
estudos envolvendo pacientes adultos, nos
quais existe uma grande preocupação com
lesões induzidas pelo balonete do tubo
traqueal. Para a faixa etária pediátrica, a
fisiopatologia das lesões em via aérea inferior
está principalmente relacionada a trauma pela
extremidade do tubo ou traumatismo da
mucosa em decorrência de aspirações do tubo
traqueal, uma vez que tubos com balonete são
raramente utilizados. Lesões do tipo traqueíte
e/ou bronquite não estão diretamente relacio-
nadas ao tubo traqueal, mas provavelmente à
pressão positiva do respirador, concentração
de oxigênio, umidade e temperatura do ar
inspirado30. A baixa incidência de lesões repor-
tadas abaixo da subglote pode ser devida a viés
de observação uma vez que os autores
pesquisados se concentraram em reportar a
incidência de estenose subglótica ou pelo fato
de que tubos sem balonete são geralmente
recomendados para crianças menores de oito
a 10 anos31. Essa recomendação se baseia nos
conhecimentos da anatomia da via aérea nesta
faixa etária. INCIDÊNCIA E CARACTERÍSTICAS ENDOSCÓPICAS DE LESÕES A região subglótica que corres-
ponde ao anel da cartilagem cricóide repre-
senta o ponto de maior estreitamento da via
aérea, atuando como “balonete funcional”. No
entanto, essa prática tem sido revista em fun-
ção de estudos que mostraram incidência de
estridor pós-extubação similar entre pacientes
que utilizaram tubos com e sem balonete32,33. O desenvolvimento de lesões secundárias DISCUSSÃO Alguma lesão em via aérea de pacientes
submetidos à intubação é um achado esperado
em função do conhecimento da fisiopatologia
dessas lesões. O desenvolvimento de lesões 90 Rev Assoc Med Bras 2004; 50(1): 87-92 Rev Assoc Med Bras 2004; 50(1): 87-92 INCIDENCE AND ENDOSCOPIC CHARAC-
TERISTICS OF AIRWAY INJURIES ASSOCIATED
WITH
ENDOTRACHEAL
INTUBATION
IN
CHILDREN gy
3. Donnelly WH. Histopathology of endotra-
cheal intubation. An autopsy study of 99 cases. Arch Pathol 1969; 88:511-20. 4. Joshi VV, Mandavia SG, Stern L, Wiglesworth
FW. Acute lesions induced by endotracheal
intubation. Occurrence in the upper respiratory
tract of newborn infants with Respiratory Distress
Syndrome. Am J Dis Child 1972; 124:646-9. 23. Parkin JL, Stevens MH, Jung AL. Acquired and
congenital subglottic stenosis in the infant. Ann
Otol Rhinol Laryngol 1976; 85 (pt 1): 573-81. g
g
Otol Rhinol Laryngol 1976; 85 (pt 1): 573-81. OBJECTIVE. describe the incidence and
endoscopic characteristics of airway injuries in
children submitted to intubation. 24. Jones R, Bodnar A, Roan Y, Johnson D. Sub-
glottic stenosis in newborn intensive care unit
graduates. Am J Dis Child 1981; 135:367-8. y
J
5. Bishop MJ, Weymuller EA Jr, Fink BR. Laryn-
geal effects of prolonged intubation. Anesth
Analg 1984; 63:335-42. METHODS. during a two-year period (october/
1999-october/2001) we conducted a pros-
pective study in which all patients that required
intubation, excluding those who deceased before
extubation and newborns (NB) weighing less
than 1.250g, were submitted to airway endos-
copy at extubation. The endoscopic findings were
classified as minor, moderate or severe. Descri-
ptions were made through proportions and
medians, comparisons were done through
chi-square for proportions. 25. Papsidero MJ, Pashley NR. Acquired stenosis
of the upper airway in neonates. An increasing
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gology head and neck surgery. 2nd ed. St
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tube and neonatal acquired subglottic stenosis. Study Group for Neonatology and Pediatric
Emergencies in the Parisian Area. Arch Otolar-
yngol Head Neck Surg 1993; 119:815-9. 7. Weymuller EA Jr, Bishop MJ, Fink BR, Hibbard
AW, Spelman FA. Quantification of intralaryngeal
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g
27. Walner DL, Loewen MS, Kimura RE. Neonatal
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factors for neonatal acquired subglottic ste-
nosis. Ann Otol Rhinol Laryngol 1986; 95 (pt
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tracheal damage after prolonged intubation in
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TERISTICS OF AIRWAY INJURIES ASSOCIATED
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IN
CHILDREN Neonatal intensive care. St. Louis:
Mosby Year Book; 1976. p.253-70. q
p p
RESULTS. we studied 61 NB and 154 children. In 89.8%, 55 NB and 138 children (P=0.89), it
was detected at least one lesion in a total of 507. Patients with minor lesions were 54.8%
(IC95%:48.1–61.5), those with moderate were
24.2% (IC95%:18.5–30.0) and severe injury
occurred in 10.7% (IC95%:6.6–14.8). Lesions
at the glottis (48.1% of lesions) and subglottis
(35.1%) presented the highest incidence. Erosions had the highest incidence in both age
groups (P=0.88). Vocal folds edema was the
main moderate lesion in both groups (P=0.96),
followed by ulcerations (P=0.92). Fibrous
nodules at vocal folds and adhesions were the
main severe injuries in both groups (P=0.12). Subglottic stenosis was detected in 2.8% of
the patients without difference between
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stenosis in the very-low birth-weight infant. Am J Dis Child 1983; 137:40-3. 9. Sherman JM, Lowitt S, Stephenson C, Ironson,
G. Factors influencing acquired subglottic
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nates. AACN Clin Issues 2000; 11:283-99. 10. Nicklaus PJ, Crysdale WS, Conley S, White AK,
Sendi K, Forte V. Evaluation of neonatal
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Barber CS, Fink R. Prospective study of subglo-
ttic stenosis in intubated neonates. Ann Otol
Rhinol Laryngol 1990; 99(5 pt 1): 390-5. 32. Deakers TW, Reynolds G, Stretton M, Newth
CJ. Cuffed endotracheal tubes in pediatric
intensive care. J Pediatr 1994; 125:57-62. 12. Downing GJ, Kilbride HW. Evaluation of
airway complication in high-risk preterm
infants: application of flexible airway endos-
copy. Pediatrics 1995; 95:567-72. 33. Khine HH, Corddry DH, Kettrick RG, Martin
TM, McCloskey JJ, Rose JB, et al. Comparison
of cuffed and uncuffed endotracheal tubes in
young children during general anesthesia. Anesthesiology 1997; 86:627-31. 13. Silva O, Stevens D. Complications of airway
management in very-low-birth-weight infants. Biol Neonate 1999; 75:40-5. 34. King BR, Baker MD, Braitman LE, Seidl-
Friedman J, Schreiner MS. Endotracheal tube
selection in children: a comparison of four
methods. Ann Emerg Med 1993; 22:530-4. 14. Fan LL, Flynn JW, Pathak DR. Risk factors
predicting laryngeal injury in intubated neo-
nates. CONCLUSÕES Nessa população de 215 pacientes ob-
servamos elevada incidência de lesões em
vias aéreas secundárias à intubação
traqueal, sem diferença estatisticamente
significante entre os grupos etários para
nenhuma das características estudadas. Houve predomínio de lesões classificadas
como leves, de acometimento da glote e
lesões caracterizadas por erosão, edema e
ulceração. No entanto, lesões moderadas e
graves estiveram presentes. O conheci-
mento da distribuição das lesões quanto
à gravidade e localização anatômica pode
auxiliar na redução de lesões com potencial
de evolução desfavorável bem como das
seqüelas de intubação. Estudos de acompa-
nhamento clínico e/ou endoscópico de
pacientes com lesões moderadas à extu-
bação são necessários para definir o prog-
nóstico associado a essa forma de lesão. Outro aspecto que dificulta comparações
é o denominador a partir do qual a incidência
de estenose subglótica é reportada. Alguns
autores reportam seus dados a partir do
número de recém-nascidos admitidos en-
quanto outros reportam a partir do número de
recém-nascidos submetidos à intubação
traqueal, e ainda a partir do número de pacien-
tes intubados por determinado período9,11,12,. Incidência de 4,9% de estenose subglótica
em recém-nascidos encontrada na nossa
pesquisa teve como denominador todos os O desenvolvimento de lesões secundárias
à intubação é certamente um fenômeno
multifatorial, desse modo diversas variáveis Rev Assoc Med Bras 2004; 50(1): 87-92 91 CORDEIRO AMG ET AL. INCIDENCE AND ENDOSCOPIC CHARAC-
TERISTICS OF AIRWAY INJURIES ASSOCIATED
WITH
ENDOTRACHEAL
INTUBATION
IN
CHILDREN Crit Care Med 1983; 11:431-3. 35. Van den Berg AA, Mphanza T. Choice of
tracheal tube size for children: finger size or age-
related formula? Anaesthesia 1997; 52:701-3. CONCLUSIONS. we observed a high incidence of
airway injury, without statistical significant
difference between age groups in regard to the
incidence and characteristics of the injuries. Minor injury was detected in the majority of the
population. Lesions were mainly noticed at the
glottis and were characterized by erosions,
edema and ulcerations. [Rev Assoc Med Bras
2004; 50(1): 87-92] 15. Strong RM, Passy V. Endotracheal intubation. Complications in neonates. Arch Otolaryngol
1977; 103:329-35. 36. Hofer CK, Ganter M, Tucci M, Klaghofer R,
Zollinger A. How reliable is length-based deter-
mination of body weight and tracheal tube size
in the paediatric age group? The Broselow tape
reconsidered. Br J Anaesth 2002; 88:283-5. 16. Hawkins DB. Glottic and subglottic stenosis
from endotracheal intubation. Laryngoscope
1977; 87:339-46. 17. Blahova O, Brezovsky P. Stenosing processes
due to endotracheal intubation and tracheos-
tomy in children. Int J Pediatr Otorhinolaryngol
1981; 3:199-203. 37. Eck JB, De Lisle DG, Phillips-Bute BG,
Ginsberg B. Prediction of tracheal tube size in
children using multiple variables. Paediatr
Anaesth 2002, 12:495-8. 18. Volpi D, Lin PT, Kuriloff DB, Kimmelman CP. Risk factors for intubation injury of the larynx. Ann Otol Rhinol Laryngol 1987; 96:684-6. 38. Zaritsky AL, Nadkari VM, Hickey RW, Schex-
nayder SM, Berg RA. Airway, ventilation and
management of respiratory distress and failure. Pediatric advanced life support. Dallas: AHA;
2002. p.81-126. Key words : Intratracheal intubation. Child. Newborn. Airway injury. Subglottic stenosis. Key words : Intratracheal intubation. Child. Newborn. Airway injury. Subglottic stenosis. y g
19. Woo P, Kelly G, Kirshner P. Airway compli-
cations in the head injured. Laryngoscope
1989; 99(7 pt 1):725-31. REFERÊNCIAS 20. Thomas R, Kumar EV, Kameswaran M, Shamin
A, Al Ghamdi S, Mummigatty AP, et al. Post
intubation laryngeal sequelae in an intensive
care unit. J Laryngol Otol 1995; 109:313-6. 1. Nilsson E. On treatment of barbiturate
poisoning. Acta Med Scand 1951; 253:7-127. Artigo recebido: 11/02/03
Aceito para publicação: 03/06/03 y g
21. Pollack MM, Ruttimann UE, Getson PR. Pediatric Risk of Mortality (PRISM) Score. Crit 2. Dwyer CS, Kronenberg S, Saklad M. The endo-
tracheal tube: a consideration of its traumatic 2. Dwyer CS, Kronenberg S, Saklad M. The endo-
tracheal tube: a consideration of its traumatic Rev Assoc Med Bras 2004; 50(1): 87-92 92
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The MRCPsych examinations
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The bulletin of the Royal College of Psychiatrists/Bulletin of the Royal College of Psychiatrists
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Obituary Editor: Henry R. Rollin VOJTECHADALBERTKRAI., Professor Emeritus of
ClinicalPsychiatry,Universityof WesternOntario,
Canada. Following their liberation, the Krals returned to
Czechoslovakia but, after the Communist takeover,
they fled to Canada. From 1952 to 1972, he served
first as lecturer and ultimately as Associate Professor
at McGill University, having achieved specialist cer
tification
in both
neurology
and
psychiatry
in
Canada. Thereafter, he moved to London. Ontario,
and continued to work and teach psychogeriatrics. Dr Vojtcch Adalbert Krai, a distinguished Canadian
psychiatrist and pioneer of psychogcriatrics, known
to his family and friends as Bertie, died on 6 June. 1988during a visit to Israel. g
Dr Kral was born on 5 February, 1903 in what is
now Czechoslovakia and received his education in
the
German-speaking
state
high school
(Gym
nasium) in Prague and at the German University of
Prague, graduating MD in March 1927. He studied
further with the leaders of psychiatry and neurology
in Vienna, Zurich, and Munich and, from 1935 to
1938, was a privat-dozent,
corresponding
to an
Associate Professor, in the Department of Neurology
and Psychiatry at his alma mater. p y
g
Dr Kral published some 180 papers or chapters
of books on scientific topics. His initial studies were
on the biochemistry of the cerebrospinal
fluid in
different illnesses of the nervous system. From 1933
onwards,
he studied
memory
function
in such
disorders. In 1956,he described benign senescent for-
gctfulncss as a common phenomenon of the elderly,
not to be confused with dementing
illnesses like
Alzheimer's disease. His recognition of this harmless
syndrome was widely appreciated
and helped to
reassure many ageing individuals. He undertook
further studies on Alzheimer's disease, neurotropic
medication, and pseudo-dementia of the aged. y
y
He and his wife, Katherine, (nee Neumark), an
ophthalmologist,
as Jews, were both imprisoned in
Thcresicnstadt Concentration Camp throughout the
Nazi period. There, he participated in the medical care
of the prisoners and he and his wife were saved from
deportation to Auschwitz and extermination because
of the Nazi need for an expert opinion on an out
break of encephalitis amongst prisoners which might
have affected the staff. He subsequently described
this epidemic in 1947 in the Journal of Nervous and
Mental Diseases, as well as in a Czech journal. The M RC PSYCHexaminâtions 2. Dr O. Daly's article The Membership Examin
ation—OneCandidate's View Point' (p 231)makes a
reference to a variable percentage contribution
of
marks from each section of the Examination. In the June 1988 issue of the Bulletin (112). several
articles relating to the examinations appeared under
the section. Trainees Forum. We would
like to draw
your attention
to two
inaccuracies: In fact the four parts of the Membership Examin
ation (MCQ, Essay, Clinical and Oral) have equal
weight in terms of marks. Although candidates must
pass the Clinical Examination, the other three parts
should
be seen as complementary
rather
than
secondary to it. 1. Drs Armstrong and Loosmore's article 'How to
run the MRCPsych Part II Examination' (p 229) re
fers to the clinical and oral section of the examin
ation. It states that the examination includes a viva
lasting 15minutes. Professor A. C. P. SIMS,Dean
Professor H. G. MORGAN,Chief Examiner This information is incorrect. The PMP Oral Section
of the MRCPsych Part II lasts 30 minutes. The College The M RC PSYCHexaminâtions Professor A. C. P. SIMS,Dean
Professor H. G. MORGAN,Chief Examiner 395 395 The College Obituary Dr Kral received the Allan Award and Gold
Medal of the American Geriatric Society, as well as
an honorary award from the Ontario Psychogcriatric
Association and the annual award of the Psychiatric
Out-patients
Center of America. He was elected a
Fellow of the British Royal College of Psychiatrists
and a Fellow of the American Psychiatric Associ- https://doi.org/10.1192/S0140078900021878 Published online by Cambridge University Press
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The association between genetic polymorphism of glutathione peroxidase 1 (rs1050450) and keratoconus in a Turkish population
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Arquivos brasileiros de oftalmologia
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A associação entre o polimorfismo genético da glutationa-peroxidase 1
(rs1050450) e pacientes com ceratocone em uma população turca Abdullah Ilhan1
, Salih Altun2, Irfan Durukan1, Umit Yolcu1, Uzeyir Erdem2
1. Department of Ophthalmology, ERA Eye Center, Ankara, Turkey. 2. Department of Ophthalmology, Gulhane Military Medical Academy, Turkey. Abdullah Ilhan1
, Salih Altun2, Irfan Durukan1, Umit Yolcu1, Uzeyir Erdem2
1. Department of Ophthalmology, ERA Eye Center, Ankara, Turkey. Abdullah Ilhan1
, Salih Altun2, Irfan Durukan1, Umit Yolcu1, Uzeyir Erdem2 Abdullah Ilhan1
, Salih Altun2, Irfan Durukan1, Umit Yolcu1, Uze
1. Department of Ophthalmology, ERA Eye Center, Ankara, Turkey. 2. Department of Ophthalmology, Gulhane Military Medical Academy, Turkey. 1. Department of Ophthalmology, ERA Eye Center, Ankara, Turkey. 2. Department of Ophthalmology, Gulhane Military Medical Academy, Turkey. Keywords: Keratoconus; Glutathione peroxidase; Catalase;
Superoxide dismutase; Polymorphism, genetic ABSTRACT | Purpose: To investigate the potential associa-
tions between keratoconus and catalase rs1001179, superoxide
dismutase 2 rs4880, and glutathione peroxidase 1 rs1050450
gene polymorphisms in a Turkish population. Methods: The
study group included 121 unrelated keratoconus patients and
94 unrelated healthy controls. Blood samples (200 µl) were
collected from all patients and controls to isolate genomic DNA. Genotyping was performed to identify rs1001179, rs4880,
and rs1050450 using real-time polymerase chain reaction
(PCR). Genotype and allele frequencies were calculated; their
associations with keratoconus risk were assayed, and the
association with keratoconus risk and demographic factors
was examined. Results: Glutathione peroxidase 1 rs1050450
polymorphism was present in 41% cases compared with 29%
controls (OR=1.66; 95% CI=1.11-2.50; p=0.014). No asso-
ciation was observed between catalase rs1001179 and SOD2
rs4880 polymorphisms and keratoconus (for all, p>0.05). Conclusions: This study evaluated possible relationships
between rs1050450, rs1001179, and rs4880 polymorphisms
and keratoconus susceptibility. We found a possible association
between glutathione peroxidase 1 rs1050450 polymorphism
and an increased risk of keratoconus. However, the genotype
and allele frequencies were identical in the catalase rs1001179
and superoxide dismutase 2 rs4880 polymorphisms. Further
studies are needed to analyze the effect of such variations
in identifying keratoconus susceptibility. RESUMO | Objetivo: Investigar as possíveis associações entre o
ceratocone e os polimorfismos rs1001179 da catalase, rs4880 da
superóxido-dismutase 2 e rs1050450 da glutationa-peroxidase
1 rs1050450 em uma população turca. Métodos: O grupo de
estudo incluiu 121 pacientes com ceratocone não relacionados
e 94 controles saudáveis também sem parentesco. Amostra de
sangue (200 μL) foram coletadas de todos os pacientes e controle
para isolar o DNA genômico. A associação entre o polimorfismo genético da glutationa-peroxidase 1
(rs1050450) e pacientes com ceratocone em uma população turca A genotipagem foi realizada para
identificar rs1001179, rs4880 e rs1050450 utilizando a reação
em cadeia da polimerase (PCR) em tempo real. As frequências
de genótipos e alelos foram calculadas, suas associações com o
risco de ceratocone foram avaliadas, e a associação com risco
de ceratocone e fatores demográficos foi examinada. Resul-
tados: O polimorfismo da glutationa-peroxidase 1 rs1050450
estava presente em 41% dos casos, comparado com 29% dos
controles (OR=1,66, IC 95%=1,11-2,50; p=0,014). Não foi
observada associação entre o ceratocone e os polimorfismos
rs1001179 e SOD2 rs4880 da catalase (para todos, p>0,05). Conclusões: Este estudo avaliou possíveis relações entre os poli-
morfismos rs1001179, rs4880 e suscetibilidade a ceratocone. Encontramos uma possível associação entre polimorfismo da
glutationa-peroxidase 1 rs1050450 e um risco aumentado de
ceratocone. No entanto, o genótipo e as frequências alélicas
foram idênticas nos polimorfismos rs1001179 da catalase e
superóxido-dismutase 2 rs4880. Mais estudos são necessários
para esclarecer o efeito dessas variações na detecção da sus
cetibilidade ao ceratocone. Descritores: Ceratocone; Glutationa peroxidase; Catalase; Su
peróxido dismutase; polimorfismo genético Accepted for publication: February 5, 2019 Submitted for publication: June 20, 2017 A r q u i v o s b r a s i l e i r o s d e Original Article ■http://dx doi org/10 5935/0004-2749 20190102
This content is licensed under a Creative Commons Attributions 4.0 Interna
Submitted for publication: June 20, 2017
Accepted for publication: February 5, 2019
Funding: No specific financial support was available for this study.
Disclosure of potential conflicts of interest: None of the authors have any potential
conflicts of interest to disclose.
Corresponding author: Abdullah Ilhan.
Yayla Mah. Şehit Mustafa Ercigez Cad. No: 21 - Kecioren - Ankara - 06220, Ankara,
Turkey - E-mail: dzilhan@hotmail.com
Approved by the following research ethics committee: Ataturk University Faculty
of Medicine (#B.30.2.ATA.0.01.00/73). Patients and controls Our study enrolled 121 unrelated Turkish patients
with KC (aged 14 to 62 years) and 94 age-matched indi-
viduals with no history of KC as the control group. Each
subject granted informed consent before study parti-
cipation. The principles of the Declaration of Helsinki
were strictly followed during all stages of the study. Under physiological conditions of aerobic organisms, re-
active oxygen species (ROS) generated during the course
of metabolism is restricted by antioxidant enzymes. Superoxide, the primary ROS, is converted to hydrogen
peroxide (H2O2), mainly by superoxide dismutase (SOD). H2O2 is decomposed by catalase (CAT) or glutathione pe-
roxidase (GPX), releasing water and molecular oxygen. Any malfunction, i.e., in genetic polymorphisms, in the-
se enzymes could lead to a shift in the balance between
ROS production and the antioxidant defense mechanism
resulting in oxidative stress(8). SOD, CAT, and GPX have
been described in aqueous humor, indicating their sig-
nificance in the balance of oxidative status in the eyes(9). All patients and controls were evaluated at the De-
partment of Ophthalmology, Gulhane Military Medical
Academy. The diagnosis of KC was made based on the cli-
nical signs (Munson sign, protrusion, Vogt striae, corneal
thickness, scarring, Fleischer rings, refractive errors) and
topographic findings obtained using Oculus Pentacam
(OCULUS Optikgeräte GmbH, Wetzlar, Germany) and
OPD-Scan ARK-10000 (NIDEK, Tokyo, Japan). The patients with a confirmed diagnosis of KC were
included in the study group. Control subjects underwent
the same examinations to eliminate mild cases that
could be overlooked in regular examinations. None of
the participants reported any genetic disorder. SOD has 3 subtypes. SOD1 and SOD2 are intracellu-
lar subtypes, and SOD3 is extracellular; all are present
in the cornea(4,10). SOD1 has been evaluated numerous
times, and many studies have shown potential causa-
tive roles of SOD1(11,12). CAT and GPX are upregulated
in KC corneas, emphasizing their importance in de
creasing ROS(13,14). INTRODUCTION Keratoconus (KC) is a progressive degenerative colla-
gen disorder distinguished by stromal thinning, corneal Arq Bras Oftalmol. 2019;82(6):501-6 501 The association between genetic polymorphism of glutathione peroxidase 1 (rs1050450) and keratoconus in a Turkish population Many studies have revealed phenotype associations
for SOD2 (Val16Ala, rs4880) including heart diseases,
several malignancies, fatty liver disease, and age-related
macular degeneration. Polymorphism studies of the
CAT (-262C>T, rs1001179) gene have been conducted
in many diseases including diabetes, systemic lupus
erythematosus, Alzheimer disease, and several malig-
nancies. The GPX-1 (Pro198Leu, rs1050450) polymor-
phism has also been extensively studied, with significant
results being reported in panic disorder, kidney disease,
morbid obesity, and cardiovascular diseases. However,
no reports have described these common SNPs in KC
patients. In fact, no study has yet reported on SOD2,
GPX, and CAT SNPs in KC. steepening, and irregular astigmatism leading to deterio-
rated visual acuity and impaired quality of life. It mainly
occurs in the second decade of life at an incidence of
approximately 1 per 2000 in the general population. KC affects all ethnicities and both genders(1). g
The etiology of KC remains unclear. Various factors
contribute to the pathophysiological processes of
KC, including environmental, behavioral, and genetic
factors. As 6% to 10% of KC patients have familial
KC, genetic susceptibility has been widely studied for
years. However, no specific genetic defect for KC has
been described. In addition, several investigations have
acknowledged the role of oxidative stress in KC patho-
genesis. KC corneas accumulate cytotoxic end products
of the nitric oxide and lipid peroxidation pathways
and have impaired antioxidant enzymes and increased
mitochondrial DNA damage(2-7). These findings suggest
that oxidative stress is significantly involved in the
pathogenesis of KC. However, additional genetic and
molecular studies are needed to thoroughly define the
underlying problem. We aimed to determine whether the presence of these
SNPs in key antioxidant enzymes is associated with an
increased risk of KC. 502
Arq Bras Oftalmol. 2019;82(6):501-6 GPX1 rs1050450, SOD2 rs4880, and CAT
rs1001179 genotyping In KC patients, the GPX1 C/T poly-
morphism was present in 43 (36%) wild-type for the C/C
genotype, 59 (49%) heterozygous for the C/T genotype,
and 19 (16%) homozygous for the T/T genotypes. Sta-
tistical analysis demonstrated a possible harmful effect
of the GPX1 C/T+TT and TT genotypes (OR=1.89, 95%
CI=1.05-3.40; p=0.022; OR=2.65; 95% CI=1.05-6.67;
p=0.035, respectively) in the development of KC and
a possible protective effect of the C/C genotype in con-
trols. Table 3 presents the comparative analysis of the
demographic data and the SNPs. GPX1 rs1050450, SOD2 rs4880, and CAT
rs1001179 genotyping Recently, more attention has been given to the im-
portance of genomic variations between individuals. Single nucleotide polymorphism (SNP) can be detected
at certain positions in the genome. SNPs in the gene
coding may affect metabolic pathways by altering the
enzyme structure or substrate affinity and/or activity
and impairing antioxidant capacity. Peripheral blood samples were collected in EDTA tu-
bes and stored at -20°C before use. Genomic DNA was
extracted from the blood samples (200 µl) using the EZ1
DNA Blood 200 µl Kit by EZ1 Advanced XL automated
purification system (Qiagen, Hilden, Germany) accor-
ding to the manufacturer’s instructions. Ilhan A, et al. The SNP identification assay (LightSNiP; Tib-MolBiol,
Berlin, Germany), which is based on simple probe melting
curve analysis, was used to detect GPX1 (rs1050450),
SOD2 (rs4880), and CAT (rs1001179) polymorphisms. Both PCR and melting curve analysis were conducted on
the Rotor-Gene Q (Qiagen). The reaction for each SNP
was performed in a final volume of 20 μl and included
2 μl DNA, 1 μl LightSNiP reagent mix (Tib-MolBiol),
2 μl LightCycler FastStart DNA Master HybProbe (Roche
Diagnostics GmbH, Mannheim, Germany), 1.6 μl Mg Cl2
(25 mM), and 14.4 µl H2O. PCR consisted of initial
denaturation at 95°C for 10 minutes came next by 45
cycles, consisting of denaturation at 95°C for 10 seconds,
annealing at 60°C for 10 seconds, and elongation at 72°C
for 15 seconds. After the last cycle, samples were held at
95°C for 30 seconds before the temperature was lowered
to 45°C. Next, melting was conducted by steadily raising
the temperature in 0.5°C increments every 2 seconds
from 45°C to 75°C. Distributions of the GPX1 rs1050450 C/T, SOD2
rs4880 T/C, and CAT rs1001179 C/T genotypes con-
sistent with the Hardy-Weinberg equilibrium among
patients (p=0.867, 0.554, 0.199, respectively) and
controls (p=0.901, 0.921, 0.839, respectively). We exa-
mined the genetic polymorphisms in the GPX1, SOD2,
and CAT genes, and allele and genotype distributions
are shown in table 2. No significant difference existed between the SOD2
(rs4880) and CAT (rs1001179) genotypes (p=0.936 and
0.443, respectively) or allele frequencies (p=0.854 and
0.627, respectively) of the KC patients and the controls. The difference in the allele frequency of the variant
T allele of the GPX1 C/T polymorphism was statistically
significant in the KC patients (41%) when compared
with the controls (29%) (OR=1.66; 95% CI=1.11-2.50;
p=0.014), thus the T allele was a statistically significant
risk factor for KC. RESULTS The frequency distribution of selected characteristics
of the cases and controls is shown in table 1. No statis-
tically significant difference was seen in sex or age dis-
tribution between the cases and control (both p>0.05). Statistical analysis Hardy-Weinberg equilibrium was evaluated using the
chi-squared (χ2) test. The χ2 test and two-way Student’s
t-test were used to conduct statistical comparisons of
genotypic and allelic distributions between the control
and KC patients. All statistical analyses were performed
using SPSS for Windows, version 16.0 (SPSS Inc., Chi
cago, IL, USA). A p value ≤0.05 was considered statis
tically significant. In our study, the GPX1 (rs1050450), SOD2 (rs4880),
and CAT (rs1001179) polymorphisms did not impact
the risk of KC with respect to sex (p=0.550, 0.870,
0.303, respectively) and age (p=0.927, 0.686, 0.523,
respectively). DISCUSSION Our main concern in researching CAT rs1001179,
SOD2 rs4880, and GPX1 rs1050450 polymorphisms in
KC pathogenesis stems from their distinct enzymatic pro-
perties. They are essential to limiting ROS accumulation
and inhibiting premature apoptosis in eukaryotic cells. In addition, they exist in almost all living organisms that
require oxygen. SOD2, GPX1, and CAT share a common
metabolic detoxification pathway(15). Dysfunctions of
GPX1, SOD2, and CAT are shown to play an integral
role in the pathogenesis of various diseases. In addition,
previous studies have reported oxidative stress and
oxidative stress-induced premature apoptosis. Thus, an
evaluation of these antioxidant enzymes would provide Table 1. Characteristics of keratoconus cases and controls
Variable
Cases
Controls
P
(n=121) (%)
(n=94) (%)
Sex
Male
83 (68.6%)
74 (78.7%)
0.097
Female
38 (31.4%)
20 (21.3%)
Age (years)
<20
14 (11.6%)
12 (12.8%)
0.061
20-29
41 (33.9%)
49 (52.1%)
30-39
46 (38.0%)
25 (26.6%)
40-49
17 (14.0%)
7 (7.4%)
≥50
3 (2.5%)
1 (1.1%) Table 1. Characteristics of keratoconus cases and controls 503 Arq Bras Oftalmol. 2019;82(6):501-6 The association between genetic polymorphism of glutathione peroxidase 1 (rs1050450) and keratoconus in a Turkish population Table 2. Genotype and allele distribution of rs1050450, rs4880, and rs1001179 polymorphisms in keratoconus patients and controls
KC patients (n=121)
Control group (n=94)
OR (95% CI)
p
Number (freq)
Number (freq)
GPX1 (rs1050450)
Genotypes
CC (Pro/Pro)
43 (0.36)
48 (0.51)
0.049
CT (Pro/Leu)
59 (0.49)
38 (0.40)
TT (Leu/Leu)
19 (0.16)
8 (0.09)
CT + TT versus CC
1.89 (1.05-3.40)
0.022
CT versus CC
1.73 (0.97-3.09)
0.062
TT versus CC
2.65 (1.05-6.67)
0.035
TT versus CT
1.53 (0.61-3.84)
0.364
Alleles
T (Leu)
97 (0.41)
54 (0.29)
1.66 (1.11-2.50)
0.014
C (Pro)
145 (0.59)
134 (0.71)
SOD2 (rs4880)
Genotypes
TT (Val/Val)
45 (0.37)
33 (0.35)
0.936
TC (Val/Ala)
55 (0.46)
45 (0.48)
CC (Ala/Ala)
21 (0.17)
16 (0.17)
Alleles
T (Val)
145 (0.60)
111 (0.59)
1.04 [0.70-1.53]
0.854
C (Ala)
97 (0.40)
77 (0.41)
CAT (rs1001179)
Genotypes
CC
85 (0.70)
65 (0.69)
0.443
CT
35 (0.29)
26 (0.28)
TT
1 (0.01)
3 (0.03)
Alleles
C
205 (0.85)
156 (0.83)
1.14 [0.68-1.91]
0.627
T
37 (0.15)
32 (0.17)
OR= odds ratio; CI= confidence interval. e and allele distribution of rs1050450, rs4880, and rs1001179 polymorphisms in keratoconus patients and controls Table 3. 504
Arq Bras Oftalmol. 2019;82(6):501-6 REFERENCES 1. Gordon-Shaag A, Millodot M, Shneor E, Liu Y. The genetic
and environmental factors for keratoconus. BioMed Res Int. 2015;2015:795738. 2. Buddi R, Lin B, Atilano SR, Zorapapel NC, Kenney MC, Brown DJ. Evidence of oxidative stress in human corneal diseases. J Histo-
chem Cytochem. 2002;50(3):341-51. 3. Gondhowiardjo TD, van Haeringen NJ, Völker-Dieben HJ, Beekhuis
HW, Kok JH, van Rij G, et al. Analysis of corneal aldehyde dehydro-
genase patterns in pathologic corneas. Cornea. 1993;12(2):146-54. 4. Behndig A, Karlsson K, Johansson BO, Brännström T, Marklund
SL. Superoxide dismutase isoenzymes in the normal and diseased
human cornea. Invest Ophthalmol Vis Sci. 2001;42(10):2293-6. 5. Kenney MC, Chwa M, Atilano SR, Tran A, Carballo M, Saghizadeh
M, et al. Increased levels of catalase and cathepsin V/L2 but de-
creased TIMP-1 in keratoconus corneas: evidence that oxidative
stress plays a role in this disorder. Invest Ophthalmol Vis Sci. 2005;46(3):823-32. 6. Atilano SR, Coskun P, Chwa M, Jordan N, Reddy V, Le K, et al. Accumulation of mitochondrial DNA damage in keratoconus
corneas. Invest Ophthalmol Vis Sci. 2005;46(4):1256-63. 7. Göncü T, Akal A, Adıbelli FM, Çakmak S, Sezen H, Yılmaz OF. Tear
Film and Serum Prolidase Activity and Oxidative Stress in Patients
With Keratoconus. Cornea. 2015;34(9):1019-23. No statistically significant association existed between
the rs1001179 polymorphism of the CAT gene, rs4880
polymorphism of the SOD2 gene, and KC risk. Many
studies on different diseases have reported both signi-
ficant and insignificant results for both SNPs(25-28). The
genotype distribution of these SNPs fluctuates between
populations(29,30). These discrepancies are probably due
to the limited number of subjects and population and/or
ethnicity differences. 8. Lubrano V, Balzan S. Enzymatic antioxidant system in vascular
inflammation and coronary artery disease. World J Exp Med. 2015;
5(4):218-24. 9. Abu-Amero KK, Kondkar AA, Mousa A, Osman EA, Al-Obeidan SA. Analysis of catalase SNP rs1001179 in Saudi patients with primary
open angle glaucoma. Ophthalmic Genet. 2013;34(4):223-8. 10. Liu C, Ogando D, Bonanno JA. SOD2 contributes to anti-oxidative
capacity in rabbit corneal endothelial cells. Mol Vis. 2011;17:
2473-81. 11. Moschos MM, Kokolakis N, Gazouli M, Chatziralli IP, Droutsas
D, Anagnou NP, et al. Polymorphism Analysis of VSX1 and SOD1
Genes in Greek Patients with Keratoconus. Ophthalmic Genet. 2015;36(3):213-7. As the genotype and allele frequency of both
polymorphisms were identical, it is assumed that the
rs1001179 polymorphism of the CAT gene and rs4880
polymorphism of the SOD2 gene are not involved in KC
pathogenesis. DISCUSSION Demographic characteristics of keratoconus patients with GPX1 (rs1050450), SOD2 (rs4880), and CAT (rs1001179) genotypes
GPX1 (rs1050450)
SOD2 (rs4880)
CAT (rs1001179)
CC
CT
TT
TT
TC
CC
CC
CT
TT
Sex
Male n
65
74
18
3
43
111
25
78
54
%
41.4%
47.1%
11.5%
1.9%
27.4%
70.7%
15.9%
49.7%
34.4%
Female n
26
23
9
1
18
39
12
22
24
%
44.8%
39.7%
15.5%
1.7%
31.0%
67.2%
20.7%
37.9%
41.4%
p
0.550
0.870
0.303
Age
<20 n
13
10
3
0
11
15
3
11
12
%
50.0%
38.5%
11.5%
0.0%
42.3%
57.7%
11.5%
42.3%
46.2%
20-29 n
34
45
11
1
23
66
16
39
35
%
37.8%
50.0%
12.2%
1.1%
25.6%
73.3%
17.8%
43.3%
38.9%
30-39 n
30
32
9
2
19
50
14
39
18
%
42.3%
45.1%
12.7%
2.8%
26.8%
70.4%
19.7%
54.9%
25.4%
40-40 n
12
9
3
1
6
17
3
10
11
%
50.0%
37.5%
12.5%
4.2%
25.0%
70.8%
12.5%
41.7%
45.8%
≥50 n
2
1
1
0
2
2
1
1
2
%
50.0%
25.0%
25.0%
0.0%
50.0%
50.0%
25.0%
25.0%
50.0%
P
0.927
0.686
0.523 504
Arq Bras Oftalmol. 2019;82(6):501-6 Ilhan A, et al. To the best of our knowledge, this is the first report
to investigate the potential associations between the
rs1050450, rs1001179, and rs4880 polymorphisms and
KC susceptibility. In conclusion, we confirmed a possi-
ble association of the GPX1 rs1050450 polymorphism
with KC in the Turkish population. However, we did not
observe a significant difference in the CAT rs1001179
and SOD2 rs4880 polymorphisms between the study
and control groups. Further studies with large cohorts
of multiple nationalities and ethnicities are needed to
clarify the association and significance of this metabolic
pathway. essential data(5). Finally, after extensively searching the
literature, we did not find a report on the role of SNPs
associated with these enzymes in KC pathogenesis. These factors motivated us to evaluate the possible asso-
ciation of these SNPs with KC patients of Turkish origin. In the present study, we evaluated the role of rs1050450
(GPX1), rs4880 (SOD2), and rs1001179 (CAT) polymor-
phisms in detecting KC susceptibility. We chose to assess
the most prominently researched polymorphisms that
were compatible with multiple disorders(16-18). We identified a significant association between the
rs1050450 polymorphism of GPX1 genotype and KC. GPX1 is involved in the decomposition H2O2 into water
and oxygen. DISCUSSION It is one of the most significant antioxidant
enzymes in higher vertebrates and humans and is pre-
sent in both cytoplasm and mitochondria. Brigelius-Flohé
et al. reported that GPX1 could not compensate for
other selenoproteins in the elimination of systemic oxi-
dative stress, making it a primary antioxidant enzyme
in vivo(19). GPX1 also accelerates the reduction of lipid
peroxides and hydrogen as well as peroxynitrite(20). The-
refore, GPX1 dysfunction may cause the accumulation
of peroxynitrite and lipid hydroperoxides. The extreme
amount of reactive oxygen and nitric species may da-
mage nucleic acids, membrane lipids, mitochondrial
DNA, and proteins. Various studies have reported that
this metabolite accumulation may induce apoptosis(21,22). Further, keratocyte apoptosis is seen in keratoconic
corneas(23,24). Based on these facts and our results, we
propose that GPX1 dysfunctions should be considered
in KC pathogenesis. REFERENCES Our study was conducted in the Turkish
population, and similar studies are needed in different
populations to precisely define the relationship between
these SNPs and KC. 12. Udar N, Atilano SR, Brown DJ, Holguin B, Small K, Nesburn AB,
et al. SOD1: a candidate gene for keratoconus. Invest Ophthalmol
Vis Sci. 2006;47(8):3345-51. 13. Chwa M, Atilano SR, Reddy V, Jordan N, Kim DW, Kenney MC. Increased stress-induced generation of reactive oxygen species and
apoptosis in human keratoconus fibroblasts. Invest Ophthalmol Vis
Sci. 2006;47(5):1902-10. Arq Bras Oftalmol. 2019;82(6):501-6 505 The association between genetic polymorphism of glutathione peroxidase 1 (rs1050450) and keratoconus in a Turkish population 22. Das UN. Essential fatty acids, lipid peroxidation and apoptosis. Prostaglandins Leukot Essent Fatty Acids. 1999;61(3):157-63. 14. Leema G, Muralidharan AR, Annadurai T, Kaliamurthy J, Geraldine
P, Thomas PA. Oxidative stress in experimental rodent corneas
infected with aflatoxigenic and nonaflatoxigenic Aspergillus flavus. Cornea. 2013;32(6):867-74. 23. Palamar M, Onay H, Ozdemir TR, Arslan E, Egrilmez S, Ozkinay
F, et al. Relationship between IL1β-511C>T and ILRN VNTR poly-
morphisms and keratoconus. Cornea. 2014;33(2):145-7. 15. Bastaki M, Huen K, Manzanillo P, Chande N, Chen C, Balmes JR,
et al. Genotype-activity relationship for Mn-superoxide dismutase,
glutathione peroxidase 1 and catalase in humans. Pharmacogenet
Genomics. 2006;16(4):279-86. 24. Wójcik KA, Błasiak J, Kurowska AK, Szaflik J, Szaflik JP. [Oxidative
stress in the pathogenesis of keratoconus]. Klin Oczna. 2013;
115(4):311-6. Review. Polish. 16. Sousa VC, Carmo RF, Vasconcelos LR, Aroucha DC, Pereira LM,
Moura P, et al. Association of Catalase and Glutathione Peroxidase
1 Polymorphisms with Chronic Hepatitis C Outcome. Ann Hum
Genet. 2016;80(3):145-53. 25. Rajić V, Aplenc R, Debeljak M, Prestor VV, Karas-Kuzelicki N,
Mlinaric-Rascan I, et al. Influence of the polymorphism in candi-
date genes on late cardiac damage in patients treated due to acute
leukemia in childhood. Leuk Lymphoma. 2009;50(10):1693-8. 17. Kidir V, Uz E, Yigit A, Altuntas A, Yigit B, Inal S, et al. Manganese
superoxide dismutase, glutathione peroxidase and catalase gene
polymorphisms and clinical outcomes in acute kidney injury. Ren
Fail. 2016;38(3):372-7. 26. Góth L, Nagy T, Kósa Z, Fejes Z, Bhattoa HP, Paragh G, et al. Effects
of rs769217 and rs1001179 polymorphisms of catalase gene on
blood catalase, carbohydrate and lipid biomarkers in diabetes
mellitus. Free Radic Res. 2012;46(10):1249-57. 18. Kakkoura MG, Demetriou CA, Loizidou MA, Loucaides G, Neo-
phytou I, Malas S,et al. 506
Arq Bras Oftalmol. 2019;82(6):501-6 REFERENCES MnSOD and CAT polymorphisms modulate
the effect of the Mediterranean diet on breast cancer risk among
Greek-Cypriot women. Eur J Nutr. 2016;55(4):1535-44. 27. Kondo N, Bessho H, Honda S, Negi A. SOD2 gene polymorphisms
in neovascular age-related macular degeneration and polypoidal
choroidal vasculopathy. Mol Vis. 2009;15:1819-26. 28. Méplan C, Hughes DJ, Pardini B, Naccarati A, Soucek P, Vodickova
L, et al. Genetic variants in selenoprotein genes increase risk of
colorectal cancer. Carcinogenesis. 2010;31(6):1074-9. 19. Brigelius-Flohé R, Maiorino M. Glutathione peroxidases. Biochim
Biophys Acta. 2013;1830(5):3289-303. Review. 29. Silig Y, Tas A. Manganese-superoxide dismutase (MnSOD) polymor-
phisms. Turk J Biochem. 2015;40(2):163-8. 20. Tan SM, Stefanovic N, Tan G, Wilkinson-Berka JL, de Haan JB. Lack
of the antioxidant glutathione peroxidase-1 (GPx1) exacerbates
retinopathy of prematurity in mice. Invest Ophthalmol Vis Sci. 2013;54(1):555-62. 30. Sutton A, Khoury H, Prip-Buus C, Cepanec C, Pessayre D, Degoul
F. The Ala16Val genetic dimorphism modulates the import of hu-
man manganese superoxide dismutase into rat liver mitochondria. Pharmacogenetics. 2003;13(3):145-57. 21. Yang WS, Stockwell BR. Ferroptosis: Death by Lipid Peroxidation. Trends Cell Biol. 2016;26(3):165-76. 6
Arq Bras Oftalmol. 2019;82(6):501-6 506
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A preliminary simulation-based qualitative study of healthcare students’ experiences of interprofessional primary care scenarios
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Advances in simulation
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cc-by
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A preliminary simulation-based qualitative study of healthcare students’ experiences of
interprofessional primary care scenarios A preliminary simulation-based qualitative study of healthcare students’ experiences of
interprofessional primary care scenarios Lunde, Lene; Moen, Anne; Jakobsen, Rune; Møller, Britta; Rosvold, Elin ; Brænd, Anja Lunde, Lene; Moen, Anne; Jakobsen, Rune; Møller, Britta; Rosvold, Elin ; Brænd, Anja Published in:
Advances in Simulation DOI (link to publication from Publisher):
10.1186/s41077-022-00204-5 Citation for published version (APA):
Lunde, L., Moen, A., Jakobsen, R., Møller, B., Rosvold, E., & Brænd, A. (2022). A preliminary simulation-based
qualitative study of healthcare students’ experiences of interprofessional primary care scenarios. Advances in
Simulation, 7(1), Article 9. https://doi.org/10.1186/s41077-022-00204-5 General rights
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Lunde, L., Moen, A., Jakobsen, R., Møller, B., Rosvold, E., & Brænd, A. (2022). A preliminary simulation-based
qualitative study of healthcare students’ experiences of interprofessional primary care scenarios. Advances in
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ely and investigate your claim. Lunde et al. Advances in Simulation (2022) 7:9
https://doi.org/10.1186/s41077-022-00204-5 Abstract Background: Introducing interprofessional education (IPE) in healthcare curricula can prepare students for health-
care practices that have become increasingly complex. The use of simulation is promoted to support IPE. This study
explores healthcare students’ experiences of participating in common, sub-acute patient scenarios that routinely
occur in clinical practice in primary care. More specifically, it looks at how sub-acute patient scenarios from primary
care can help develop interprofessional collaborative competence. Methods: Medical students (N = 10), master’s students in advanced geriatric nursing (N = 8) and bachelor’s students
in nursing (N = 9) participated in the simulations. The students were in their last or second-to-last year of education. We conducted five semi-structured focus group interviews with the participants’ directly after the simulation training
to elicit experiences related to the scenarios, the simulation and interprofessional collaboration. The transcripts were
analysed using systematic text condensation. To supplement the focus group interviews, the students also completed
the interprofessional collaborative competency attainment survey (ICCAS), which measures the students’ self-assessed
interprofessional competence. Results: Three main themes emerged from the analysis of the focus group interviews: realism, uncertainty and
reflection. The students emphasised the importance of authentic and recognisable scenarios. They said the vague
and unspecific patient symptoms created uncertainty in the situation, making it difficult to understand the patient’s
diagnosis. Despite that uncertainty, they described the experience as positive. Further, the students expressed that
the simulation increased their confidence in interprofessional collaboration and prepared them for future work. The
results from the ICCAS questionnaire showed that the students reported a subjective positive change in their inter-
professional competence after participating in the scenarios. Conclusions: This study showed that simulation-based IPE with sub-acute primary care scenarios contributes to
develop interprofessional collaborative competence in healthcare education. Sub-acute scenarios can supplement
the more common approaches with acute care scenarios and aid in developing the collaborative competence
required to work in healthcare teams. Keywords: Simulation, Interprofessional, Primary care, Healthcare students, Sub-acute scenarios, Focus group © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the
original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or
other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this
licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco
mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Background Interprofessional education (IPE) is a critical component
in healthcare curricula and can help prepare students
for healthcare practices that have become increasingly
complex [1, 2]. However, there is no widespread educa-
tional consensus on how to conduct IPE so that it better *Correspondence: lene.lunde@medisin.uio.no
1 Department of Nursing Science, Institute of Health and Society, Faculty
of Medicine, University of Oslo, Oslo, Norway
Full list of author information is available at the end of the article As a
result, healthcare education needs to find approaches
that expose students to interprofessional collaboration
(IPC).h The use of simulation provides learning experiences
where the students are placed in realistic and safe clini-
cal situations [6]. A growing body of research promotes
simulation as an educational strategy to support IPE in
healthcare education [7–10]. Most simulation-based
IPE experiences have focused on life-threatening, time-
critical acute-care scenarios [11–14]. While it is impor-
tant for healthcare students to learn and practice how to
respond to severe, acute care scenarios, everyday clinical
situations are rife with IPC. Shorter hospital stays and
an increased emphasis on home care and ageing in place
suggest that more patients with increasingly complex
needs will require treatment in a primary care setting
[15]. In contrast to most acute care algorithm-based sce-
narios, sub-acute patient scenarios in primary care pro-
vide the students with more time to solve a problem, but
the actual clinical situation may be more complex. Intro-
ducing simulation training of scenarios typical of primary
care can therefore contribute to the students’ learning
experiences of IPC. The simulations took place in a research laboratory at
the University of Oslo. The simulation units were set up
like rooms in nursing homes. The scenarios were cre-
ated in collaboration with primary care health profes-
sionals and comprised common medical conditions from
primary care: an older patient convalescing at a nursing
home following surgery for a hip fracture. The patient
then developed symptoms of either a urinary tract infec-
tion or pneumonia.h The students participated in both scenarios described
in Additional file 2 during the simulation-based training. Two scenarios were conducted during each simulation-
based training activity each preceded by a briefing and
immediately followed by a debriefing [20]. The briefing
provided an introduction to the simulation room, the
available (technical) equipment and the patient simulator
SimMan® by Laerdal Medical [21], as well as a reminder
about confidentiality and an introduction to the sce-
nario [22]. During the simulation, facilitators acted as
the patient’s voice and answered the questions directed
towards the patient. We instructed the students to act
according to their distinct professional roles and future
responsibilities. Each scenario lasted approximately 30
min. The debriefing took place directly after each sce-
nario and lasted on average 25 min [23]. With this in mind, we developed simulation-based IPE
with sub-acute patient scenarios that would commonly
occur in clinical practice. © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the
original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or
other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this
licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco
mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Lunde et al. Advances in Simulation (2022) 7:9 Page 2 of 12 Page 2 of 12 Lunde et al. Advances in Simulation IPE simulation we developed is not yet implemented in
our healthcare curricula. FG interviews were considered
well suited to elicit experiences and views from the par-
ticipants and encourage group dialogue after participat-
ing in a joint experience such as simulation-based IPE
[16]. A series of questions addressing experiences related
to the scenarios, the simulation and IPC acted as a guide
for the semi-structured interviews. The participants were
encouraged to elaborate on topics they considered rel-
evant and important (Additional file 1). In addition, to
supplement the FG interviews, the students completed
the Norwegian version of the interprofessional collabo-
rative competency attainment survey (ICCAS). ICCAS
captures the students’ self-assessment of their interpro-
fessional competence and is validated across various set-
tings and countries, including Norway [17–19].h prepares students to collaborate across healthcare disci-
plines. Traditionally, healthcare students are educated
in professional silos [3, 4]. As such, traditional teach-
ing does not promote students’ interactions with other
healthcare professions. It is a common assumption that
students’ exposure to, and involvement in, teamwork
occurs naturally in clinical practice and, consequently,
prepares the students for working in interprofessional
teams. However, there is no guarantee that without pur-
poseful organisation, students will experience exemplary
teamwork or even collaborate with other healthcare pro-
fessionals or students during clinical practice [5]. Participants We recruited medical students, master’s students in
advanced geriatric nursing and bachelor’s students in
nursing through purposeful sampling. Educational lead-
ers in universities in central Eastern Norway facilitated
the recruitment. The inclusion criteria were healthcare
students in the final semester of their last or second-to-
last year of education because they had completed most
of their clinical practice rotation and thus presumably
would have skills competence sufficient to be capable of
participating in IPC. Potential participants that met our The main aim of this article is
to explore healthcare students’ experiences of participat-
ing in the sub-acute patient scenarios. Specifically, we
aimed to understand how the use of sub-acute patient
scenarios from primary care could support the develop-
ment of interprofessional collaborative competence. Strategies to enhance trustworthiness in the analysish Strategies to enhance trustworthiness in the analysis
The authors are nurses (LL, AM), medical doctors (RBJ,
EOR, AMB) and an educator (BM). Collectively, our
experience combines primary care and medical educa-
tions, as well as research, teaching, curriculum planning,
workplace learning and simulation-based training. Our
backgrounds might have influenced our preunderstand-
ing of the simulation setting, the scenarios and the stu-
dents’ experiences. However, having co-authors with
different, yet complementary backgrounds might also
help in ensuring the legitimacy of our interpretations
[26]. By reporting the process of analysis and providing
examples of codes, construction of condensates, syn-
theses and themes in Table 2, we have brought a cer-
tain transparency to the process. Through the research
group’s collective reading and analysis, we have worked
to enhance the trustworthiness of the results [26]. i
The interviewers observed the simulations from behind
a one-way mirror in the control room and did not inter-
act with the students during the simulation. The FG
interviews were audio-recorded and were exported to a
secure data storage facility at the University of Oslo, then
transcribed verbatim by LL. Research design and setting We conducted a qualitative study, using focus group (FG)
interviews to capture experiences from students par-
ticipating in IPE simulation sessions. This is part of an
exploratory study exploring different aspects of simula-
tion as a strategy for training healthcare students in IPC
in future curricula development. We developed scenarios
comprised of sub-acute situations from primary care. The Page 3 of 12 Lunde et al. Advances in Simulation (2022) 7:9 discussed, modified themes, reviewed abstractions and
syntheses until reaching a consensus. inclusion criteria received information about the study
from contact persons at the different universities. The
lead author LL was also invited to several lectures to talk
about the project to recruit participants. A total of 27
healthcare students agreed to participate, ranging from
21 to 49 years of age (mean 31), and 21 were female and
six were male. All participants in the simulation train-
ing took part in the FG interviews. Table 1 presents the
details regarding the participants. We used the software NVivo12 to organise and struc-
ture the data. As the analysis progressed, we organised
the material into tables. Table 2 shows an example of the
analysis. The ICCAS questionnaires were analysed using IBM SPSS
Statistics Version 27. ICCAS comprised the interprofes-
sional competency communication, collaboration, patient-
and family-centred care, roles and responsibilities and
conflict management. Since prior validation studies recom-
mend analysing ICCAS at an overall level to address change
in interprofessional competence [17, 18], we used paired
t-test to determine the difference in perceived abilities in
the mean overall pre- and post-score (range 1–5). We ana-
lysed the differences in terms of Cohen d standardised effect
size (“large” = values of ≥ 0.8, “moderate” = values between
0.79 and 0.50 and “small” values between 0.2 and 0.49) and
95% confidence limits [25]. To maintain the anonymity of the participants, gender
and name were excluded from the transcripts, and abbre-
viations were used, as can be seen in Table 1. The partici-
pants were numbered in the order they appeared in the
interviews (e.g. NS1). The FG interviews were numbered
in the order they were conducted (e.g. FG1). Data analysis
Th The transcripts from the FG interviews were analysed
by systematic text condensation, in a four-step process
[24]. First, we read the transcripts independently to get
an overview and total impression and to identify prelimi-
nary themes. Secondly, we collaboratively identified and
sorted the meaning units into code groups. In the third
step, we abstracted condensates from each code group. Finally, we created synthesised descriptions by recon-
ceptualising the condensates and chose the quotes that
would best represent the synthesised description (golden
quotes). The initial steps were conducted by LL and AMB
independently (step 1) and in collaboration (step 2). Then, LL drafted the first versions of condensates (step
3) and synthetisation (step 4) and translated the quotes
into English. For each step of the analysis, the research
group read the material independently, collaboratively Resultsh Three main themes emerged from the analysis of the FG
interviews: realism, uncertainty and reflection. Within
uncertainty, the sub-themes “unspecific situations”, “time
to collaborate” and “room for communication” became
apparent. In reflection, the sub-themes “opportunities not
present in practice”, “developing confidence” and “better
prepared for the future” emerged. Data collection
d
d We conducted the FG interviews in April 2019, just
after the students had finished the simulations and com-
pleted the ICCAS questionnaire, to avoid conflicts with
study schedules. Each student was a member of one of
10 interprofessional teams during the simulations. Two
teams participated in the simulation each day, and they
joined the same FG, resulting in five FG interviews. The
lead interviewers were members of the research group
with experience in qualitative research and with doctoral
degrees in nursing (AM) and medicine (AMB, EOR). Each FG interview lasted between 60 and 90 min and had
five or six participants.h Unspecific situationh Although there was enough time to work on it, the
clinical problem itself was less clear-cut. The students
reported that they could not take any shortcuts because
the symptoms were so vague. They had to discuss what
they were unsure of and do a full clinical examination. The students expressed that in prior simulation experi-
ences, they were usually provided with predefined ways
of solving the problem, either through algorithms or
checklists. In these scenarios, however, they experienced
an ambiguous situation, where the right solution did not
clearly stand out. They described it as they knew something
was going on, but the unspecific clinical signs made the
situation difficult to grasp and therefore difficult to analyse. AGN3 (FG2): If it is a cardiac arrest, pretty much
everyone knows what to do, and you cooperate. But
when it is so vague, you get a discussion of everyone’s
knowledge, and it’s completely different. You get to
use each other’s competence in a completely differ-
ent way than if it was a very specific and dramatic
situation. AGN3 (FG2): Very often it starts with the fact that
you realise that there is something going on. Without
having anything specific, everything is a bit vague. That’s what it’s often like. In contrast, the students found that a simulation solv-
ing an acute care situation such as a cardiac arrest where
they follow a predefined algorithm was more rehearsed
and explicit as they would know what to do and how to
react. Sub-acute scenarios provided the students with
an opportunity to use each other’s competences in new
ways. The vagueness, the students said, consequently
led to another kind of insight of what the other students
knew and how they could contribute, as they had to share
their knowledge to expand on the problem. The simula-
tion seemed to contribute to increased understanding of
the competence the different educations provided, and
how they could complement each other. Thus, when they
combined their different perspectives, it helped reduce
the uncertainty. This indicated that adding different The realisation that the patient’s situation was chang-
ing encouraged the students to pay careful attention to
the vague and undefinable signs that were found in the
clinical examination. The students explained that espe-
cially with elderly patients, the clinical signs might not
be as apparent or lead to textbook solutions. Realism of the scenarioh The students recognised the scenarios as realistic,
authentic and likely to be encountered in healthcare and,
specifically, in primary care. MS9 (FG5): I especially think about the fact that it MS9 (FG5): I especially think about the fact that it Page 4 of 12 Lunde et al. Advances in Simulation (2022) 7:9 Lunde et al. Advances in Simulation (2022) 7:9 was so relevant. The topics were important, and the
situations ones that you would often experience. The students expressed that they were unaccustomed
to these assessments, especially because there was no
quick fix or easy solution. However, the students per-
ceived this experience as positive because, in nursing
homes, and primary care in general, they would often
experience vague clinical situations. As such, it seemed
that the scenarios were recognised as important learn-
ing activities to prepare for real-life situations. The use of
sub-acute scenarios shifted their focus to the inherently
complex health services that are provided in primary
care on a daily basis and appeared to renew the students’
understanding of the many different challenges that can
occur. Furthermore, the students described the nursing home
setting as recognisable and representative. They noted
that to make the simulations authentic, you needed to
have such surroundings. The students seemed to manage
to conceptualise the scenario in a clearer way based on
the information provided and the environmental set-up. AGN6 (FG4): And I also think that it was very good
that we were told immediately that this is a nurs-
ing home and this is the available equipment in the
nursing home, and the doctor is present one day a
week. This made it realistic. Time to collaborateh As such, the students emphasised that it was important
to have authentic, recognisable scenarios and that having
the setting and equipment described beforehand allowed
them to better envision the scenario. Together, these
statements illustrated that including information about
setting, surroundings and available equipment as well as
scenario description in the pre-briefing was important to
prepare the students for the simulation. The students emphasised that having an adequate time
to practice scenarios together in a calm setting offered
the opportunity to ask additional questions, listen to
one another and engage in group discussions to solve
problems. MS4 (FG2): When you have so much time and it is
quite calm, you have the opportunity to listen, and
to ask, “What do you think? Is there anything we
have not thought of?” Room for communication Based on their prior experience with simulation, the stu-
dents said they expected an extraordinary situation even
though they were prepared for sub-acute scenarios. The
fact that the clinical condition in the scenario did not
overwhelm them was highlighted as positive. Thus, it was
possible to focus on the team’s interactions and commu-
nication, which they deemed important. The students explained that it was not always possible
to take an active part in collaboration in clinical prac-
tice, and the possibility to interact with other students or
healthcare personnel could be limited or non-existent. In clinical practice, they experienced that there was lit-
tle time given over to reflect together with others. This
appeared to illustrate that profession-specific learning
goals in clinical practice are still the most common and
that interprofessional activities where the students have
time to reflect with others are scarce. MS5 (FG3): When it’s not medically precarious and
acute, you get a little more time to actually commu-
nicate. And that’s what’s most important. The students appreciated that the clinical condition did
not decline rapidly, as it gave them more time to react
and collaborate. They pointed out that in a medically
complicated scenario, you could just as well end up with
a situation where one team member dominates. NS9 (FG5): We know that, in practice, we can call
the priest, social worker, or nutritionist and get them
up there and then talk to them. But you may not
know how you would collaborate with them in that
meeting. You are doing that in here. What we do
here is very important in shedding light on how we
should collaborate. NS9 (FG5): We know that, in practice, we can call
the priest, social worker, or nutritionist and get them
up there and then talk to them. But you may not
know how you would collaborate with them in that
meeting. You are doing that in here. What we do
here is very important in shedding light on how we
should collaborate. NS10 (FG5): If it gets too complicated and there’s a
dispute between the professions, and the one who
speaks loudest overrides the rest of the group. Some
just cave in and heed to the one who has the strong-
est opinions. As the students pointed out, complicated cases could
negatively affect the communication and collaboration. Unspecific situationh The presen-
tation of vague and unspecific symptoms made the stu-
dents think more broadly in their clinical assessments, as
a symptom could be interpreted in several ways. Conse-
quently, they were less certain of the patient’s diagnosis. MS6 (FG3): Because there were vague symptoms,
you had to think a bit more broadly. You think there
can always be something more to it. And, that this
kind of assessment feels a little unfamiliar. Page 5 of 12 Lunde et al. Advances in Simulation (2022) 7:9 Lunde et al. Advances in Simulation professional perspectives enhanced the joint discussion
and thus increased their learning outcome. awareness and understanding of the situation together
without merely pointing out what went wrong. MS10 (FG5): I absolutely believe that training in
controlled settings where you get time to reflect after-
wards has great value that is difficult to include in
practice. Because in practice, you are dependent on
a supervisor taking time to include reflection and a
department with suitable conditions for reflection
with others MS10 (FG5): I absolutely believe that training in
controlled settings where you get time to reflect after-
wards has great value that is difficult to include in
practice. Because in practice, you are dependent on
a supervisor taking time to include reflection and a
department with suitable conditions for reflection
with others Room for communication By sharing experiences and reflecting together, the stu-
dents indicated that they got to know the competences of
the other healthcare professions first hand. This was per-
ceived as important for managing collaboration. The stu-
dents described the simulation setting as a good way to
become more aware of the roles and responsibility they
would assume in their future work life. It also gave room
to reflect on how to collaboratively solve problems, not
just on the idea that collaboration was necessary. Expe-
riencing the benefits of IPC may also lead to enhanced
respect for each other’s profession. As such, the students
voiced the importance of participating in training that
enhances the quality of IPC. Opportunities not present in practice Several students talked about the simulation as being
similar to practice and yet not so, especially regarding
time to reflect during the simulation and in the debrief-
ing. They highlighted that in these scenarios, they had
time to talk through the clinical picture of the patient
together and really listen to each other. In real-life prac-
tice settings, they said it might be busy and chaotic,
and opportunities for reflecting together and share pro-
fession-specific knowledge about the patient were less
available. AGN3 (FG2): I learned a lot from seeing what
the others reflected on. Here you do the reflection
together. You see what the different students see;
there is not always room for that when you work. Developing confidence
Th
d
id
h The students said that participating in the simulation
made them more aware of themselves for better or worse,
in terms of how they behaved and dealt with situations. They described the experience as discovering themselves
in a new way. Consequently, the experience appeared to
develop their confidence to engage more actively in IPC. The students also emphasised that having the oppor-
tunity to sit down together in the debriefing and reflect
on what they did enhanced the learning outcome. In the
debriefing, the students appreciated the possibility to talk
about how they communicated and collaborated in the
simulation sessions individually and as a team. They par-
ticularly pointed out that they valued the focus on raising AGN4 (FG3): With simulation, I see that if I can talk
to the medical student, then maybe I can talk to a
real doctor. You see proof that it’s actually possible to AGN4 (FG3): With simulation, I see that if I can talk
to the medical student, then maybe I can talk to a
real doctor. You see proof that it’s actually possible to Page 6 of 12 Lunde et al. Advances in Simulation (2022) 7:9 Discussioni In the findings reported here, the students emphasised
the importance of authentic and recognisable scenarios. They described that the vague and unspecific symptoms
in the scenarios created an uncertain situation where
it was difficult to find a clear direction. The students
repeatedly emphasised, however, that this experience
was positive. They acknowledged, with some surprise,
the complexity the sub-acute scenarios presented and the
opportunity that arose for them to focus on collabora-
tion and communication. Further, the students reported
increased confidence and preparedness for future work. Our results from ICCAS also supported that partici-
pating in the scenarios led to a positive change in self-
assessed interprofessional competence. Furthermore, we
discuss the potential for the sub-acute scenarios to pro-
mote interprofessional collaborative learning opportuni-
ties for healthcare students. Self‑reported interprofessional competence In addition to the material from the FG interviews, all 27
participants completed the ICCAS questionnaire. The
results from the ICCAS questionnaire showed that after
participating in the scenarios, the students reported a
positive change in self-assessed interprofessional com-
petence. There was a statistically significant increase in
the mean sum score from pre-scores (mean = 3.64, SD =
0.65) to post-scores (mean = 4.4, SD = 0.3), t (26) = 6.67,
p < .001 (two-tailed). The mean difference, 0.76, 95% CI
[0.53, 0.99], represented a large effect of d = 1.29. NS5 (FG3): I learned today that I don’t have to be
afraid. If I have some knowledge or something that
I think of, with the patient in mind, I will just say it. NS5 (FG3): I learned today that I don’t have to be
afraid. If I have some knowledge or something that
I think of, with the patient in mind, I will just say it. The students explained that when they discussed
together, they realised that they had an important role to
play. Thus, the joint problem-solving activities the sce-
narios provided seemed to increase their experience of
themselves as important contributors to the interprofes-
sional discussion. Consequently, the simulation experi-
ence led to newfound confidence in the students’ abilities
to participate and voice their opinions. This confidence
appeared to reassure the students in their own role as
healthcare professionals. When reassured in their own
role, they managed to benefit from the others’ compe-
tence and mutually create joint knowledge. talk to other professional groups. education. Consequently, this type of scenarios could
prepare the students for future IPC. The students found that as the simulation progressed,
they got more comfortable with expressing their opinion
with the team, which made it possible to have a clinical
conversation across professions. Solving the scenario,
they explained, provided an opportunity for participating
in discussions in a safe environment as equals. The crea-
tion of a safe environment allowed the students to dare to
present their perspectives and express their opinions. Better prepared for the futureh The students indicated that the experiences from the sce-
narios would be long lasting because the simulation cre-
ated practical memories they could recall later. MS5 (FG3): It’s the kind of experience that you can
come back to and reflect on. You can call on it in dif-
ferent settings and think, “Oh, yes, we did this that
time.” Collaborative problem solving in a realistic setting
A
fi d
f
h
d
h Collaborative problem solving in a realistic setting
An important finding from this study was the students’
positive response to the sub-acute scenarios, especially
their seeing scenarios as authentic and realistic learn-
ing situations. The recognisable scenarios, together with
information about the setting and available equipment,
were important factors in getting students to engage in
the simulation. Considering the simulation activity as a
social practice where learning is constructed in interac-
tion between the participants, environment and equip-
ment, it highlights the importance of pre-briefing to
create a safe and recognisable environment for the stu-
dents to interact in [22, 27, 28]. Thus, they seemed to
manage to utilise the resources available in the room
and frame the simulated situations into something
manageable.hi The students said that taking part in the simulations
would help them deal with similar situations in the
future. Facing such issues in a safe environment dur-
ing education gave the students a sense of assurance for
future work. NS6 (FG4): If you could act through it in advance
and be trained beforehand, you can handle it better
later, in terms of how to talk to each other. Thus, the students reported that interprofessional col-
laboration could become something familiar and man-
ageable because of prior training. Participating in the
scenarios seemed to provide the students with a clearer
frame of reference for problem-solving in future situ-
ations. Having useful experiences to refer could pro-
vide security since they had faced such issues during This supports the findings showing that IPE has to be
meaningful and relevant, with authentic activities, to be
able to support interprofessional learning [4, 29]. Further, Page 7 of 12 Lunde et al. Advances in Simulation (2022) 7:9 for a learning experience to be of value and to prepare the
students for future teamwork, structured opportunities
for active engagement need to be made available [11, 30]. Thus, IPC experiences involving engagement and oppor-
tunities to interact, rather than passive observation of
teamwork, are found to have more impact on interpro-
fessional learning and competence development [31–33].hi in the simulations. For students to be prepared for the
expected collaboration, educators have to create spaces
to train for IPC in healthcare education [15]. Learning opportunitiesh Through IPE-based simulation training of sub-acute
situations, this study shows that the following learning
potentials can be realised: establishing greater confidence
in handling uncertain, sub-acute situations through IPC,
understanding their own and others’ perspectives and
competencies and strengthened confidence in their own
IPC competencies and contributions for future work. These practice spaces for IPC emerge during the joint
examination of the clinical situation and is strengthened
through reflection.l l
Reflecting on the simulation experience, especially the
debriefing, is seen as a cornerstone in simulation-based
training for students to reconstruct their experience into
learning [27]. There are several ways of facilitating sce-
nario debriefing [41, 42], making it important for educa-
tors to make well-considered choice of debriefing strategy
beforehand. In this study, the facilitators were instructed
to follow the debriefing framework proposed by Rudolph
et al. [23] where the focus is enhancing awareness and
understanding of the situation. The framework highlights
creating a safe learning environment where the students
feel comfortable discussing successes and failures to
understand and learn of their actions. The students in our
study appreciated that the facilitators did not solely focus
on what went wrong, but prompted questions, thoughts
and opinions that engaged the students to contribute
actively with their own reflections and perspectives on
collaboration and communication. i
When developing scenarios for simulation-based train-
ing, careful consideration of the level of difficulty and
complexity is necessary to optimise the learning oppor-
tunities [27, 28, 36]. It is important to take into account
that the students participating in the scenarios are there
to train on competence they have not yet fully acquired
[37]. Thus, a mismatch between the difficulty and com-
plexity of the scenario and the students’ capacity to make
sense of the scenario could compromise the learning
opportunities. As the students explained, complicated
cases can breed poor communication, as one team mem-
ber may dominate. As such, scenarios where the patient’s
condition is stable seem to provide students with more
time and opportunity to emphasise team collaboration
[38, 39].h In our study, the realistic but vague and unspecific
signs and symptoms in the scenarios without a clear
conclusion created uncertainty that challenged the stu-
dents’ competence, their role understanding and task
sharing. However, the uncertainty also mobilised their
resources as they resolved the uncertainty by communi-
cation and joint reflection in the simulation and during
the debriefing. Collaborative problem solving in a realistic setting
A
fi d
f
h
d
h The founda-
tion for fruitful learning spaces have to be laid in the pre-
briefing to get the students to engage in the simulation
and interact with the participants, scenario and environ-
ment [22]. Without these spaces, it is difficult for health-
care students to get to know one another and find ways of
working together [40]. The practice space for IPC in the
sub-acute scenarios seems to provide the opportunity for
healthcare students to explore one another’s perspectives
and use one another’s competencies interprofessionally. The unspecific symptoms presented in the scenarios
created an uncertain situation for the students, where
the patient’s problem or diagnosis was unclear. As such,
the sub-acute scenarios exposed the students to the
complexity often presented by this patient group, where
accurate diagnosis can be difficult due to atypical symp-
toms [34]. Since there was no detailed algorithm to fol-
low, the outcome depended on the students’ capacity to
discuss, identify signs and symptoms and use relevant
knowledge to solve the patients’ main concerns. Students
who actively share information, discuss and draw on one
another’s resources and competencies seem to manage
defining the patients’ concerns and prepare for future
care in collaboration [35]. In our study, the students high-
lighted that the relaxed pace of the scenarios, combined
with a reasonable amount of time to complete them,
made it possible to focus on the interactions and commu-
nication within the team, to ask each other questions and
discuss and reflect together without being overwhelmed. When students recognise the simulation-based activity as
a safe environment, it can motivate them to perform at
the edge of their expertise [22], which might enable them
to expand on the learning activity and enhance their
knowledge. In our scenarios, the students recognised the
setting as a safe environment, which made them willing
to ask questions, listen to reflections from others and
contemplate on the best way forward together, although
it might highlight skills deficiencies. Learning opportunitiesh As such, the development of IPC com-
petence took place both during the scenario and in the
debriefing. The quality of the debriefing seems as impor-
tant for the development of IPC competence as the The students’ experiences of a collaborative learning
potential in simulation seemed to come from the combi-
nation of a realistic scenario and a practice space for IPC Lunde et al. Advances in Simulation (2022) 7:9 Page 8 of 12 Page 8 of 12 quality of the scenario since the debriefing is where the
participants shift their perspective from the action to
the reflection on actions and common experiences from
the scenario [23]. This study suggests that the scenarios
allowed for discussion and joint reflection and that the
simulation training may lead to enhanced understand-
ing of one another’s sense of competence and scope of
practice. Most especially, the simulation provided an
opportunity for equal discussions in a safe environment. This supports the findings suggesting that feeling safe
in a learning situation fosters confidence in one’s role
and willingness to participate in a team [8, 9, 12, 14]. Moreover, our results may indicate that the scenarios
provided safe ways of developing interprofessional col-
laborative competence where different perspectives
are valued. Unequal power relations and hierarchical
structures are seen as barriers for learning [43, 44]. We
highlighted that everyone’s knowledge and perspective
were necessary to solve the problem which seemed to
promote a non-hierarchical learning environment and
strengthen the students’ confidence in their interprofes-
sional competence. which means that the necessary level of realism should
be evaluated to create the required learning environ-
ment [28]. In our study, we have shown that the stu-
dents valued the authentic and realistic scenarios. Although the simulation was conducted in a simula-
tion centre with a SimMan as the older nursing home
patient, the student perceived the situation as realistic
due to the authentic scenario description, convincing
access to equipment, presentation of vague clinical
signs and credible information provided in the medical
record. This highlights that to create a realistic simu-
lation experience—or the right amount of fidelity—it
has to contain physical elements but also situations the
students manage to make sense of and experience as
relevant [28, 39]. Learning opportunitiesh In our study, albeit the fact that not
everything was identical to practice, the abovemen-
tioned factors contributed to create a context where
the students experienced a sense of recognisability
and, thus, engaged in the scenarios. Systematic IPE could be an advantage for future team-
work, as the students explained that having experienced
IPC, they felt prepared to contribute in future IPC situ-
ations. The positive change in the students’ self-reported
competence score supported that participating in the
scenarios prepared the students for collaborative prac-
tice. Other studies have also found that IPC training
develops competence and enhances the ability to engage
in future interprofessional teamwork in clinical practice
[33]. Exposing healthcare students to IPE during educa-
tion can result in more graduates with IPC competence,
which in turn can promote a positive change towards fur-
ther interprofessional collaborative healthcare practice. Thus, the IPC learning outcomes the students achieved
in these scenarios could be transferable to other settings
and situations. The students said that though similar to clinical
practice, the simulated setting was also different. The
joint discussions and reflections about the patients’
clinical picture they experienced during the simula-
tion session were not usually encountered in clinical
practice or work, neither was the structured debrief-
ing. This might be seen as an educational paradox, in
which students participate in IPE to prepare for future
interprofessional practice that rarely takes place. Thus, it can be challenging to prepare students with
IPC competencies if they do not find opportunities to
practice in clinical work. Consequently, those students
might not consider IPC as important in real-life clini-
cal work [5]. At the same time, education institutions
have a responsibility to include high-quality IPE, and
thereby contribute to the quality of IPC in the future. Otherwise, newly graduated students risk entering
their professions without the interprofessional col-
laborative competence needed to work efficiently in
future healthcare teams [2]. Strengths and limitations A strength of this study is that it expands simulation-
based IPE as a strategy to prepare healthcare students
for future IPC and shows the potential of adding sim-
ulations of sub-acute primary care scenarios to IPE. We acknowledge that the participating students might
be more positive about simulation and IPC than other
students might. Reasons for non-participation were,
however, mainly the lack of time and not getting time
off from work or clinical practice. The simulations
and FG interviews were conducted in 1 day to facili-
tate participation and avoid study schedule conflicts. We do not know if the students would have shared the
same viewpoints in the FG interview had they had time
to process the experience over a longer period. How-
ever, FG interviews provided us with detailed and rich
descriptions of the students’ immediate experiences. The simulation-based experience offered a frame of
reference for future problem solving. Thus, it seems
that the realistic setting not only enhances learning,
but also makes it more transferable for future situ-
ations, confirming existing research which suggests
that authentic, interactive and competence-building
IPE experiences create lasting impressions [29, 30]. However, it is important to highlight that realism—or
fidelity—does not mean that everything must be as
found in practice, without exception. Simulation fidel-
ity relates to the educational value of the simulation Page 9 of 12 Lunde et al. Advances in Simulation (2022) 7:9 Lunde et al. Advances in Simulation Fig. 1 Simulation set-up and available equipment Fig. 1 Simulation set-up and available equipment Fig. 1 Simulation set-up and available equipment The students have not had the opportunity to comment
on our interpretations, and we acknowledge that their
interpretations or explanations of the transcripts may
differ from ours. Although research promotes the use
of simulation to support IPE, there are few studies with
sub-acute scenarios from primary care. Thus, our study
contributes to a new perspective on how to facilitate
for IPE in healthcare education. These scenarios seem
to be feasible for implementation in healthcare educa-
tion. Adding observers with specific tasks related to
observation of the simulation activity could be one way
to scale up to accommodate real student numbers and
consequently avoid inactive participants in the scenar-
ios. Step 4 Step 4 Acknowledgements g
We wish to thank all the medical students, master’s students in advanced geri-
atric nursing and bachelor’s students in nursing who participated in this study. We also wish to thank Ragnhild Hellesø and Astri Letnes Janson for assisting in
the focus group interviews. Abbreviations IPE: Interprofessional education; IPC: Interprofessional collaboration; MS:
Medical student; AGN: Master’s students in advanced geriatric nursing; NS:
Bachelor’s students in nursing; FG: Focus group. Conclusionsh The present study shows that simulation-based IPE with
sub-acute primary care scenarios in healthcare educa-
tion contributes to the development of the collaborative
competence. The students valued the authentic scenarios
and expressed that solving the scenarios increased their
competence in IPC and prepared them for future work. The sub-acute scenarios, although complex in relation to
the unspecific and vague symptoms, promoted collabo-
rative learning opportunities for the students due to the
authenticity and sufficient time to discuss and reflect. Introducing simulation-based IPE with a focus on pri-
mary care scenarios can supplement more common acute
care simulation approaches for developing the collabora-
tive competence required to work in healthcare teams. Additional file 1. Additional file 2. Supplementary Information The online version contains supplementary material available at https://doi.
org/10.1186/s41077-022-00204-5. Authors’ contributions LL, AMB, AM, EOR and RBJ contributed to the study conception, design, mate-
rial preparation and data collection. All authors contributed to the analysis. LL wrote the first draft of the manuscript, and all authors contributed to the
subsequent versions and the final manuscript. The authors read and approved
the final manuscript. Step 3 Construction of artificial quotations
(condensates) summarising several
meaning units (first person) Identification and coding of mean-
ing units (first person) Construction of artificial quotations
(condensates) summarising several
meaning units (first person) Syntheses of contents into main
themes and sub-categories
Choice of golden quotes (third
person) Very realistic and similar to practice MS1 (FG1): “I am in nursing home
practice now and had my first day
yesterday. This could have been yes-
terday! And it could be tomorrow.”
NS2 (FG2): “I think they were really
good cases. Because it’s the type of
patient you would actually meet.”
MS9 (FG5): “I especially think about
the fact that it was so relevant. The
topics were important, and the situ-
ations ones that you would often
experience.” MS1 (FG1): “I am in nursing home
practice now and had my first day
yesterday. This could have been yes-
terday! And it could be tomorrow.”
NS2 (FG2): “I think they were really
good cases. Because it’s the type of
patient you would actually meet.”
MS9 (FG5): “I especially think about
the fact that it was so relevant. The
topics were important, and the situ-
ations ones that you would often
experience.” I am in practice at a nursing home
and this could have been yesterday,
or tomorrow. It felt very realistic
and relevant for primary care. This is
also the kind of patient you would
typically meet in healthcare. We
were told immediately that this is a
nursing home and what equipment
we had access to. Having been in
a nursing home, the resources and
their availability felt realistic. You
need to have surroundings that
feel realistic to make the simulation
believable. Main theme: realism
Sub category: recognition of realistic
scenario and setting Strengths and limitations Advances in Simulation Table 2 Example of analysis
Step 1
Step 2
Step
General impression and identifica-
tion of preliminary themes
Identification and coding of mean-
ing units (first person)
Cons
(con
mea Table 2 Example of analysis Main theme: realism
Sub category: recognition of realistic
scenario and setting Main theme: realism
Sub category: recognition of realistic
scenario and setting The students recognised the
scenarios as realistic, the situations
as authentic situations and ones
that they would likely encounter in
healthcare, and specifically in primary
care. The students also described
the setting in a nursing home as
recognisable and realistic. They high-
lighted the necessity to have realistic
surroundings that would make the
simulation authentic. AGN6 (FG4): “And I also think that
it was very good that we were told
immediately that this is a nurs-
ing home and this is the available
equipment in the nursing home,
and that the doctor is present one
day a week. This made it realistic.”
MS7 (FG4): “You need those sur-
roundings to make it is as believable
as possible.” Golden quotes: Golden quotes: MS9 (FG5): “I especially think about
the fact that it was so relevant. The topics were important, and the
situations ones that you would often
experience.” AGN6 (FG4): “And I also think that
it was very good that we were told
immediately that this is a nursing
home and this is the available equip-
ment in the nursing home, and that
the doctor is present one day a week. This made it realistic.” another scenario. The scenarios also have potential to
be expanded to include other healthcare professions,
which would have been an interesting opportunity for
further study. Abbreviations Strengths and limitations Then, the students could take turn in taking part
in a scenario and observing their peers taking part in The students have not had the opportunity to comment
on our interpretations, and we acknowledge that their
interpretations or explanations of the transcripts may
differ from ours. Although research promotes the use
of simulation to support IPE, there are few studies with
sub-acute scenarios from primary care. Thus, our study
contributes to a new perspective on how to facilitate
for IPE in healthcare education. These scenarios seem
to be feasible for implementation in healthcare educa-
tion. Adding observers with specific tasks related to
observation of the simulation activity could be one way
to scale up to accommodate real student numbers and
consequently avoid inactive participants in the scenar-
ios. Then, the students could take turn in taking part
in a scenario and observing their peers taking part in An interesting follow-up study could be to investigate
how the students experienced the simulation after
having entered healthcare as healthcare professionals. Although ICCAS added the students’ individual and
anonymous assessment of their own competence, we
have too small a sample size to evaluate the effect of the
sub-acute scenarios. We performed the FG interviews
with the whole interprofessional group and not divided
by professions. This might have inhibited some partici-
pants to speak freely, since they might be influenced by
how they think they are expected to act in their future
professional roles. However, since the students were
in these groups for 1 day only, we consider it unlikely
that this was a major problem. The analysis was based
on the researchers’ interpretations of the transcripts. Table 1 Description of the participants
a The AGN students have a minimum of 2 years of clinical experience as staff nurses before entering into the master’s programme
N (%)
Prior participation in simulation
Prior participation in
interprofessional simulation
Yes, N (%)
No, N (%)
Yes, N (%)
No, N (%)
Total
27 (100)
22 (82)
5 (18)
7 (26)
20 (74)
Medical students (MS)
10 (37)
8 (80)
2 (20)
2 (20)
8 (80)
Master’s students in adv. geriatric nursing
(AGN)a
8 (30)
6 (75)
2 (25)
2 (25)
6 (75)
Bachelor’s students in nursing (NS)
9 (33)
8 (88.9)
1 (11.1)
3 (33.3)
6 (66.7) Page 10 of 12 Lunde et al. Advances in Simulation (2022) 7:9 Lunde et al. Very realistic and similar to practice Funding
Th F
l g
The Faculty of Medicine, University of Oslo, funded this study. Page 11 of 12 Lunde et al. Advances in Simulation (2022) 7:9 (2022) 7:9 Lunde et al. Advances in Simulation Consent for publication The students and facilitator in Fig. 1 have consented to the use of the picture
in this article. y
p
15. Thistlethwaite J. Interprofessional education: a review of context, learning
and the research agenda. Med Educ. 2012;46(1):58–70. y
15. Thistlethwaite J. Interprofessional education: a review of c
and the research agenda. Med Educ. 2012;46(1):58–70. 16. Malterud K. Fokusgrupper som forskningsmetode for medisin og
helsefag [Focus groups as a research method for medicine and health
sciences]. Oslo: Universitetsforlaget; 2012. 16. Malterud K. Fokusgrupper som forskningsmetode for medisin og
helsefag [Focus groups as a research method for medicine and health
sciences]. Oslo: Universitetsforlaget; 2012. Ethics approval and consent to participate 13. Akselbo I, Killingberg H, Aune I. Simulation as a pedagogical learning
method for critical paediatric nursing in Bachelor of Nursing pro-
grammes: a qualitative study. Adv Simul. 2020;5(1):24. 13. Akselbo I, Killingberg H, Aune I. Simulation as a pedagogical learning
method for critical paediatric nursing in Bachelor of Nursing pro-
grammes: a qualitative study. Adv Simul. 2020;5(1):24. The Norwegian Centre for Research Data approved the study (project number
60867). We obtained written, informed consent from the participants after
providing oral and written information. 14. Costello M, Prelack K, Faller J, Huddleston J, Adly S, Doolin J. Student
experiences of interprofessional simulation: findings from a qualitative
study. J Interprof Care. 2018;32(1):95–7. Received: 17 May 2021 Accepted: 6 March 2022 Received: 17 May 2021 Accepted: 6 March 2022 Received: 17 May 2021 Accepted: 6 March 2022 20. INACSL Standards Committee, Watts PI, McDermott DS, Alinier G,
Charnetski M, Ludlow J, et al. Healthcare Simulation Standards of Best
Practice™: simulation design. Clin Simul Nurs. 2021;58:14–21. 21. Laerdal Medical. SimMan 3G Plus Stavanger: Norway; 2020. Avail-
able from: https://www.laerdal.com/no/doc/86/SimMan. [cited 2020
November 5] Availability of data and materials respiratory therapy students: a mixed-method research study. Nurse Educ
Today. 2021;99:104816. respiratory therapy students: a mixed-method research study. Nurse Educ
Today. 2021;99:104816. The datasets generated and/or analysed during the current study are not
publicly available due to the data corpus still being subject to analysis but are
available from the corresponding author on reasonable request. Additional
file 1: Interview guide; Additional file 2: Scenario description. y
11. Nichols A, Wiley S, Morrell BLM, Jochum JE, Moore ES, Carmack JN, et al. Interprofessional healthcare students’ perceptions of a simulation-based
learning experience. J Allied Health. 2019;48(3):159–66. 11. Nichols A, Wiley S, Morrell BLM, Jochum JE, Moore ES, Carmack JN, et al. Interprofessional healthcare students’ perceptions of a simulation-based
learning experience. J Allied Health. 2019;48(3):159–66. 12. Washington VL, Zakrajsek A, Myler L, Seurynck K, Holt S, Scazzero J. Blending interprofessional education and simulation learning: a mixed-
methods study of an interprofessional learning experience with nursing
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methods study of an interprofessional learning experience with nursing
and occupational therapy students. J Interprof Care. 2021:1–6. The authors declare that they have no competing interests. 17. Schmitz CC, Radosevich DM, Jardine P, Macdonald CJ, Trumpower D,
Archibald D. The interprofessional collaborative competency attain-
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https://openalex.org/W2776198988
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https://childshealth.zaslavsky.com.ua/index.php/journal/article/download/739/931
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Ukrainian
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Особливості діагностики захворювань органів дихання у дітей на сучасному етапі
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Zdorovʹe rebenka
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Êë³í³÷íà ïåä³àòð³ÿ / Clinical Pediatrics Êë³í³÷íà ïåä³àòð³ÿ / Clinical Pediatrics УДК 616.2-07-053.2
ÄÓÊÀ Ê.Ä., ªÔÀÍÎÂÀ À.Î. ÄÇ «Äí³ïðîïåòðîâñüêà ìåäè÷íà àêàäåì³ÿ ÌÎÇ Óêðà¿íè» Âñòóï тологію, є: інтоксикація, лихоманка, кашель, утруд-
нення дихання, ціаноз і типові фізикальні дані над
легенями. Для ГРЗ провідним є температура тіла <
38 °С з тенденцією до її нормалізації протягом трьох
днів. При пневмонії температура тіла > 38 °С із три-
валістю понад 3 дні (виняток — атипові форми в
перші місяці життя), тоді як при бронхітах зазвичай
температура тіла буває < 38 °С або вона протягом
1–3 днів знижується до цього рівня [1, 4]. Захворювання органів дихання залишаються
актуальною медико-соціальною проблемою, що
пов’язано з їх високою частотою, складністю діа-
гностики, ризиком формування хронічної респіра-
торної патології, що призводить до інвалідизації. Захворювання органів дихання мають доміную-
че положення в структурі захворюваності дітей. Так,
частота бронхітів коливається в межах 7,5–25 %,
пневмоній — 5–12 % [6]. У нозологічній структурі
гострих бронхітів у дітей на обструктивні бронхіти
припадає близько 20–25 %, частіше такий варіант
перебігу бронхіту реєструється у дітей раннього
віку, що певною мірою обумовлено анатомо-фізіо-
логічними особливостями, факторами преморбід-
ного фону, станом імунної системи та характером
відповіді макроорганізму [2, 5]. Перше завдання в діагностиці — виявити у ди-
тини з ознаками ГРЗ ураження нижніх дихальних
шляхів, для яких характерною є наявність хоча б од-
нієї з таких ознак: задишка з утрудненням (обструк-
цією) дихання, скорочення перкуторного звуку, на-
явність вологих хрипів різного калібру. Друге завдання — відрізнити пневмонію від
бронхіту, переважно вірусного ураження верхніх
дихальних шляхів. Важливий симптом пневмонії —
збільшення частоти дихання, що спостерігається
тим частіше, чим більше площа ураження легень і
чим менший вік дитини. Проте ця ознака значуща
тільки за відсутності симптомів обструкції, що ха- За офіційними даними, в Україні за останні 10
років питома вага дітей, які часто хворіють на гострі
респіраторні захворювання (ГРЗ), зросла до 50–
90 %. Встановлено, що сприятливий прогноз при
захворюваннях органів дихання у дітей залежить від
ранньої діагностики й адекватної етіотропної тера-
пії. При цьому своєчасне їх виявлення базується на
результатах детального та послідовного аналізу клі-
ніко-анамнестичних і рентгенологічних даних. Адреса для листування з авторами:
Дука Катерина Дмитрівна
E-mail: pdb05@i.ua
© Дука К.Д., Єфанова А.О., 2015
© «Здоров’я дитини», 2015
© Заславський О.Ю., 2015 ÎÑÎÁËÈÂÎÑÒ² IJÀÃÍÎÑÒÈÊÈ ÇÀÕÂÎÐÞÂÀÍÜ ÎÐÃÀͲÂ
ÄÈÕÀÍÍß Ó Ä²ÒÅÉ ÍÀ ÑÓ×ÀÑÍÎÌÓ ÅÒÀϲ Резюме. У наш час захворювання органів дихання мають домінуюче положення в структурі захворюва-
ності у дітей, при цьому клініка різних захворювань органів дихання має схожі риси. Саме тому важли-
вою є якісна діагностика захворювань органів дихання у дітей для вибору правильної тактики лікування. Під час дослідження нами було виявлено, що діагностика гострих респіраторних захворювань в обсте-
жених дітей відповідає діагностичним критеріям, затвердженим у клінічних протоколах. Діагностика
вірусних інфекцій повинна включати клінічні прояви та параклінічні дослідження: визначення антигену
вірусу й виявлення антитіл до вірусу, а діагностика бактеріальних інфекцій дихальних шляхів повинна
базуватися на визначенні рівня прокальцитоніну. Ключові слова: органи дихання діти діагностика Ключові слова: органи дихання, діти, діагностика. Êë³í³÷íà ïåä³àòð³ÿ / Clinical Pediatrics
® Êë³í³÷íà ïåä³àòð³ÿ / Clinical Pediatrics
® Ðåçóëüòàòè òà îáãîâîðåííÿ У більшості дітей захворювання розпочиналось
із підвищення температури тіла до фебрильних
цифр, появою кашлю, закладеності носа та виділень
із нього. Тривалість і характер лихоманки залежали
від нозології захворювання (рис. 1). Оскільки діагноз пневмонії за вказаними вище
фізикальними даними вдається встановити мен-
ше ніж у половини випадків, слід використовува-
ти алгоритм, створений на підставі запропонова-
них ВООЗ ознак. Алгоритм звільняє педіатра від
необхідності встановлювати діагноз при нестачі
інформації й дозволяє знизити гіпердіагностику
пневмонії і скоротити клькість необґрунтованих
рентгенівських знімків і призначень антибіотиків;
його чутливість — 94 %, специфічність — 95 % [7]. Підвищення температури тіла до фебрильних
цифр спостерігалось у 65,5 % дітей із ГРЗ та пнев-
моніями, тривалість гарячкового періоду відповід-
но становила від 3 до 4,5 доби. Субфебрильна лихо-
манка була діагностована у 27,5 % дітей з ураженням
верхніх дихальних шляхів і бронхітами та зберігалась
протягом 3 діб. У 5,5 % дітей із ГРЗ, пневмоніями та
бронхітами з обструктивним синдромом реєструва-
лась піретична лихоманка, тривалість якої була не
більше 2 діб. В 1,5 % дітей з ураженнями органів ди-
хання не було відзначено підвищення температури. Якою мірою допомагають діагностиці лабора-
торні дані? Лейкоцитоз 10–15 • 109/л спостерігаєть-
ся в перші дні у половини хворих на пневмонію, але
також і у третини хворих на ГРЗ, гострий бронхіт. Так що сам по собі він не свідчить про бактеріаль-
ну інфекцію і не вимагає призначення антибіотиків. Але й число лейкоцитів нижче від 10 • 109/л у ряді
випадків не виключає пневмонії. Це може бути ха-
рактерно для пневмоній, викликаних гемофільною
паличкою і мікоплазмою, а також нерідко спосте-
рігається при пневмонії в перші дні хвороби іншої
етіології. Цифри лейкоцитозу вище від 15 • 109/л (і/
або абсолютне число нейтрофілів понад 10 • 109/л
і/або паличкоядерних форм понад 1,5 • 109/л) ро-
блять діагноз пневмонії досить імовірним. Це сто-
сується і підвищення ШОЕ понад 30 мм/год. Більш
низькі цифри не виключають пневмонію, але вони
нерідкі і при бронхітах. Аналіз анамнестичних даних дітей, які були під
спостереженням, показав, що в 71,5 % випадків спо-
стерігався ускладнений перебіг вагітності у матері,
в основному через гестоз, загрозу переривання ва-
гітності та анемію; 3,5 % вагітних хворіли на ГРЗ, у
4,5 % відзначалось загострення хронічної патології. Характер вигодовування на першому році жит-
тя: на грудному вигодовуванні було 57,5 % дітей, на
частковому грудному середнього рівня — 17,5 %, на
штучному вигодовуванні — 20,5 %; 4,5 % дітей зо- Рисунок 1. Ðåçóëüòàòè òà îáãîâîðåííÿ Характер і тривалість лихоманки
у дітей із патологією органів дихання
Примітки: по осі абсцис — тривалість лихоманки
(кількість днів), по осі ординат — температура тіла
дитини. 35,5
36
36,5
37
37,5
38
38,5
39
1
2
3
4
5
Пневмонія
Бронхіт
ГРЗ Р
1 Х
і
і
35,5
36
36,5
37
37,5
38
38,5
39
1
2
3
4
5
Пневмонія
Бронхіт
ГРЗ Серед додаткових маркерів бактеріальної ін-
фекції у діагностиці допомагає С-реактивний білок
(СРБ), рівні якого понад 30 мг/л і рівні прокаль-
цитоніну вище від 2 нг/мл дозволяють на 90 % ви-
ключити вірусну інфекцію. Однак низькі рівні цих
маркерів можуть спостерігатися при пневмоніях,
частіше атипових, отже, їх негативна прогностична
цінність щодо діагнозу пневмонії недостатня [8]. Рисунок 1. Характер і тривалість лихоманки Рисунок 1. Характер і тривалість лихоманки
у дітей із патологією органів дихання у дітей із патологією органів дихання
Примітки: по осі абсцис — тривалість лихоманки
(кількість днів), по осі ординат — температура тіла
дитини. ˳òåðàòóðíèé îãëÿä © Дука К.Д., Єфанова А.О., 2015
© «Здоров’я дитини», 2015
© Заславський О.Ю., 2015 Провідними клінічними симптомами, що до-
зволяють запідозрити у дитини бронхолегеневу па- 27 ¹ 8 (68) • 2015 www.mif-ua.com Êë³í³÷íà ïåä³àòð³ÿ / Clinical Pediatrics в педіатричному відділенні було проліковано 3139
дітей. У структурі захворюваності дітей, які перебу-
вали на лікуванні в умовах педіатричного відділен-
ня, питома вага дітей із ГРЗ у середньому становила
36,5 %, гострими бронхітами — 33,4 %, гострими
обструктивними бронхітами — 18,1 %, з пневмоні-
ями — 12 % дітей. рактерна для крупу і бронхітів, у тому числі обструк-
тивного. Параметри частоти дихання (за 1 хв), за
даними ВООЗ, характерні для пневмонії, становлять
понад 60 у дітей віком 0–2 міс., понад 50 у дітей віком
2–12 міс., понад 40 — у дітей віком 1–4 роки [7]. ,
у
р
[ ]
Скорочення перкуторного звуку також харак-
терно для пневмонії, але зустрічається лише в по-
ловини випадків, тому його відсутність сьогодні не
виключає пневмонії. Це стосується і локалізованих
над місцем ураження дрібнопухирчастих або кре-
пітуючих хрипів та ослабленого або бронхіального
дихання. Їх виявлення дозволяє встановити діагноз
пневмонії, але відсутність їх такий діагноз не ви-
ключає. У половини всіх хворих із типовою пневмо-
нією в гострому періоді хрипи можуть бути відсутні. Хрипи, що рівномірно вислуховуються в обох леге-
нях, як і обструкція, характерні для бронхітів, але не
для пневмонії: сухі хрипи виявляються у 10 %, а роз-
сіяні вологі — у 25 % хворих, найчастіше вони аси-
метричні [3]. До основної групи увійшли 124 дитини віком від
1 до 7 років, більшість із них (85 %) надійшли до від-
ділення вперше, 15 % дітей — повторно, як правило,
це діти віком від 2 до 3 років життя. 30 % дітей було
госпіталізовано до стаціонару на 1-шу — 2-гу добу
захворювання, 48 % — на 3-тю добу, 22 % — у 1–2-й
тиждень захворювання. Ìàòåð³àëè òà ìåòîäè Зміни в загальному аналізі крові дітей
із захворюваннями органів дихання
Примітки: по осі абсцис — тип захворювання
органів дихання: 1 — гостре респіраторне захво-
рювання; 2 — бронхіт; 3 — пневмонія; по осі орди-
нат — кількість дітей (%). 0
10
20
30
40
50
60
70
80
%
1
2
3
Вікова норма
Абсолютний лімфоцитоз
Абсолютний нейтрофільоз Інфільтрація
легеневої тканини
12 %
Ознаки
емфізематозу
13 %
Посилення судинного
рисунка
75 % Фізикальні зміни над легенями характеризува-
лись жорстким диханням, при пневмонії дихання
було послабленим. На фоні послабленого дихан-
ня на 3-тю — 5-ту добу від початку захворювання
вислуховувались вологі дрібнопухирчасті хрипи
та крепітація. При гострому бронхіті на фоні жор-
сткого дихання вислуховувались сухі хрипи, що в
динаміці ставали вологими, різнопухирчастими. Для гострого бронхіту з бронхообструктивним син-
дромом характерною була наявність у 42,5 % дітей
сухих хрипів із подовженим видихом, для 57,5 % —
наявність вологих дрібнопухирчастих хрипів з обох
легеневих сторін. При рентгенологічному дослідженні у 75,5 % об-
стежених дітей виявлено посилення судинного ри-
сунка, у 12,5 % — ознаки емфізематозу, у 12 % дітей
на фоні посиленого бронхосудинного рисунка ви-
значалась інфільтрація легеневої тканини (рис. 3). Рисунок 3. Особливості рентгенограм дітей
із захворюваннями органів дихання Рисунок 3. Особливості рентгенограм дітей
із захворюваннями органів дихання у
р
р
д
із захворюваннями органів дихання на фоні посиленого бронхосудинного рисунка ви
значалась інфільтрація легеневої тканини (рис. 3). Рисунок 2. Розподіл дітей за типом
вигодовування на першому році життя
Примітки: по осі абсцис — кількість дітей (%); по
осі ординат — тип вигодовування на першому році
життя: 1 — діти, які не отримували грудне молоко
від народження; 2 — частково грудне вигодову-
вання середнього рівня; 3 — штучне вигодовуван-
ня; 4 — грудне вигодовування. 0
10
20
30
40
50
60
1
2
3
4
у
р
р
із захворюваннями органів дихання
Рисунок 4. Зміни в загальному аналізі крові дітей
із захворюваннями органів дихання
Примітки: по осі абсцис — тип захворювання
органів дихання: 1 — гостре респіраторне захво-
рювання; 2 — бронхіт; 3 — пневмонія; по осі орди-
нат — кількість дітей (%). 0
10
20
30
40
50
60
70
80
%
1
2
3
Вікова норма
Абсолютний лімфоцитоз
Абсолютний нейтрофільоз 0
10
20
30
40
50
60
70
80
%
1
2
3
Вікова норма
Абсолютний лімфоцитоз
Абсолютний нейтрофільоз 0
10
20
30
40
50
60
1
2
3
4 Рисунок 2. Ìàòåð³àëè òà ìåòîäè Дослідження проводилось у педіатричному від-
діленні МДКЛ № 5 у 2011–2014 рр. За чотири роки 28 ¹ 8 (68) • 2015 ¹ 8 (68) • 2015 Çäîðîâüå ðåáåíêà, ISSN 2224-0551 Êë³í³÷íà ïåä³àòð³ÿ / Clinical Pediatrics всім не отримували грудне молоко від народження
через тяжкий стан (рис. 2). У більшості дітей, хворих на ГРЗ, у загальному
аналізі крові спостерігався лейкоцитоз, у третини
хворих — через підвищення кількості нейтрофілів,
в інших — через підвищення рівня лімфоцитів, а у
більше ніж 30 % дітей показники крові відповіда-
ли віковій нормі. У дітей із гострими бронхітами
абсолютний нейтрофільоз діагностувався у 42,5 %,
абсолютний лімфоцитоз — у 35,5 %, збільшена
ШОЕ — у 30,5 % дітей. При пневмонії у загальному
аналізі крові у значної кількості хворих діагносту-
вався абсолютний нейтрофільоз із зсувом лейкоци-
тарної формули вліво, у третини хворих — збільше-
на ШОЕ (рис. 4). Найбільш тяжкий перебіг захворювань органів
дихання відзначався у дітей 1-ї та 2-ї груп. Обтяже-
ний алергологічний анамнез було виявлено у більше
ніж половини дітей внаслідок наявності різних ви-
дів алергозів у батьків. У кожної четвертої дитини на
першому році життя відзначались алергічні реакції
у вигляді шкірних висипів, свербіння, у третини ді-
тей — прояви дисбіозу кишечника. При первинному огляді в стаціонарі загальний
стан середньої тяжкості діагностовано у 102 дітей, у
22 дітей — тяжкий стан через дихальні розлади, ін-
токсикацію, виражені фізикальні зміни над легеня-
ми. Кашель спостерігався у 100 % дітей. На початку
захворювання він був частим, малопродуктивним. На 3-тю добу терапії кашель ставав продуктивним
у 62,5 % дітей, наприкінці першого тижня захворю-
вання він рідшав, а на 10-ту добу залишався лише у
22 % дітей. У 49,1 % дітей діагностували різні роз-
лади дихання. Серед них задишка експіраторного
характеру спостерігалась у 39 %, інспіраторна — у
42 %, змішана — у 19 % дітей. У дітей із тяжким і середнім ступенем перебігу
пневмонії спостерігалось суттєве підвищення рівня
СРБ, а також рівня прокальцитоніну вище від 2 нг/мл,
що свідчило про бактеріальну етіологію процесу. Отже, для якісної діагностики захворювань орга-
нів дихання у дітей необхідно враховувати комплекс Рисунок 3. Особливості рентгенограм дітей
із захворюваннями органів дихання
Інфільтрація
легеневої тканини
12 %
Ознаки
емфізематозу
13 %
Посилення судинного
рисунка
75 %
Рисунок 4. Âèñíîâêè 1. Важливим критерієм оцінки тяжкості перебігу
пневмонії слід вважати відсутність грудного вигодо-
вування, а саме частково грудне та штучне вигодо-
вування. 1. Важливим критерієм оцінки тяжкості перебігу
пневмонії слід вважати відсутність грудного вигодо-
вування, а саме частково грудне та штучне вигодо-
вування. 3. Заплатников А.Л. Внебольничные пневмонии у детей:
принципы диагностики и этиотропной терапии / А.Л. Заплат-
ников // Современная педиатрия. — 2010. — № 4(32). — С. 161-
165. 2. Діагностика ГРЗ в обстежених дітей відповідає
діагностичним критеріям, затвердженим у клініч-
них протоколах. 4. Клименко В.А. Особливості перебігу гострого обструктив-
ного бронхіту у дітей грудного віку з перинатальним ураженням
центральної нервової системи / В.А. Клименко, О.В. Давиден-
ко // Педіатрія, акушерство та гінекологія. — 2012. — № 2. —
С. 19-21. 3. Діагностика гострих вірусних інфекцій повин-
на включати клінічні прояви та параклінічні до-
слідження: визначення антигену вірусу у змиві з
носоглотки за допомогою ІФА, виявлення антитіл
до вірусу за допомогою реакції зв’язування компле-
менту. 5. Мизерницкий Ю.Л. Дифференциальная диагностика и
принципы дифференцированной терапии бронхообструктивного
синдрома при ОРИ у детей / Ю.Л. Мизерницкий // Здоровье ре-
бенка. — 2009. — № 1. 6. Полковниченко А.Н. Применение статистического ана-
лиза в изучении заболеваемости детей бронхолегочной патоло-
гией / Полковниченко А.Н., Гальченко В.Я., Гейвах В.С., Черепа-
хин Ю.А. // Укр. мед. альманах. — 2010. — Т. 13, № 2. — С. 17-19. у
4. Диференціальна діагностика вірусних і бакте-
ріальних інфекцій дихальних шляхів повинна базу-
ватись на визначенні рівня прокальцитоніну, вміст
якого суттєво підвищується при дії бактеріальних
токсинів. у
4. Диференціальна діагностика вірусних і бакте-
ріальних інфекцій дихальних шляхів повинна базу-
ватись на визначенні рівня прокальцитоніну, вміст
якого суттєво підвищується при дії бактеріальних
токсинів. 7. Таточенко В.К. Пневмония у детей: диагностика и ле-
чение / В.К. Таточенко // Современная педиатрия. — 2009. —
№ 3(25). — С. 10-11. 8. Тихомирова А.Р. Клинико-иммунологическая характери-
стика детей младшего возраста с бронхообструктивным син-
дромом: Автореф. дис… канд. мед. наук / А.Р. Тихомирова. —
СПб, 2008. — 24 с. Ìàòåð³àëè òà ìåòîäè Розподіл дітей за типом
вигодовування на першому році життя д
у
р
у р ц
Примітки: по осі абсцис — кількість дітей (%); по
осі ординат — тип вигодовування на першому році
життя: 1 — діти, які не отримували грудне молоко
від народження; 2 — частково грудне вигодову-
вання середнього рівня; 3 — штучне вигодовуван-
ня; 4 — грудне вигодовування. Рисунок 4. Зміни в загальному аналізі крові дітей
із захворюваннями органів дихання р
р
д
Примітки: по осі абсцис — тип захворювання
органів дихання: 1 — гостре респіраторне захво-
рювання; 2 — бронхіт; 3 — пневмонія; по осі орди-
нат — кількість дітей (%). 29 ¹ 8 (68) • 2015 www.mif-ua.com Êë³í³÷íà ïåä³àòð³ÿ / Clinical Pediatrics обструктивний бронхіт / О.Є. Абатуров, О.О. Русакова // Пе-
ринатология и педиатрия. — 2012. — № 1(49). — С. 106-108. обструктивний бронхіт / О.Є. Абатуров, О.О. Русакова // Пе-
ринатология и педиатрия. — 2012. — № 1(49). — С. 106-108. скарг, дані анамнезу життя та хвороби, об’єктивні
клінічні дані й результати параклінічних обстежень. скарг, дані анамнезу життя та хвороби, об’єктивні
клінічні дані й результати параклінічних обстежень. 2. Безрукава Л.А. Предупреждение повторных бронхооб-
структивных состояний у детей, перенесших респираторно-
синцитиальную и аденовирусную инфекции: Автореф. дис…
канд. мед. наук / Л.А. Безрукава. — Тюмень, 2009. — 21 с. Çäîðîâüå ðåáåíêà, ISSN 2224-0551 Ñïèñîê ë³òåðàòóðè ð
óð
1. Абатуров О.Є. Клінічні особливості перебігу та стан міс-
цевого імунітету в дітей раннього віку, які хворіють на гострий Отримано 04.11.15 Duka K.D., Yefanova A.O. State Institution «Dnipropetrovsk Medical Academy of the
Ministry of Healthcare of Ukraine», Dnipropetrovsk, Ukraine Äóêà Å.Ä., Åôàíîâà À.À.. ÃÓ «Äíåïðîïåòðîâñêàÿ ìåäèöèíñêàÿ àêàäåìèÿ
ÌÇ Óêðàèíû» Äóêà Å.Ä., Åôàíîâà À.À.. ÃÓ «Äíåïðîïåòðîâñêàÿ ìåäèöèíñêàÿ àêàäåìèÿ
ÌÇ Óêðàèíû» Duka K.D., Yefanova A.O. State Institution «Dnipropetrovsk Medical Academy of the
Ministry of Healthcare of Ukraine», Dnipropetrovsk, Ukraine ÎÑÎÁÅÍÍÎÑÒÈ ÄÈÀÃÍÎÑÒÈÊÈ ÇÀÁÎËÅÂÀÍÈÉ
ÎÐÃÀÍÎÂ ÄÛÕÀÍÈß Ó ÄÅÒÅÉ ÍÀ ÑÎÂÐÅÌÅÍÍÎÌ ÝÒÀÏÅ Summary. Nowadays, respiratory diseases occupy a domi-
nant position in the structure of morbidity in children, and
clinic picture of various respiratory diseases has similar features. That’s why the qualitative diagnosis of respiratory diseases in
children is so important for choosing the right treatment. Dur-
ing the study, we have found that the diagnosis of acute respi-
ratory diseases in surveyed children meets diagnostic criteria
approved by clinical protocols. Diagnosis of viral infections
should include clinical manifestations and paraclinical re-
searches: detection of virus antigen and antibodies to the virus,
and diagnosis of bacterial respiratory infections should be based
on the determination of procalcitonin levels. Резюме. В настоящее время заболевания органов ды-
хания занимают доминирующее положение в структуре
заболеваемости у детей, при этом клиника различных за-
болеваний органов дыхания имеет схожие черты. Поэтому
так важна качественная диагностика заболеваний органов
дыхания у детей для выбора правильной тактики лечения. В результате проведенного исследования нами выявлено,
что диагностика острых респираторных заболеваний у об-
следованных детей соответствует диагностическим крите-
риям, утвержденным в клинических протоколах. Диагно-
стика вирусных инфекций должна включать клинические
проявления и параклинические исследования: определе-
ние антигена вируса и выявление антител к вирусу, а диа-
гностика бактериальных инфекций дыхательных путей —
базироваться на определении уровня прокальцитонина. Key words: respiratory system, children, diagnosis. Ключевые слова: органы дыхания, дети, диагностика. 30 ¹ 8 (68) • 2015 Çäîðîâüå ðåáåíêà, ISSN 2224-0551
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https://openalex.org/W4256096337
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https://bg.copernicus.org/articles/6/2227/2009/bg-6-2227-2009.pdf
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English
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Long term changes in the ecosystem in the northern South China Sea during 1976–2004
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Long term changes in the ecosystem in the northern South China
Sea during 1976–2004 X. Ning1,2,3,†, C. Lin3, Q. Hao2,3, C. Liu1,2,3, F. Le2,3, and J. Shi3
1State Key Lab of Satellite Ocean Environment Dynamics, China
2SOA Key Lab of Marine Ecosystems and Biogeochemistry, China
3Second Institute of Oceanography (SIO), State Oceanic Administration (SOA), Hangzhou, Zhejiang, 310012, China
†deceased State Key Lab of Satellite Ocean Environment Dynamics, China
2SOA Key Lab of Marine Ecosystems and Biogeochemistry, China
3Second Institute of Oceanography (SIO), State Oceanic Administration (SOA), Hangzhou, Zhejiang, 310012, China
†deceased Received: 22 July 2008 – Published in Biogeosciences Discuss.: 12 September 2008
Revised: 26 May 2009 – Accepted: 6 October 2009 – Published: 26 October 2009 Abstract. Physical and chemical oceanographic data were
obtained by seasonal monitoring along a transect (Transect
N) in the northern South China Sea (nSCS) during 1976–
2004. Fluctuations of DIN (dissolved inorganic nitrogen),
seawater temperature (SST and Tav – average temperature
of the water column), N:P ratio and salinity (Sav and S200
– salinity at the 200 m layer) exhibited an increasing trend,
while those of T200, DO, P, Si, Si:N and SSS exhibited a de-
creasing trend. The annual rates of change in DIN, DO, T
and S revealed pronounced changes, and the climate trend
coefficients, which was defined as the correlation coefficient
between the time series of an environmental parameter and
the nature number (namely 1,2,3,......n), were 0.38 to 0.89
and significant (p≤0.01 to 0.05). Our results also showed
that the ecosystem has obviously been influenced by the pos-
itive trends of both SST and DIN, and negative trends of both
DO and P. For example, before 1997, DIN concentrations in
the upper layer were very low and N:P ratios were less than
half of the Redfield ratio of 16, indicating potential N lim-
itation. However after 1997, all Si:P ratios were >22 and
the Nav:Pav was close to the Redfield ratio, indicating poten-
tial P limitation, and therefore N limitation has been reduced
after 1997. induced by not only anthropogenic activities, but also global
climate change, e.g. ENSO. The effects of climate change
on the nSCS were mainly through changes in the monsoon
winds, and physical-biological oceanography coupling pro-
cesses. In this study physical-chemical parameters were systemic
maintained, but the contemporaneous biological data were
collected from various sources. Regional response to global
climate change is clearly a complicated issue, which is far
from well understood. Long term changes in the ecosystem in the northern South China
Sea during 1976–2004 This study was made an attempt to
tackle this important issue. For the aim these data were valu-
able. Biogeosciences, 6, 2227–2243, 2009
www.biogeosciences.net/6/2227/2009/
© Author(s) 2009. This work is distributed under
the Creative Commons Attribution 3.0 License. Biogeosciences, 6, 2227–2243, 2009
www.biogeosciences.net/6/2227/2009/
© Author(s) 2009. This work is distributed under
the Creative Commons Attribution 3.0 License. Biogeosciences Published by Copernicus Publications on behalf of the European Geosciences Union. 1
Introduction Due to the strong northeast monsoon; during the flood sea-
son in summer, the river plume extends well into the nSCS,
and its southeastward and southward tongue can reach up to
17◦00′ N, 112◦E, about 5 degrees, in latitude, away from the
river mouth (Cai et al., 2007; Xue et al., 2001a, b). However, the long term changes in environmental condi-
tions and the responses of the ecosystem in this region have
not been well documented yet. The objective of this study
was to analyze the 29 y time series of multidisciplinary ob-
servational data obtained during 1976–2004, aiming at un-
derstanding how the environment has changed and how the
ecosystem and biological resources have responded to the en-
vironmental changes in the nSCS. It must be pointed out that the data set we adopted in
present analysis is large and from various sources (using
data in this study were systemic maintained for the physical-
chemical parameters, but for contemporaneous biological
data were collected from various sources, and we have col-
lected data from the same period of investigation as best as
one can. Inevitably, there are some mismatches between the
scales of physical and chemical parameters reflecting the pro-
cesses of environmental change and the spatial and temporal
dimensions of biological investigations, due to the less fre-
quency for the latter). Although the quality of data might
vary throughout the long period of observation, these data
were valuable for the long term changes in environmental
conditions and the responses of the ecosystem in this region. Using these data, we can still find some disciplines about the
response of the ecosystem to the environmental change in the
SCS. Regional response to global climate change is clearly a
complicated issue, which is far from well understood. This
study made an attempt to tackle this important issue. And
in order to improve our understanding, additional long-term
study is mandatory. The meteorological forcing over the nSCS is dominated
by the East Asian Monsoon (Sadler et al., 1985). The upper
ocean circulation follows closely the alternating monsoons
(Wyrtki, 1961). During winter northeast monsoon, along the
northern boundary, the warm and saline Kuroshio Current
water with oligotrophic properties intrudes through Luzon
Strait and flows westward along the continental margin of
China to become the deep-water mass of the nSCS (Nitani,
1972; Shaw, 1991). 1
Introduction The South China Sea (SCS) is the largest semi-enclosed
marginal sea in Southeast Asia with an area of about
3.5×106 km2. Our study area is the northern SCS (nSCS),
bounded by the mainland of China on the north and north-
west sides, Taiwan Strait on the northeast, Taiwan Island and
Bashi Strait on the east side, and the Hainan Island on the
west side. The nSCS is connected to the East China Sea
through Taiwan Strait, and it is connected to the open ocean
through Luzon Strait, where a deep sill (>2000 m) allows
effective water exchange with the western Pacific. The to-
pography of the area is characterized by the incline from
the coast of mainland China towards the southeast, with a
gradient from the coastal zone (<50 m), continental shelf
(<200 m), the slope and open sea (>200 m), to the deep sea
(>3000 m) (Fig. 1). Ecological investigation shows that there have been some
obvious responses of the ecosystems to the long-term envi-
ronmental changes in the nSCS. Chlorophyll-a concentra-
tion, primary production, phytoplankton abundance, benthic
biomass, cephalopod catch and demersal trawl catch have in-
creased. But phosphorus depletion in upper layer may be re-
lated to the shift in the dominant species from diatoms to di-
noflagellates and cyanophytes. The ecosystem response was The runoff from 29 rivers, with different sized input into
the nSCS with total drainage area of 5.5×105 km2, and
an annual fresh water discharge of 3.8×1011 m3 (Han et Published by Copernicus Publications on behalf of the European Geosciences Union. 2228 X. Ning et al.: Long term changes in the northern South China Sea X. Ning et al.: Long term changes in the northern South China Sea al., 1998). Among them, the Pearl River is the largest
with a drainage area of 4.3×105 km2 and a discharge of
3.3×1011 m3 y−1 (Han et al., 1998). It carries a large quan-
tity of suspended solids (8.3×107 t y−1, Han et al., 1998)
and dissolved nutrients (N=8.6×104 t y−1; P=1.2×104 t y−1;
Si=184.3×104 t y−1, before 1998, Wang and Peng, 1996; and
N=19.14×104 t y−1, P=0.8×104 t y−1 after 1998, SOAC,
2000, 2001, 2002, 2003, 2004) into the nSCS. The Pearl
River plume extends offshore to cover a large area of the
nSCS (Yin et al., 2001). During the dry season in winter, the
river plumes extend westward along the coast of Guangdong. 1
Introduction The coastal water of the East China Sea
flows southwestward through Taiwan Strait into the nSCS
(Fang et al., 1998; Xue et al., 2004). On the contrary, dur-
ing the summer southwest monsoon, the Guangdong Coastal
Current flows eastward along the southern coast of main-
land China, which eventually flows into the East China Sea
through Taiwan Strait. The southwesterly winds also induce
Ekman transport toward offshore and coastal upwelling. The
deep water upwells and mixes with the upper water to form
the SCS intermediate water, which flows out of the nSCS into
the northwestern Pacific Ocean through Luzon Strait (Gong
et al., 1992). X. Ning et al.: Long term changes in the northern South China Sea 2229 X. Ning et al.: Long term changes in the northern South China Sea Fig. 1
China
Vietnam
Luzon
Island
South China Sea
110
112
114
116
118
120
18
20
22
24
China
Taiwan
Island
Luzon
Island
Pearl River
Hainan
Island
1
2
3
4
5
6
100m
1000m
2000m
N
E
N
Fig. 1. Geographical locations of the transect and stations and the circulation in the northern South China Sea (nSCS) (modified from Su,
1998; Xue et al., 2004). Transect N from the Pearl River Estuary towards the southeastern nSCS is the main transect with 6 stations (full
circle). 18 Fig. 1
Fig. 1. Geographical locations of the transect and stations and the circulation in the northern South China Sea (nSCS) (modified from Su,
1998; Xue et al., 2004). Transect N from the Pearl River Estuary towards the southeastern nSCS is the main transect with 6 stations (full
circle). analyses were conducted on time series (Chen and Ma, 1991)
and climate trend coefficients (Rxt) were estimated. Rxt was
used to assess whether there was a significant linear climate-
trend in a time series (Shi et al., 1995). This coefficient was
defined as the correlation coefficient between the time series
of an environmental parameter, {Xi}, and the nature num-
ber {i}, i=1, 2, 3 ..., n. In this study, n is the total span of
the years covered by the data. The coefficient was computed
from the following equation: (Strickland and Parsons, 1972). Photosynthetic pigments
(Chl-a) were measured by the acetone extraction and fluo-
rescence method (Holm-Hansen et al., 1965). Annual mean values were the average for winter and sum-
mer which were derived from observations during February
and August, respectively. The regional average was the av-
erage value for the all stations illustrated in Fig. 1. First, we
took the values at the sea surface (SS), the depth of 200 m
and the average through the water column for 0–200 m (inte-
grated) for each parameter for each station, since at the depth
of 200 m concentrations of biogenic elements and other prop-
erties were relatively stable and much less influenced by the
upper layers. Second, the regional means for each parameter
on an annual scale were calculated. 2
Data and methods In this study, data were obtained from winter and summer
monitoring along transect N (Fig. 1, an observation tran-
sect, including six stations, crossing the nSCS, from the
northwestern to southeastern), maintained by the survey team
of the State Oceanic Administration (SOA), China during
1976–2004. These data include physical [seawater tempera-
ture (T) and salinity (S)] and chemical parameters [dissolved
oxygen (DO), phosphate (PO4-P), silicate (SiO3-Si), dis-
solved inorganic nitrogen (DIN, including NO3-N, NO2-N
and NH4-N)]. The parameters of T, S and DO data collection
started from 1976; and the nutrients data (PO4-P, SiO3-Si,
NO3-N, NO2-N and NH4-N) collection started from 1989. Seawater samples were collected using Nansen bottles from
the surface, 5, 10, 15, 20, 25, 30, 35, 50, 75, 100, 150 and
200 m for T and S, and at the surface, 10, 20, 30, 50, 75, 100,
150 and 200 m for biogenic element determination. Seawater
temperature was measured by using a reversing thermometer
attached to the Nansen bottle, and salinity was measured us-
ing induction salinometer, according to SOAC (1975) and
NBTS (1991). Nutrients (nitrate, phosphate and silicate)
were analyzed by standard spectrophotometric method, and
dissolved oxygen (DO) was analyzed by the Winkler method In the nSCS, the thermocline occurs all the year round, and
the interannual change in its strength is pronounced (Yuan
and Deng, 1997a, b; Shi et al., 2001). Previous studies have
examined on variations in seawater temperature and salinity
distributions (Yang and Liu, 1998; Yuan and Deng, 1998),
dissolved oxygen distribution (Lin and Han, 1998), pollution
status along the coast of the nSCS (Li and Chen, 1998) and
the fisheries environment in the nSCS (Jia et al., 2005). Fur-
thermore, it has been found that due to the combined effects
of monsoons, topography, shape of the coastal line and the in-
ertial effects, mesoscale eddies (Zeng et al., 1989; Xu et al.,
2001; Li et al., 2003; Chen et al., 2005). Recent studies re-
vealed that the effects of coupling between physical – chem-
ical – biological oceanographic processes on phytoplankton
biomass and production are important for understanding the
influence on the long-term environmental changes and the
ecosystem dynamics of the SCS (Liu et al., 2002, 2007; Ning
et al., 2004). Biogeosciences, 6, 2227–2243, 2009 www.biogeosciences.net/6/2227/2009/ X. Ning et al.: Long term changes in the northern South China Sea Variation trends in seawater salinity in the nSCS during
1976–2004. (a), (b) and (c) show the annual means of sea surface
salinity (SSS), water column average salinity in the upper 200 m
(Sav) and salinity at 200 m (S200), respectively. The lines are linear
regressions. 1975
1985
1995
2005
33.6
33.8
34.0
34.2
34.4
34.6
(b)
Salinity
Salinity
(b) Fi
3
1975
1985
1995
2005
33.6
33.8
34.0
34.2
34.4
34.6
(b)
Salinity
34.3
34.4
34.5
34.6
34.7
34.8
1975
1985
1995
2005
(c)
34.2
Salinity
Salinity
Salinity
(b)
(c) 1975
1985
1995
2005
12
13
14
15
16
17
(c)
Temperature( ε )
Temperature (oC)
(c) Fig. 3
Fig. 3. Variation trends in seawater salinity in the nSCS during
1976–2004. (a), (b) and (c) show the annual means of sea surface
salinity (SSS), water column average salinity in the upper 200 m
(Sav) and salinity at 200 m (S200), respectively. The lines are linear
regressions. Fig. 2
Fig. 2. Variation trends of seawater temperature in the nSCS dur-
ing 1976–2004. (a), (b) and (c) show the annual mean of the sea
surface temperature (SST), the water column average temperature
in the upper 200 m (Tav), and the temperature at the depth of 200 m
(T200). The lines are the linear regressions. (BB) was measured gravimetrically after removing the body
surface water in the lab (SOAC, 1975; NBTS, 1991). The
nekton samples were collected by using a cystoid net with a
mesh size of 20 mm, towed by a pair of boats at a speed of
3–4 kn for 1 h at each station (SOAC, 1975; NBTS, 1991). 35
(1952) and modified for scintillation counting by Wolfe and
Schelske (1967). Phytoplankton samples were collected by
vertical haul using a Judy net with a mesh size of 76 µm. The samples were preserved with Lugol’s solution, and the
species identification and cell counts were made using a
microscope to get phytoplankton abundance (PA, Sournia,
1978). Zooplankton samples were collected by vertical haul
using a plankton net with a mesh size of 505 µm, and the
samples were preserved with neutral formaldehyde solution
(5%). The species identification and individual counts were
made using a stereo microscope, and the wet weight biomass
(ZB) was measured by an electronic balance after removing
the body surface water in the lab, according to SOAC (1975)
and NBTS (1991). X. Ning et al.: Long term changes in the northern South China Sea The average value for
the water column was computed, according to the following
equation: Rxt =
nP
i=1
(xi −¯x)(i −¯t)
s
nP
i=1
(xi −¯x)2
nP
i=1
i −¯t
2
(2) (2) Where t=(n+1)/2. Its significance level is determined from
the Student t-test. A positive/ negative value of Rxt indicates
that the time series, {Xi}, has a linear positive/negative trend. In order to compare the environmental change rates between
coastal/shelf waters with the slope/open sea, data for water
depths <200 and >200 m, respectively, were analyzed. Where t=(n+1)/2. Its significance level is determined from
the Student t-test. A positive/ negative value of Rxt indicates
that the time series, {Xi}, has a linear positive/negative trend. In order to compare the environmental change rates between
coastal/shelf waters with the slope/open sea, data for water
depths <200 and >200 m, respectively, were analyzed. 34
Xav = 1
b
Z b
0
X(z)dz
(1) 4(1) Where X is an environmental parameter; b is the water depth
(200 m, or 2 m above bottom if the water depth is shallower
than 200 m) and z is the observation depth. p
p
y
y
Biological oceanography data, such as chlorophyll-a,
phytoplankton abundance, primary production, zooplankton
biomass, benthos biomass, cephalopod catch, etc. were ob-
tained during the period by marine ecosystem surveys con-
ducted by the South China Sea Fisheries Research Insti-
tute (SCSFRI), the South China Sea Institute of Oceanog-
raphy, Chinese Academy of Sciences and the Second Insti-
tute of Oceanography, SOA. For the observational methods,
Chl-a was determined by acetone extraction fluorescence
method (Holm-Hansen et al., 1965) and often calibrated by
spectrophotometry. Primary productivity was measured us-
ing the 14C tracer method established by Steemann-Nielsen In order to show the interannual changes in environmental
parameters in the nSCS, the time series of various parameters
was determined. The parameters include physical parame-
ters, such as SST, Tav, T200 m, SSS, Sav, S200, and chemical
parameters, such as SSDO, DOav, DO200, SSP, Pav, P200,
SSSi, Siav, Si200, SSDIN, DINav, DIN200, SSNO2-N, NO2-
Nav, NO2-N200, SSNO3-N, NO3-Nav, NO3-N200, SSNH4-
N, NH4-Nav, NH4-N200, and the ratios of the chemical pa-
rameters SSN:SSP, Nav:Pav, N200:P200, SSSi:SSN, Siav:Nav,
Si200:N200, where av = average for the whole water column
and 200=200 m depth. Statistical test and linear regression Biogeosciences, 6, 2227–2243, 2009 www.biogeosciences.net/6/2227/2009/ X. Ning et al.: Long term changes in the northern South China Sea 2230 2230
X
Fig. X. Ning et al.: Long term changes in the northern South China Sea 2
(a)
1985
1995
2005
Temperature(ε )
1975
27
26
25
24
23
1975
1985
1995
2005
20
21
22
25
24
23
Temperature(ε )
(b)
1975
1985
1995
2005
12
13
14
15
16
17
(c)
Temperature( ε )
Temperature (oC)
Temperature (oC)
Temperature (oC)
(a)
(b)
(c)
Fig. 2. Variation trends of seawater temperature in the nSCS dur-
ing 1976–2004. (a), (b) and (c) show the annual mean of the sea
surface temperature (SST), the water column average temperature
in the upper 200 m (Tav), and the temperature at the depth of 200 m
(T200). The lines are the linear regressions. g
g
g
Fig. 2
(a)
1985
1995
2005
Temperature(ε )
1975
27
26
25
24
23
1975
1985
1995
2005
20
21
22
25
24
23
Temperature(ε )
(b)
1975
1985
1995
2005
12
13
14
15
16
17
(c)
Temperature( ε )
Temperature (oC)
Temperature (oC)
Temperature (oC)
(a)
(b)
(c)
Fig. 2. Variation trends of seawater temperature in the nSCS dur-
ing 1976–2004. (a), (b) and (c) show the annual mean of the sea
surface temperature (SST), the water column average temperature
in the upper 200 m (Tav), and the temperature at the depth of 200 m
(T200). The lines are the linear regressions. Fig. 3
1975
1985
1995
2005
32.0
32.5
33.0
33.5
34.0
34.5
Salinity
(a)
1975
1985
1995
2005
33.6
33.8
34.0
34.2
34.4
34.6
(b)
Salinity
34.3
34.4
34.5
34.6
34.7
34.8
1975
1985
1995
2005
(c)
34.2
Salinity
Salinity
Salinity
Salinity
(a)
(b)
(c)
Fig. 3. Variation trends in seawater salinity in the nSCS during
1976–2004. (a), (b) and (c) show the annual means of sea surface
salinity (SSS), water column average salinity in the upper 200 m
(Sav) and salinity at 200 m (S200), respectively. The lines are linear
regressions. (a)
1985
1995
2005
Temperature(ε )
1975
27
26
25
24
23
1975
1985
1995
2005
20
21
22
25
24
23
Temperature(ε )
(b)
Temperature (oC)
Temperature (oC)
(a)
(b) 1975
1985
1995
2005
32.0
32.5
33.0
33.5
34.0
34.5
Salinity
(a)
Salinity
(a) Fig. 3
1975
1985
1995
2005
32.0
32.5
33.0
33.5
34.0
34.5
Salinity
(a)
1975
1985
1995
2005
33.6
33.8
34.0
34.2
34.4
34.6
(b)
Salinity
34.3
34.4
34.5
34.6
34.7
34.8
1975
1985
1995
2005
(c)
34.2
Salinity
Salinity
Salinity
Salinity
(a)
(b)
(c)
Fig. 3. X. Ning et al.: Long term changes in the northern South China Sea Benthic macrofauna samples were col-
lected by using a grab with a sampling area of 0.1 m2, and
the animals were sorted after removing the mud by elutria-
tion. The species identification and individual counts were
made using a stereo microscope, and the wet weight biomass Biogeosciences, 6, 2227–2243, 2009 3.1.2
Concentration of dissolved oxygen (DO) 1976 and 2004 (Fig. 2, Table 1). During this period, the SST
and Tav increased by 2.26 and 2.61◦C, respectively, while
T200 decreased by 3.10◦C (Fig. 2, Table 1). The Rxt of the
time series of SST, Tav and T200 were 0.89, 0.85 and −0.71,
respectively, and the variation trends for this time series were
significant (p≤0.01). A significant (p≤0.01) decrease in DO concentrations was
observed (Fig. 4; Table 1), and the annual rate was between
−0.91 and −1.93 µmol L−1 y−1. The regional mean of DO
of the sea surface, the water column average in the upper
200 m and at 200 m significantly decreased by 26.5, 55.8 and
47.9 µmol L−1. The Rxt values of time series for DO ranged
−0.48 to −0.81 (p≤0.01, Table 1). 37
During the observation period the annual rate of SSS ex-
hibited a decreasing trend (−0.022 y−1), while the annual
rates of Sav and S200 exhibited increasing trend (0.007 y−1
and 0.005 y−1, respectively, Fig. 3, Table 1). The SSS de-
creased by 0.653, while the Sav and S200 increased by 0.206
and 0.151. The Rxt values of time series of SSS, Sav and S200
were −0.41(P≤0.05), 0.38 (P≤0.05) and 0.44 (p≤0.02), re-
spectively (Table 1). X. Ning et al.: Long term changes in the northern South China Sea (a), (b) and (c) show the annual means of sea surface P
(SSP), water column average P in the upper 200 m (Pav) and P in the
200 m layer (P200), respectively. The lines are linear regressions. Fig. 4
DO(μmol/L)
1975
1985
1995
2005
250
300
400
450
500
350
(b)
DO(μmol/L)
1975
1985
1995
2005
(a)
250
300
350
400
450
500
550
400
DO(μmol/L)
1975
1985
1995
2005
(c)
300
200
150
250
350
450
DO (μmol L-1)
DO (μmol L-1)
DO (μmol L-1)
(a)
(b)
(c)
Fig. 4. Variation trends in dissolved oxygen (DO) concentration
in the nSCS during 1976–2004. (a), (b) and (c) show the annual
means of sea surface DO (SSDO), water column average in the up-
per 200 m DO (DOav) and DO at the 200 m (DO200), respectively. The lines are linear regressions. 0.2
0.4
0.6
Po4-P(μmol/L)
1992
1996
2000
2004
1988
(a)
0.1
0.3
0.5
0.4
0.6
0.8
Po4-P(μmol/L)
1992
1996
2000
2004
1988
0.3
0.5
0.7
0.9
(b)
PO4-P (μmol L-1)
PO4-P (μmol L-1) DO(μmol/L)
1975
1985
1995
2005
(a)
250
300
350
400
450
500
550
DO (μmol L-1)
(a) 0.2
0.4
0.6
Po4-P(μmol/L)
1992
1996
2000
2004
1988
(a)
0.1
0.3
0.5
PO4-P (μmol L-1) (b)
(b) Fig. 5
1.8
0.8
1.0
1.2
1.4
1.6
Po4-P(μmol/L)
1988
1992
1996
2000
2004
(c)
PO4-P (μmol L-1) g
Fig. 4. Variation trends in dissolved oxygen (DO) concentration
in the nSCS during 1976–2004. (a), (b) and (c) show the annual
means of sea surface DO (SSDO), water column average in the up-
per 200 m DO (DOav) and DO at the 200 m (DO200), respectively. The lines are linear regressions. g
Fig. 5. Variation trends in P concentrations in the nSCS during
1989–2004. (a), (b) and (c) show the annual means of sea surface P
(SSP), water column average P in the upper 200 m (Pav) and P in the
200 m layer (P200), respectively. The lines are linear regressions. X. Ning et al.: Long term changes in the northern South China Sea X. Ning et al.: Long term changes in the northern South China Sea 2231 Fig. 4
DO(μmol/L)
1975
1985
1995
2005
250
300
400
450
500
350
(b)
DO(μmol/L)
1975
1985
1995
2005
(a)
250
300
350
400
450
500
550
400
DO(μmol/L)
1975
1985
1995
2005
(c)
300
200
150
250
350
450
DO (μmol L-1)
DO (μmol L-1)
DO (μmol L-1)
(a)
(b)
(c)
Fig. 4. Variation trends in dissolved oxygen (DO) concentration
in the nSCS during 1976–2004. (a), (b) and (c) show the annual
means of sea surface DO (SSDO), water column average in the up-
per 200 m DO (DOav) and DO at the 200 m (DO200), respectively. The lines are linear regressions. Fig. 5
0.2
0.4
0.6
Po4-P(μmol/L)
1992
1996
2000
2004
1988
(a)
0.1
0.3
0.5
1.8
0.8
1.0
1.2
1.4
1.6
Po4-P(μmol/L)
1988
1992
1996
2000
2004
(c)
0.4
0.6
0.8
Po4-P(μmol/L)
1992
1996
2000
2004
1988
0.3
0.5
0.7
0.9
(b)
PO4-P (μmol L-1)
PO4-P (μmol L-1)
PO4-P (μmol L-1)
Fig. 5. Variation trends in P concentrations in the nSCS during
1989–2004. (a), (b) and (c) show the annual means of sea surface P
(SSP), water column average P in the upper 200 m (Pav) and P in the
200 m layer (P200), respectively. The lines are linear regressions. Fig. 4
DO(μmol/L)
1975
1985
1995
2005
250
300
400
450
500
350
(b)
DO(μmol/L)
1975
1985
1995
2005
(a)
250
300
350
400
450
500
550
400
DO(μmol/L)
1975
1985
1995
2005
(c)
300
200
150
250
350
450
DO (μmol L-1)
DO (μmol L-1)
DO (μmol L-1)
(a)
(b)
(c)
Fig. 4. Variation trends in dissolved oxygen (DO) concentration
in the nSCS during 1976–2004. (a), (b) and (c) show the annual
means of sea surface DO (SSDO), water column average in the up-
per 200 m DO (DOav) and DO at the 200 m (DO200), respectively. The lines are linear regressions. Fig. 5
0.2
0.4
0.6
Po4-P(μmol/L)
1992
1996
2000
2004
1988
(a)
0.1
0.3
0.5
1.8
0.8
1.0
1.2
1.4
1.6
Po4-P(μmol/L)
1988
1992
1996
2000
2004
(c)
0.4
0.6
0.8
Po4-P(μmol/L)
1992
1996
2000
2004
1988
0.3
0.5
0.7
0.9
(b)
PO4-P (μmol L-1)
PO4-P (μmol L-1)
PO4-P (μmol L-1)
Fig. 5. Variation trends in P concentrations in the nSCS during
1989–2004. 3.1.1
Seawater temperature and salinity 36
The annual means of SST and Tav (the average temperature
in the water column) exhibited highly significant increasing
trends (p≤0.01), their annual rates were 0.078 and 0.090◦C
y−1, respectively. However, the temperature at 200 m (T200)
showed a significant decreasing trend with an annual rate of
−0.108◦C y−1 during the 29 years of observation between Biogeosciences, 6, 2227–2243, 2009 www.biogeosciences.net/6/2227/2009/ X. Ning et al.: Long term changes in the northern South China Sea
2231 X. Ning et al.: Long term changes in the northern South China Sea X. Ning et al.: Long term changes in the northern South China Sea X. Ning et al.: Long term changes in the northern South China Sea 2232 Table 1. The annual rate, climate trend coefficients (Rxt) and the amplitude of fluctuations of the environmental parameters in the nSCS
during 1976–2004 (units: ◦C y−1 for annual change in temperature, and µmol L−1 y−1for DO and nutrients). X. Ning et al.: Long term changes in the northern South China Sea Annual rate
Raxt
Amplitude of fluctuationb
Mean±SDc
Mean
Mean
Mean
Mean
Mean
Mean
Mean
Mean
Mean
Mean
Mean
Mean
<200 m
>200 m
whole area
<200 m
>200 m
whole area
<200 m
>200 m
whole area
<200 m
>200 m
whole area
SST
0.078
0.063
0.078
0.84A
0.80A
0.89A
2.67
3.28
2.97
24.75±1.82
26.06±0.67
25.63±0.74
Tav
0.089
0.095
0.090
0.80A
0.85A
0.85A
4.07
4.41
3.86
23.49±0.97
22.42±0.95
22.29±0.89
T d
200
0.045
−0.107
0.45B
0.71A
2.90
3.94
20.36±0.86
15.41±1.30
SSS
−0.007
−0.015
−0.022
−0.19
−0.35
−0.41C
3.61
2.30
2.20
33.10±0.78
34.01±0.40
33.55±0.48
Sav
0.008
0.005
0.007
0.32
0.34
0.38C
0.97
0.71
0.74
33.89±0.30
34.22± 0.19
34.05±0.21
Sd
200
0.005
0.005
0.30
0.44B
0.42
0.48
34.34±0.20
34.59±0.10
SSDO
−0.964
−0.927
−0.913
−0.46B
−0.52A
−0.48A
80
85
83
426.5±76.0
425.4±15.0
434.6±15.9
DOav
−1.857
−2.037
−1.926
−0.67A
−0.82A
−0.81A
102
92
94
422.6±33.0
384.9±21.0
402.3±20.3
DOd
200
−2.230
−1.653
−0.70A
−0.63A
99
104
376.3±28.0
317.2±22.4
SSP
−0.011
−0.004
−0.008
−0.41
−0.30
−0.31
0.54
0.76
0.45
0.31±0.14
0.32±0.11
0.32±0.12
Pav
−0.008
−0.007
−0.009
−0.29
−0.35
−0.37
0.32
0.63
0.50
0.39±0.12
0.69±0.17
0.52±0.13
P d
200
−0.006
−0.017
−0.22
−0.50D
0.48
0.78
0.57±0.17
1.19±0.21
SSSi
−0.642
−0.284
−0.071
−0.19
−0.30
−0.22
12.19
12.90
11.50
9.93±3.46
10.33±4.81
10.20±3.71
Siav
−0.284
−0.240
−0.161
−0.24
−0.19
−0.16
17.84
21.46
17.86
11.51±6.05
15.15±7.40
12.64±7.17
Sid
200
−0.038
−0.387
−0.06
−0.19
17.57
32.50
14.98±5.16
26.80±9.53
SSDIN
0.613
0.391
0.504
0.70A
0.63A
0.74A
17.27
9.69
11.85
4.44±4.24
3.73±3.00
4.09±3.29
DINav
0.416
0.343
0.381
0.75A
0.61A
0.73A
9.68
10.53
10.53
4.71±2.63
6.33±2.65
6.71±2.60
DINd
200
0.721
0.813
0.73A
0.64A
15.71
20.13
7.61±4.68
16.08±6.03
SSNO3
0.457
0.342
0.456
0.70A
0.54D
0.66A
12.05
9.56
9.74
2.62±3.09
2.45±2.99
2.55±2.85
NO3av
0.345
0.324
0.335
0.71A
0.58B
0.67A
8.46
9.85
8.13
2.98±2.31
5.09±2.68
4.01±2.37
NOd
3200
0.582
0.711
0.68A
0.55C
13.57
20.55
5.57±4.05
14.53±6.17
SSDIN:SSP
3.61
1.89
2.75
0.55C
0.54D
0.63A
126.55
52.65
84.02
20.5±30.7
14.9±16.8
17.8±21.2
DINav:Pav
1.66
0.75
1.21
0.70A
0.57B
0.69A
40.26
23.24
28.65
14.0±11.3
10.7±6.3
12.3±8.4
DIN200:Pd
200
1.51
1.04
0.74A
0.64A
31.26
27.3
13.8±8.6
14.0±7.8
SSSi:SSDIN
−0.52
−0.46
−0.44
−0.75A
−0.39
−0.67A
12.32
22.68
9.69
4.1±3.3
5.4±5.7
4.3±3.2
Siav:DINav
−0.46
−0.15
−0.22
−0.71A
−0.46
−0.70A
13.05
5.62
5.41
3.4±3.1
2.9±1.6
2.8±1.5
Si200:DINd
200
−0.32
−0.10
−0.75A
−0.65A
7.29
2.8
2.87±2.02
1.85±0.8
a Significance: A p≤0.01, B p≤0.02, C p≤0.03, and D p≤0.05
b The amplitude is the difference between maximal annual mean value and minimal one during the observation period. X. Ning et al.: Long term changes in the northern South China Sea It was also found that the annual rate
of DIN was higher in the shallow (<200 m) than in the deep
water (>200 m) areas (Table 1). The spatial-temporal dis-
tributions of the DINav (mean concentrations in the water
column) indicated that since 1998 the high DINav concen- (Fig. 7, Table 1). The annual rates of the increases in DIN and
NO3-N were 0.38 to 0.81 and 0.34 to 0.71 µmol L−1 y−1,
respectively. The regional means of DIN values of the sea
surface, water column average and 200 m layer increased by
8.06, 6.10 and 13.01 µmol L−1, respectively. DIN concentra-
tions in the 200 m layer were significantly higher than those
of the average for the water column and at the sea surface. Its annual rate was 1.61 times greater than that of the sea
surface. And its multi-year average value during observa-
tion period was 3.9 times higher than that of the sea surface
layer. The Rxt value for the time series of NO3-N and DIN
were between 0.55–0.74. The variation trends of the NO3-N
and DIN time series were highly significant (p≤0.01) (Ta-
ble 1), except for the time series of NO3-N200, which was
significant (p≤0.03). It was also found that the annual rate
of DIN was higher in the shallow (<200 m) than in the deep
water (>200 m) areas (Table 1). The spatial-temporal dis-
tributions of the DINav (mean concentrations in the water
column) indicated that since 1998 the high DINav concen- trations have exhibited a pronounced large area increasing,
i.e. after 1998 in the most study area, DINav concentration
>5.71 µmol L−1 (Fig. 8, the shadow shows the area), and ex-
ceeded the low limit of suitable N concentration for diatom
growth (5.71 µmol L−1, Chu, 1949). So it is favorable for
phytoplankton growth in the most study area. X. Ning et al.: Long term changes in the northern South China Sea It indicates quantita-
tive characteristics of the fluctuation of a parameter during the observation period. Standard deviation is symbolized as Xδn−1. c Mean±SD is the multi-year’s mean value and standard deviation during the observation period. d When the water depth <200 m, data for the bottom layer are used; when the water depth >200 m, data for 200 m were used. a Significance: A p≤0.01, B p≤0.02, C p≤0.03, and D p≤0.05
b g
p≤
,
p≤
,
p≤
,
p≤
b The amplitude is the difference between maximal annual mean value and minimal one during the observation period. It indicates quantita-
tive characteristics of the fluctuation of a parameter during the observation period. Standard deviation is symbolized as Xδn−1. c Mean±SD is the multi-year’s mean value and standard deviation during the observation period. d When the water depth <200 m, data for the bottom layer are used; when the water depth >200 m, data for 200 m were used. g
p≤
,
p≤
,
p≤
,
p≤
b The amplitude is the difference between maximal annual mean value and minimal one during the observation period. It indicates quantita-
tive characteristics of the fluctuation of a parameter during the observation period. Standard deviation is symbolized as Xδn−1. c Mean±SD is the multi-year’s mean value and standard deviation during the observation period. d When the water depth <200 m, data for the bottom layer are used; when the water depth >200 m, data for 200 m were used. (Fig. 7, Table 1). The annual rates of the increases in DIN and
NO3-N were 0.38 to 0.81 and 0.34 to 0.71 µmol L−1 y−1,
respectively. The regional means of DIN values of the sea
surface, water column average and 200 m layer increased by
8.06, 6.10 and 13.01 µmol L−1, respectively. DIN concentra-
tions in the 200 m layer were significantly higher than those
of the average for the water column and at the sea surface. Its annual rate was 1.61 times greater than that of the sea
surface. And its multi-year average value during observa-
tion period was 3.9 times higher than that of the sea surface
layer. The Rxt value for the time series of NO3-N and DIN
were between 0.55–0.74. The variation trends of the NO3-N
and DIN time series were highly significant (p≤0.01) (Ta-
ble 1), except for the time series of NO3-N200, which was
significant (p≤0.03). 3.1.3
Concentrations of P, Si and N Both P and Si concentrations exhibited decreasing trends
(Figs. 5 and 6, Table 1). Their annual rates were −0.008 to
−0.017 (for P) and −0.071 to −0.387 (for Si) µmol L−1 y−1,
respectively. 38
The concentrations of NO3-N, NO2-N, NH4-N and dis-
solved inorganic nitrogen (DIN = NO3 + NO2+NH4) in the
nSCS exhibited obvious increasing trends during 1989–2004 www.biogeosciences.net/6/2227/2009/ Biogeosciences, 6, 2227–2243, 2009 3.1.4
Nutrient ratios During 1989–2004, the nutrient ratios (DIN:P and Si:DIN)
changed significantly. For example, the DIN:P ratios in-
creased by 1.04–2.75 y−1, and it reached 28.1 (SSDIN:SSP)
and 18.0 (DINav:Pav) in 2004 (Fig. 9, Table 1); the Si: N ra-
tios decreased at an annual rate of −0.10∼−0.44 y−1, and it
dropped to 1.0 (SSSi:SSDIN) and 1.4 (Siav:DINav) in 2004
(Fig. 10, Table 1). The variation trends of all time series of
N: P and Si: N were significant (p≤0.01). The annual rates
of increase in DIN:P and decrease in Si:DIN were higher in
the shallow coastal waters (<200 m) than in deep continental
shelf waters (>200 m) (Table 1). Biogeosciences, 6, 2227–2243, 2009 www.biogeosciences.net/6/2227/2009/ X. Ning et al.: Long term changes in the northern South China Sea
2233 X. Ning et al.: Long term changes in the northern South China Sea 2233 Fig. 6
20
1988
1992
1996
2004
2000
(b)
5
10
15
25
30
Sio3-Si(μmol/L)
20
1988
1992
1996
2004
2000
10
30
40
50
(c)
Sio3-Si(μmol/L)
4
8
12
16
20
24
1988
1992
1996
2004
2000
(a)
Sio3-Si(μmol/L)
SiO3-Si (μmol L-1)
SiO3-Si (μmol L-1)
SiO3-Si (μmol L-1)
Fig. 6. Variation trends in Si concentrations in the nSCS during
1989–2004. (a), (b) and (c) show the annual means of sea surface
Si (SSSi), water column average Si in the upper 200 m (Siav) and
Si in the 200 m layer (Si200), respectively. The lines are linear re-
gressions. Fig. 7
8
4
0
1988
1992
1996
2000
2004
(a)
DIN(μmol/L)
12
16
10
8
6
4
2
1988
1992
1996
2000
2004
DIN(μmol/L)
12
(b)
0
10
1988
1992
1996
2000
2004
DIN(μmol/L)
(c)
5
15
20
25
30
DIN (μmol L-1)
DIN (μmol L-1)
DIN (μmol L-1)
Fig. 7. Variation trends in dissolved inorganic nitrogen (DIN) con-
centrations in the nSCS during 1989–2004. (a), (b) and (c) show
the annual means of sea surface DIN (SSDIN), water column aver-
age DIN in the upper 200 m (DINav) and DIN at the 200 m layer
(DIN200), respectively. The lines are linear regressions. 8
4
0
1988
1992
1996
2000
2004
(a)
DIN(μmol/L)
12
16
DIN (μmol L-1) 4
8
12
16
20
24
1988
1992
1996
2004
2000
(a)
Sio3-Si(μmol/L)
SiO3-Si (μmol L-1) Fig. 3.1.4
Nutrient ratios 7
8
4
0
1988
1992
1996
2000
2004
(a)
DIN(μmol/L)
12
16
10
8
6
4
2
1988
1992
1996
2000
2004
DIN(μmol/L)
12
(b)
0
10
1988
1992
1996
2000
2004
DIN(μmol/L)
(c)
5
15
20
25
30
DIN (μmol L-1)
DIN (μmol L-1)
DIN (μmol L-1)
Fig. 7. Variation trends in dissolved inorganic nitrogen (DIN) con-
centrations in the nSCS during 1989–2004. (a), (b) and (c) show
the annual means of sea surface DIN (SSDIN), water column aver-
age DIN in the upper 200 m (DINav) and DIN at the 200 m layer
(DIN200), respectively. The lines are linear regressions. 20
1988
1992
1996
2004
2000
(b)
5
10
15
25
30
Sio3-Si(μmol/L)
SiO3-Si (μmol L-1) 10
8
6
4
2
1988
1992
1996
2000
2004
DIN(μmol/L)
12
(b)
0
DIN (μmol L-1) DIN(μmo
DIN (μmol L-1) Fig. 6
20
1988
1992
1996
2004
2000
10
30
40
50
(c)
Sio3-Si(μmol/L)
SiO3-Si (μmol L-1) 10
1988
1992
1996
2000
2004
DIN(μmol/L)
(c)
5
15
20
25
30
DIN (μmol L-1) Fig. 6. Variation trends in Si concentrations in the nSCS during
1989–2004. (a), (b) and (c) show the annual means of sea surface
Si (SSSi), water column average Si in the upper 200 m (Siav) and
Si in the 200 m layer (Si200), respectively. The lines are linear re-
gressions. Fig. 7
Fig. 7. Variation trends in dissolved inorganic nitrogen (DIN) con-
centrations in the nSCS during 1989–2004. (a), (b) and (c) show
the annual means of sea surface DIN (SSDIN), water column aver-
age DIN in the upper 200 m (DINav) and DIN at the 200 m layer
(DIN200), respectively. The lines are linear regressions. 4.1
Increasing trends and the response of the ecosystem The shadow shows the area and period
of time which exceeded the low limit of suitable N concentration for diatom growth (5.71 µmol L−1, Chu, 1949). Table 2. Changes in annually average concentration (µmol dm−3) of NO3-N and dissolved inorganic nitro
the Pearl River and other rivers. age concentration (µmol dm−3) of NO3-N and dissolved inorganic nitrogen (DIN), from discharges of Table 2. Changes in annually average concentration (µmol dm−3) of NO3-N and dissolved inorganic nitrogen (DIN), from discharges of
the Pearl River and other rivers. Observation year
Location of the observation
Parameter, value
Data source
1980–1985
southern Hong Kong waters (outside Pearl River estuary)
NO3-N, 1-3
Han et al. (1990)
1986
Pearl River estuary
DIN, 19.3
He et al. (2004)
1987
Pearl River estuary
NO3-N, 28.0 DIN, 31.7
Wang and Peng (1996)
1990
Pearl River estuary
DIN, 34.6
He et al. (2004)
1995
Pearl River estuary
DIN, 36.4
He et al. (2004)
1996
Pearl River estuary
NO3-N, 39.8
Cai (2002b)
1999 (summer)
Pearl River estuary
NO3-N, 48.9 DIN, 51.9
Lin et al. (2004)
1999 (summer)
Pearl River estuary
DIN, 53.2
Guan et al. (2003)
2002
Pearl River estuary
DIN, 76.4
He et al. (2004) Observation year
Location of the observation Asia, associated with predicted large increases in population,
will increase fertilizer use, and increased industrialization. DIN from the Pearl River discharge increased by 3 times in
2002 as compared to 1986 (He et al., 2004, Table 2), and
NO3-N input from the Pearl in River Estuary was 1.7 times
in 1999 of that in 1987 (Guan et al., 2003; Wang and Peng,
1996, Table 2). SSDIN should be influenced by the increase
in DIN from the river discharges. In addition, significant in-
puts of DIN into the nSCS have also occurred through atmo-
spheric dry and wet deposition (Zhang et al., 1999) and the
upwelling of the deep waters (Zhao et al., 2005). The mitiga-
tion of N limitation in the upper layer since 1998 was clearly
related to these DIN inputs (Fig. 8). The increase in the
annual rate of DIN was higher at the 200 m layer (DIN200)
than the water column average (DINav) and at the sea surface
layer (SSDIN). This may be due to possible strengthening of
the deep water upwelling. 4.1
Increasing trends and the response of the ecosystem 40
The increase in DIN in the nSCS during 1989–2004 was
consistent with the increase in DIN along the coast of the
nSCS, such as Qinzhou Bay (Wei et al., 2002, 2003) and
Daya Bay (Qiu et al., 2005). It also shows the same trend
with the rise of DIN observed throughout the global marginal
seas (Seitzinger et al., 2002). Along with the rapid economic
development in China, DIN concentration in the Pearl River
estuary and shelf of the China Sea has been dramatically in-
creased, due to the increasing urbanization near the coastal
areas, which resulted in more municipal sewage, agricul-
tural fertilizer, mariculture waste etc. inputs (SOAC, 2001). Through analysis based on DIN and PN models, combin-
ing with spatially explicit global databases, Seitzinger et
al. (2002) showed that DIN input rates increased from ap-
proximately 21 Tg N y−1 in 1990 to 47 Tg N y−1 by 2050. The largest increases are predicted for Southern and Eastern 39
The positive increasing trends in SST and Tav in the nSCS
during 1976–2004 are consistent with the increasing trends
in the mean air temperature (AT) observed throughout the
Northern Hemisphere (Houghton et al., 1996; Fu et al.,
2006), South China (Chen et al., 1998; Chen et al., 1999;
Zhai and Ren, 1997) and the annual means of AT and SST
observed along the coast of the SCS (He et al., 2003; Martin
and Arun, 2003). The increasing trends were also in phase
with the changes in SST observed along the coast of the Yel-
low Sea and Bohai Sea (Lin et al., 2005, 2001). However,
these annual rates of water temperature change were higher
in the nSCS than in the Yellow Sea and the Bohai Sea (Lin et
al., 2005, 2001). www.biogeosciences.net/6/2227/2009/ Biogeosciences, 6, 2227–2243, 2009 X. Ning et al.: Long term changes in the northern South China Sea X. Ning et al.: Long term changes in the northern South China Sea X. Ning et al.: Long term changes in the northern South China Sea 2234 Fig. 8. Fig. 8. The spatial-temporal distributions of the DINav (mean concentrations in the water column). The shadow shows the area and period
of time which exceeded the low limit of suitable N concentration for diatom growth (5.71 µmol L−1, Chu, 1949). Fig. 8. Fig. 8. The spatial-temporal distributions of the DINav (mean concentrations in the water column). 4.1
Increasing trends and the response of the ecosystem 9
1988
1992
1996
2000
2004
0
10
20
30
40
N:P
(c)
1988
1992
1996
2000
2004
0
20
40
N:P
(a)
60
80
100
1988
1992
1996
2000
2004
0
10
20
30
40
N:P
(b)
N : P
N : P
N : P
Fig. 9. Variation trends in N: P ratios in the nSCS during 1989–
2004. (a), (b) and (c) show the annual means of sea surface DIN:P
(SSDIN:SSP), water column average DIN:P in the upper 200 m
(DINav:Pav) and DIN:P at the 200 m layer (DIN200:P200), respec-
tively. The lines are linear regressions. Fig. 10
0
2
Si:N
1988
1992
2000
2004
4
6
8
10
12
(a)
1996
0
1
2
3
4
1988
1992
2000
2004
(b)
5
Si:N
1996
0
1
2
3
4
Si:N
1988
1992
2000
2004
(c)
1996
Si : N
Si : N
Si : N
Fig. 10. Variation trends in Si: N ratios in the nSCS during 1989–
2004. (a), (b) and (c) show the annual means of sea surface Si:N
(SSSi: SSDIN), water column average Si:N in the upper 200 m
(Siav:DINav) and Si:N at the 200 m layer (Si200:DIN200), respec-
tively. The lines are linear regressions. 0
2
Si:N
1988
1992
2000
2004
4
6
8
10
12
(a)
1996
Si : N 1988
1992
1996
2000
2004
0
20
40
N:P
(a)
60
80
100
N : P Fig. 9
1988
1992
1996
2000
2004
0
10
20
30
40
N:P
(c)
1988
1992
1996
2000
2004
0
20
40
N:P
(a)
60
80
100
1988
1992
1996
2000
2004
0
10
20
30
40
N:P
(b)
N : P
N : P
N : P
Fig. 9. Variation trends in N: P ratios in the nSCS during 1989–
2004. (a), (b) and (c) show the annual means of sea surface DIN:P
(SSDIN:SSP), water column average DIN:P in the upper 200 m
(DINav:Pav) and DIN:P at the 200 m layer (DIN200:P200), respec-
tively. The lines are linear regressions. Fig. 10
0
2
Si:N
1988
1992
2000
2004
4
6
8
10
12
(a)
1996
0
1
2
3
4
1988
1992
2000
2004
(b)
5
Si:N
1996
0
1
2
3
4
Si:N
1988
1992
2000
2004
(c)
1996
Si : N
Si : N
Si : N
Fig. 10. Variation trends in Si: N ratios in the nSCS during 1989–
2004. 4.1
Increasing trends and the response of the ecosystem (a), (b) and (c) show the annual means of sea surface Si:N
(SSSi: SSDIN), water column average Si:N in the upper 200 m
(Siav:DINav) and Si:N at the 200 m layer (Si200:DIN200), respec-
tively. The lines are linear regressions. 0
1
2
3
4
1988
1992
2000
2004
(b)
5
Si:N
1996
Si : N 1988
1992
1996
2000
2004
0
10
20
30
40
N:P
(b)
N : P Fig. 9
1988
1992
1996
2000
2004
0
10
20
30
40
N:P
(c)
N : P Fi
10
0
1
2
3
4
Si:N
1988
1992
2000
2004
(c)
1996
Si : N g
Fig. 9. Variation trends in N: P ratios in the nSCS during 1989–
2004. (a), (b) and (c) show the annual means of sea surface DIN:P
(SSDIN:SSP), water column average DIN:P in the upper 200 m
(DINav:Pav) and DIN:P at the 200 m layer (DIN200:P200), respec-
tively. The lines are linear regressions. Fig. 10
Fig. 10. Variation trends in Si: N ratios in the nSCS during 1989–
2004. (a), (b) and (c) show the annual means of sea surface Si:N
(SSSi: SSDIN), water column average Si:N in the upper 200 m
(Siav:DINav) and Si:N at the 200 m layer (Si200:DIN200), respec-
tively. The lines are linear regressions. detergent ban in the late 1990s, and phytoplankton great con-
sumption. 43
et al., 2003). Particularly in 2002, the strongest typhoon rain
storm resulted in the historically greatest flood of the Pearl
River, and therefore, which induced in the lowest SSS (32.2)
in the nSCS during that study period (Lei et al., 2003; Xie
and Zhang, 2003). In 1999, the SSS was below normal due
to the influence of frequent typhoon rain storms (total of 28
typhoons, including 7 landed ones). Furthermore, in the sum-
mer of 1999, the Dongsha upwelling (Station 3–6) was weak
and the depth of the 20◦C isopleth was deeper than 105 m. However in 1998, although large floods of the Pearl River oc-
curred, the SSS did not decrease, due to the strong upwelling
as indicated by shallow (84 m) depth of the 20◦C isopleths. Chai et al. (2001) pointed out that the strength of upwelling
can be indicated by the depth of 20◦C temperature isopleth
in the SCS. In this study, the range depth of 20◦C isopleths is
84–150 m. 4.1
Increasing trends and the response of the ecosystem However, during 1989–1997, the
DINav was low, the multi-annual mean was 4.2 µmol L−1
and the lowest value of DINav was only 1.7 µmol L−1 in
1989. Since 1998, the multi-annual mean of the water col-
umn average DIN concentration has reached 7.7 µmol L−1
and the maximal value of the annual mean even reached up
to 10.8 µmol L−1 in 2001 (Fig. 7b). And therefore, the pre-
vious status of N limitation in the nSCS was significantly al- leviated. Moreover, the increases in the annual rates of DIN
and NO3-N were much higher in the nSCS than in the Yellow
Sea (Lin et al., 2005). The increase in the N:P ratio was due to an increase in
DIN and a decrease in P concentration. Before 1997, the N:P
ratios (SSN:SSP, DINav:Pav and DIN200:P200) were lower
than 10. Since 1998, these ratios have rapidly increased to
28, 18 and 16 in 2004, respectively (Fig. 9). In 2004, the
average values of DINav:Pav and DIN200:P200 were close to
the Redfield ratio (16:1), and therefore favorable to phyto-
plankton growth (Richardson, 1997; Hu et al., 1989; Jiang
and Yao, 1999). The high value of SSN:SSP (28) in 2004
was probably due to dry and wet deposition. The peak value
of SSN:SSP appeared in 2002 (up to 86, Fig. 8a), due mainly
to high rainfall as high Pearl River discharge, and this high
ratio corresponded to the lowest surface salinity (Fig. 3a). Moreover, the fact that the annual rates of DIN and DIN:P
were higher in the shallow water area (<200 m) than in the
deep water area (>200 m) (Table 1) suggested the influence
of the anthropogenic activities on the ecosystems of the shal-
low or coastal waters. These results agree well with Xu et
al. (2008) who also showed that N input from the Pearl River
has caused the nSCS to become P-limited. Phosphate de-
cline may be ascribed to the implementation of phosphate Biogeosciences, 6, 2227–2243, 2009 www.biogeosciences.net/6/2227/2009/ X. Ning et al.: Long term changes in the northern South China Sea X. Ning et al.: Long term changes in the northern South China Sea 2235 Fig. 4.1
Increasing trends and the response of the ecosystem However, the high SSS in 1993 was probably due
to the reduced Pearl River discharge (Lei et al., 2003). 4.3
Nutrient limitation The decreasing trend in DO concentration (Fig. 4) can
probably be attributed to three reasons. First, a small de-
crease in DO solubility in the seawater can be induced by
the increase in the seawater temperature (Fig. 2). Second, an
increase in DO consumption was an important reason. The
DO consumption resulted from the decomposition of organic
matter originating mainly from the Pearl River discharge and
the decay of phytoplankton blooms with increasing frequen-
cies in the coastal water was increasing (Peng, 1994; Tang et
al., 2006). Before 1998, HABs (harmful algae blooms) oc-
curred once or twice a year, but during 1998 to 2003, blooms
increased to 10 to 20 a year in the nSCS (Tang et al., 2006),
which was in phase with the dramatic decreasing trend in
DO (Fig. 4). Third, the mixing between the surface water
and the deep layers was reduced by the stronger thermocline,
due to the rapid rise in SST since 1995 (Fig. 2a), which re-
sulted in less transfer of oxygen from the atmosphere to the
deeper waters. In addition in 1998, the lowest value of DO
was probably attributed to the strongest upwelling, which oc-
curred that year. According to the compute of this study, in
1998 the depth of the 20◦C isopleths was 84 m, it show the
strongest upwelling (see above paragraph and references in
it). Since 1998, DIN concentrations have exhibited a pronounced
increasing trend, and therefore the key nutrient concentra-
tion and ratio can be divided into two phases, i.e. before
and after 1997 (Table 3). In the first phase, the average
DIN concentration was very low, SSDIN and DINav were
1.83 and 4.22 µmol L−1, respectively, which were lower than
the low limit of suitable N concentration for diatom growth
(5.71 µmol L−1, Chu, 1949). The average P concentration
was also low. SSP and Pav were 0.33 and 0.56 µmol L−1,
respectively, which is closed to the low limit of suitable P
concentration for diatom growth (0.48 µmol L−1, Zhao et
al., 2000). However, Si concentrations (SSSi and Siav were
12.38 and 14.82 µmol L−1, respectively) outclassed the low
limit of suitable for diatom growth (4.40 µmol L−1, Harvey,
1957). Therefore, Si pool was sufficient. N:P ratios were
less than half the Redfield ratio of 16 (SSN:SSP and Nav:Pav
were 5.5 and 7.5, respectively). 4.2
Decreasing trends and the response of the ecosystem 42
The decreasing trends in SSS may be related to the increase
in the freshwater discharge into the nSCS since 1990 and
less vertical mixing, because of the presence of a perma-
nent thermocline (Yuan and Deng, 1997a, b; Shi, 2001). It has previously been observed that the occurrence of low
SSS often corresponded to abnormally high discharge of the
Pearl River (Xie and Zhang, 2003; Xu et al., 2008). During
the period of 1990 to 2000, the Pearl River runoff increased
by 22.5% in comparison with the mean discharge for 1960–
1999 (Lei et al., 2003). In the 1990s (i.e. 1994, 1995, 1996,
1997, 1999) and 2002, SSS in the nSCS was especially low
(Fig. 3a), which was induced by the Pearl River floods (Lei Biogeosciences, 6, 2227–2243, 2009 www.biogeosciences.net/6/2227/2009/ 2236 X. Ning et al.: Long term changes in the northern South China Sea ton biomass and production (Table 3) and decreased the P
concentration. Hong et al. (1983) reported that diatoms can
take up 30 times more P than they need and store it for use
when P is deficient. In the most years except for 1994 and
2000, the concentrations of P in the upper layer (<75 m)
were even lower than the P concentration required for di-
atom growth (<0.1 µmol L−1, Zou et al., 1983). The de-
creasing trends in Si concentration have probably been influ-
enced by the decrease in Si concentration in the Pearl River
runoff since the 1970s (Lei et al., 2003), but still adequate
for diatom growth. At 200 m, the interannual fluctuations of
both P and Si concentrations were high (Figs. 5 and 6) and
probably attributed to the interannual changes in upwelling
of deep water. In 1996 and 1999 the depth of the 20◦isopleth
was shallow (average value from multi-stations was 93 m),
high concentrations of P and Si occurred (Pav=0.61 and
0.62 µmol L−1, P200=1.154 and 1.31 µmol L−1, Siav=25.18
and 19.55 µmol L−1, and Si200=42.12 and 44.28 µmol L−1,
respectively, in the two years). In contrast, during 2004
when the depth of the 20◦isopleth was deep ((the aver-
age value from multi-stations was 105 m), concentrations
of P (Pav=0.37 µmol L−1, and P200=1.13 µmol L−1) and Si
(Siav=8.9 µmol L−1, and Si200=20.5 µmol L−1) were obvi-
ously low. Table 3. 4.2
Decreasing trends and the response of the ecosystem Comparison of key nutrient concentration (µmol L−1) and
the ratio between the two phases (before 1997, the first phase and
after 1997, the second phase). Nutrient and ratio
mean in the
mean in the
first phase
second phase
SSDIN
1.83
5.75
DINav
4.22
7.78
DIN200
12.77
20.33
SSP
0.33
0.25
Pav
0.56
0.48
P200
0.49
0.48
SSSi
12.38
9.19
Siav
14.82
12.26
Si200
27.54
25.85
SSDIN:SSP
5.5
23.0
DINav:Pav
7.5
16.2
DIN200:P200
26.1
42.4
SSSi:SSDIN
6.3
1.6
Siav:DINav
2.5
1.6
Si200:DIN200
2.2
1.3
SSSi:SSP
37.5
36.8
Siav:Pav
26.5
25.5
Si200:P200
56.2
53.8 Nutrient and ratio
mean in the
mean in the
first phase
second phase 4.3
Nutrient limitation Whenever a medium and
strong El Nino occurs, the summer monsoon is weak (Zhang
et al., 2003; Zhu et al., 2000), which induces strengthened
cyclonic eddy, leading to strong upwelling, resulting in low
Tav and DOav, and high Sav, nutrients and Chl-a induced by
phytoplankton growth. During a La Nina event, the opposite
occurs. This is due to ENSO/La Nina events affecting the
strength of the summer monsoon related, i.e. both El Nino
and La Nina make the anomaly of Walker circulation. Dur-
ing El Nino event, the heat convection of the western Pacific
warm pool moves to the central Pacific. In contrast, dur-
ing La Nina event, the heat convection of the western Pa-
cific warm pool moves back to the western Pacific. That
makes the anomaly of Walk circumfluence. Namely, when
phase of El Nino (La Nina) fall under the influence of the
anomal Walker circulation, subsidence (ascending) air cur-
rent occur over low latitude and middle latitude of the east
Asia. If this subsidence (ascending) air current flow over
low latitude occurred in summer, and joined to west-south
monsoon, it will result in weaken (strengthen) of the sum-
mer monsoon (Wang et al., 2001; Zhang et al., 2003; Zhu et
al., 2000). The changes of ecological environment in the sea
area around Station 4 in summer respond to ENSO events,
namely, respond to the abnormity of summer monsoon. Based on the studies on the kinetics of nutrient uptake,
the thresholds of SiO3-Si, DIN and PO4-P for phytoplankton
growth have been estimated to be 2.0, 1.0 and 0.1 µmol L−1,
respectively (Justic et al., 1995). In the study area, the val-
ues of all the nutrient parameters were over these thresh-
old concentrations, except for those in the first phase, when
the mean of SSDIN was close to the threshold of N (Ta-
ble 3). According to chemical stoichiometry, in the first
phase both SSN:SSP and DINav:Pav was lower than 10,
and SSSi:SSDIN, Siav:DINav and Si200:DIN200 were all
over 1, indicating potential N limitation. In the second
phase SSDIN:SSP and DIN200:P200 were higher than 22, and
DINav:Pav was equal to the Redfield ratio. All Si:DIN ra-
tios ranged from 1.3 to 1.6, and Si:P ranged 25.5 to 53.8
(Table 3), which indicated that the potential of P limitation
increased and N limitation decreased. 4.3
Nutrient limitation In contrast, for the second phase after 1997, the aver-
age DIN concentration has been clearly increasing (SSDIN
and DINav were 5.75 and 7.78 µmol L−1 respectively, Ta-
ble 3), and exceeded the low limit of suitable N concentra-
tion for diatom growth (5.71 µmol L−1, Chu, 1949). How-
ever, the average P concentration has decreased (SSP and
Pav, were 0.25 and 0.48 µmol L−1, respectively, Table 3). Consequently, there was a rapid increase in DIN:P ratios (SS-
DIN:SSP and DINav:Pav were 23.0 and 16.2, respectively,
Table 3), which were close to the Redfield ratio (16, Richard-
son, 1997; Hutchins et al., 1998). Therefore, it is favorable to The decrease in P concentration is probably due to up-
take by phytoplankton and less P supply from deep water,
due to the presence of the permanent thermocline (Yuan and
Deng, 1997a, b; Shi et al., 2001). Furthermore, since 1998,
the reduction in N limitation has increased the phytoplank- Biogeosciences, 6, 2227–2243, 2009 www.biogeosciences.net/6/2227/2009/ 2237 X. Ning et al.: Long term changes in the northern South China Sea 0.31 psu; PO4–Pav, SiO3–Siav and DINav were higher by
0.15, 6.41 and 3.42 µmol L−1, respectively. Surface Chl-a
concentration was higher by 0.14 mg m−3, i.e. higher by 1.8
times (Table 5), even higher by 0.83 times than the average
value of normal years (1980, 1990, 2000, 2001 and 2003, for
which mean Chl-a=0.12±0.05 mg m−3). phytoplankton growth in the second phase. Si concentration
also decreased (SSSi and Siav were 9.19 and 12.26) in this
phase, resulting in rapid decrease in the ratio of SSSi:SSDIN
(Siav:DINav), from 6.3 (2.5) to 1.6 (1.6) (Table 3). The Si
concentration decrease may be due to the dam of the river
courses which significantly reduced the silica delivery to the
SCS. 0.31 psu; PO4–Pav, SiO3–Siav and DINav were higher by
0.15, 6.41 and 3.42 µmol L−1, respectively. Surface Chl-a
concentration was higher by 0.14 mg m−3, i.e. higher by 1.8
times (Table 5), even higher by 0.83 times than the average
value of normal years (1980, 1990, 2000, 2001 and 2003, for
which mean Chl-a=0.12±0.05 mg m−3). According to Takano et al. (1998) and Liao et al. (2006),
there is a cyclonic eddy in the sea area around Station 4 (near
Dongshan Islands) in summer. 4.4
Response of ecological environment to ENSO events When El Nino occurs, the warm pool of the western Pacific
Ocean moves eastward, whereas it moves westward during
La Nina (White et al., 1985; Takeuchi, 1987; Zhang and
Huang, 1993). The nSCS is located to the west of the warm
pool – however the response of the nSCS has not been well
documented. In the present study, pronounced responses to
ENSO were found. During the observation period, 9 El Nino
events (1976, 1982–1983, 1986–1987, 1991, 1993, 1994,
1997, 2002 and 2004) and 4 La Nina events (1981, 1988,
1995 and 1998–1999) occurred (Wang and Gong, 1999; Qin,
2003; Mcphaden, 2004; Levimson, 2005). In general, when-
ever El Nino/La Nina occurred, SST and Tav was low/high
in the nSCS (Fig. 2, Table 4). 4.3
Nutrient limitation Furthermore, Si was
always sufficient during the observation period, even in the
second phase when its concentration decreased (Table 3). X. Ning et al.: Long term changes in the northern South China Sea 2238 Table 4. Annual mean of SST and Tav along transect N in the nSCS in El Nino and La Nina years during 1976–2004 (unit: ◦C). El Nino Year
1976
1982 (1983)
1986 (1987)
1991
1993
1994
1997
2002
2004
Mean±SDa
SST
23.87
25.82 (25.09)
25.60 (25.73)
25.08
25.89
26.24
25.77
26.66
26.25
25.64±0.75
Tav
21.13
21.40 (23.40)
23.09 (23.32)
21.46
21.65
21.79
22.25
22.98
22.62
22.27±0.83
La Nina Year
1981
1988
1995
1998
1999
SST
25.09
25.68
25.74
26.61
26.21
25.87±0.58
Tav
23.28
23.72
24.00
24.07
24.51
23.92±0.46
a Mean±SD is the multi-year mean value ± (SD), standard deviation for the El Nino/La Nina years. an of SST and Tav along transect N in the nSCS in El Nino and La Nina years during 1976–2004 (unit: ◦C). able 4. Annual mean of SST and Tav along transect N in the nSCS in El Nino and La Nina years during 1976– a Mean±SD is the multi-year mean value ± (SD), standard deviation for the El Nino/La Nina years. Fig. 11
Fig. 11. The interannual changes of the water column average values of the seawater temperature (a), (b) salinity, (c) PO4-P, (d) SiO3-Si,
(e) DIN and (f) DO at the Station 4 of the nSCS in summer during the observation period. Fig. 11
Fig. 11. The interannual changes of the water column average values of the seawater temperature (a), (b) salinity, (c) PO4-P, (d) SiO3-Si,
(e) DIN and (f) DO at the Station 4 of the nSCS in summer during the observation period. in stock and production of low trophic levels, induced by the
alleviation in N limitation in the nSCS. In second phase our above observations were consistent
with the results obtained by the multidisciplinary investiga-
tions for assessing the environmental health status and the
fisheries environment quality in the nSCS during 1997–2002
(Jia et al., 2005). They reported that both primary produc-
tion and phytoplankton abundance were relatively high, av-
eraging 409.7 mg C m−2 d−1, and 837×103 cell m−3, respec-
tively (according to criterion of grade of primary produc-
tivity and diet organism lever of China, Jia et al., 2003). The benthic biomass (averaging 11.3 g m−2) was at nor-
mal levels (the same as above row, Jia et al., 2003). 4.5
Response of the ecosystem and living resources Although the frequency of observation on various biol-
ogy and fisheries parameters was less than that for physi-
cal and chemical parameters which reflected the long term
changes in the environmental processes, some responses of
the ecosystems to these environmental changes in the nSCS
was still evident. Comparing the biology and fisheries data between the
two phases discussed above, the average values of Chl-
a, primary production, phytoplankton abundance, benthos
biomass, cephalopod catch and demersal trawl catch in-
creased by 6.4, 1.4, 2.4, 0.7, 7.2 and 2.8 times, respectively
during the second phase, except for zooplankton abundance
which decreased by about 50% (Table 6). The decrease in
zooplankton abundance may be related to the increase in its
predators, such as fish, cephalopods, etc. (Fig. 12). The Rxt
values of the cephalopod and demersal trawl catches were
0.89 and 0.88, respectively, and highly significant (p≤0.01). Besides, the increase in both the cephalopod and demersal
trawl catches partially could be attributed to the improvement
of demersal trawl fishing techniques, and also to the increase In the area southwest of Dongsha Islands, near Station 4
(19–20◦N, 116–117◦E, Fig. 1), pronounced responses to
ENSO were observed. In general, whenever an El Nino
event occurred, Tav and DO were lower, and that the Sav,
nutrients (PO4–Pav, SiO3–Siav and DIN), sea surface Chl-
a were higher (Table 5, Fig. 11). During a La Nina event,
contrary, Tav and DO were higher, and that the Sav, nutri-
ents (PO4–Pav, SiO3–Siav and DIN), sea surface Chl-a were
lower (Table 5, Fig. 11). The fluctuations of environmental
parameters evident corresponded to El Nino/La Nina events. Furthermore, in comparison of the average values of the en-
vironmental parameters in summer of El Nino years with
those of La Nina years, Tav and DOav were lower by 1.89◦C
and 20.2 µmol L−1, respectively; while Sav was higher by www.biogeosciences.net/6/2227/2009/ Biogeosciences, 6, 2227–2243, 2009 X. Ning et al.: Long term changes in the northern South China Sea X. Ning et al.: Long term changes in the northern South China Sea Parameter
First phase (data source)
Second phase (data source)
Chl-a
0.29 (Fan 1985)b
2.16 (Cai et al., 2002a; SOAC 2003)c
PP
185.7 (Fan, 1985)b
442.5 (Ning et al., 2004; Jia et al., 2005; Hao et al., 2007)
PA
161.5 (Lin, 1985)
542.7 (Jia et al., 2005)b
ZB
44.5 (Zhang, 1984; Chen, 1985; Qian et al., 1990a, b;
Huang et al., 1990; Chen, 1992)
22.05 (Jia et al., 2005)d
BB
6.6 (Shen, 1985)
11.3 (Jia et al., 2005)
CC
1.2 (Guo and Chen, 2000)
9.8 (Guo and Chen, 2000)
DTC
89.0 (Guo and Chen, 2000)
334.8 (Guo and Chen, 2000) Parameter
First phase (data source) a The data listed in the Table are the mean of the water column of the multi-year average for each parameter from different sources, except
for BB, CC and DTC which are only treated by the multi-year average;
b The data were provided by Chen, X. from The Third Institute of Oceanography, SOA, China;
c The data was from the National Science Foundation of China (NSFC) project under the contract No. 90211021;
d The data from Jia et al. (2005) are the annual mean of the multi-year mean for each season during 1997–2002. a The data listed in the Table are the mean of the water column of the multi-year average for each parameter from different sources, except
for BB, CC and DTC which are only treated by the multi-year average; The data were provided by Chen, X. from The Third Institute of Oceanography, SOA, China; The data was from the National Science Foundation of China (NSFC) project under the contract No. 90211021
The data from Jia et al. (2005) are the annual mean of the multi-year mean for each season during 1997–2002. same as above row, Jia et al., 2003). And nutrients were
also low, i.e. the mean values of PO4–P=0.28 µmol L−1
and DIN=3.63 µmol L−1 (according to criterion of Nutrition
grade of sea water of China, Jia et al., 2003). The nutrients
values (Jia et al., 2005, nutrients) are much lower than those
we observed and the average index of ecological environ-
ment quality of fishing ground is 0.58, indicating it was in
good condition (Jia et al., 2005). X. Ning et al.: Long term changes in the northern South China Sea The
zooplankton biomass (averaging 22.1 mg m−3) was low (the In addition, after 1997, phosphorus depletion in surface
waters during summer coincided with a shift in the domi-
nant species in phytoplankton community from diatoms to
dinoflagellates and cyanophytes (Ning et al., 2004). Peng
et al. (2006) pointed out that dinoflagellates composed more
than 60% of the total phytoplankton abundance in the HK
Southeast Anti-Cyclonic Eddy and Hainan Island East Anti-
Cyclonic Eddy of the nSCS, where P concentration was near
detection limit in the summer of 2004. Biogeosciences, 6, 2227–2243, 2009 www.biogeosciences.net/6/2227/2009/ X. Ning et al.: Long term changes in the northern South China Sea 2239 Table 5. The mean of the ecological parameter for the water column (0–200 m) and sea surface Chl-a concentration at Station 4 in summer
of El Nino/La Nina years during the study period (units: ◦C for temperature, psu for salinity, µmol L−1 for DO and nutrients and mg m−3
for Chl-a). El Nino Year
1976
1982 (1983)
1986 (1987)
1991
1993
1994
1997
2002
2004
Mean±SDa
Tav
21.13
21.40 (23.40)
23.09 (23.32)
21.46
21.65
21.79
22.25
22.98
22.62
22.26±0.80
Sav
34.18
34.14 (34.16)
34.13 (34.2)
34.45
34.82
34.41
34.19
33.85
34.17
34.25±0.25
DOav
365.6
380.1 (387.3)
355.1 (364.9)
379.2
337.3
368.5
339.0
354.2
357.8
362.6±16.0
PO4–Pav
0.23
0.71
0.36
0.56
0.44
0.56
0.48±0.17
SiO3–Siav
25.36
16.22
13.38
8.96
11.65
10.56
14.36±5.94
DINav
3.20
3.85
6.19
4.29
12.51
8.12
6.36±3.50
Chl-ab
0.31
0.17
0.18
0.22±0.08
La Nina year
1981
1984
1988
1995
1998
1999
Tav
23.28
25.30
23.72
24.00
24.07
24.51
24.15±0.70
Sav
33.89
34.06
34.01
33.60
34.18
33.90
33.94±0.20
DOav
391.7
431.0
388.2
380.8
308.7
396.2
382.8±40.3
PO4–Pav
0.20
0.23
0.56
0.33±0.20
SiO3–Siav
10.00
5.20
22.32
7.95±12.45
DINav
3.50
3.00
2.31
2.94±5.98
Chl-ab
0.08, 0.06
0.08
0.09
0.08±0.01 y
y
b The Chl-a data were derived from SeaWiFS during 1998–2004, from Nimbus-7 CZCS in 1980 and 1984, provided by X. Chen, from The
Third Institute of Oceanography, SOA, China for 1982, Fan (1985) and Huang (1992) for 1990, respectively. Table 6. Comparison of the multi–year mean values of Chl-a, primary production (PP), phytoplankton abundance (PA), zooplankton biomass
(ZB), benthos biomass (BB), cephalopod catch (CC) and demersal trawl catch (DTC) in the nSCS between two periods, i.e. before and after
1997 (units: mg m−3, mg C m−2 d−1, ×103 cell m−3, mg m−3, g m−2, ×104 t, and ×104 t, respectively)a. Mean±SD is the multi-year mean value ± standard deviation for the El Nino/La Nina years. y
y
b The Chl-a data were derived from SeaWiFS during 1998–2004, from Nimbus-7 CZCS in 1980 and 1984, provided by X. Chen, from The
Third Institute of Oceanography, SOA, China for 1982, Fan (1985) and Huang (1992) for 1990, respectively. 5
Conclusion causative links, monsoon – circulation – nutrients – primary
production. It must be pointed out that although the data set we
adopted in the present analysis is large and from various
sources. As such the quality of data might vary throughout
the long period of observation. But these data were valuable
for studying the long term changes in environmental condi-
tions and the responses of the ecosystem in this region. And
using these data we can still find and understand some regula-
tions on the response of the ecosystems to the environmental
changes in the SCS. This regional response of SCS to global
climate change was investigated the first which is far from
well understood. The evolving nutrient environment may be
related to the observed ecosystem changes in the nSCS such
as increase in biological productivity. More long-time series
observations on the structure and function of ecosystems and
the relationships with environmental changes are needed in
the SCS in the future. The fluctuations in environmental parameters in the nSCS
during 1976–2004 displayed different patterns, i.e. tempera-
ture (SST and Tav), salinity (Sav and S200), dissolved inor-
ganic nitrogen (DIN) and N:P annual rates increased, while
DO, P, Si, Si:N, SSS and T200 annual rates decreased. The
climate trend coefficients, Rxt of these time series were
all over 0.38 (n=29) or 0.50 (n=16), and highly significant
(p≤0.05), except for the time series of P and Si. The increasing trends in SST and Tav were consistent with
the rise of the mean air temperature (AT) in the Northern
Hemisphere and southern China. The increase in SST and
Tav and decrease in SSS in the nSCS led to strengthening
of the thermocline and halocline, less mixing of deep wa-
ter to the surface and thus a decrease in the P supply from
deep waters. The increasing trend in DIN may have been in-
fluenced by the Pearl River discharge, and atmospheric dry
and wet deposition, (which are related to anthropogenic ac-
tivities), coastal upwelling and cyclonic eddies. X. Ning et al.: Long term changes in the northern South China Sea The first phase (before 1997) represents the initial status of
the biology and fisheries during the study period, and the sec-
ond phase (after 1997) represents the response of the ecosys-
tem to the changed environment, particularly since 1998,
when the occurrence of N limitation was significantly alle-
viated. These ecosystem responses discussed above were
clearly the result of environmental changes induced by not
only climate change, but also anthropogenic activities. www.biogeosciences.net/6/2227/2009/ Biogeosciences, 6, 2227–2243, 2009 X. Ning et al.: Long term changes in the northern South China Sea X. Ning et al.: Long term changes in the northern South China Sea X. Ning et al.: Long term changes in the northern South China Sea 2240 0
4
8
12
cephalopod catch /U104 ton
2000
1970
1980
1990
(a)
Cephalopod catch (104 t) Fig. 12
0
4
8
12
cephalopod catch /U104 ton
2000
1970
1980
1990
(a)
Cephalopod catch (104 t)
0
2000
1970
1980
1990
catch U104 ton
400
300
200
100
(b)
Fish catch (104 t)
Fig. 12. Variation trends in the catches of cephalopod and fishes of the nSCS. (a) Catches of cephalopod; (b) Catches of fishes (Data from
Guo and Chen, 2000). 0
2000
1970
1980
1990
catch U104 ton
400
300
200
100
(b)
Fish catch (104 t) Fig. 12
Fig. 12. Variation trends in the catches of cephalopod and fishes of the nSCS. (a) Catches of cephalopod; (b) Catches of fishes (Data from
Guo and Chen, 2000). 5
Conclusion The nSCS
always experienced limitation of N before 1997, but the sit-
uation in the upper layer sea water has been mitigated since
1998, due to the increase in N concentration and decrease in
P, which resulted in not only the positive trends in N:P ra-
tios which are now close to the Redfield ratio, but also the
decreasing trend in Si:N ratios, indicating of potential P lim-
itation. The decrease in DO concentration may be linked
to the increase in seawater temperature and the increase in
the concentration of organic matter inputs mainly from the
Pearl River and phytoplankton blooms, particularly since the
1990s. Chlorophyll-a, primary production, phytoplankton
abundance, benthos biomass, cephalopod catch and demer-
sal trawl catch have increased, and zooplankton abundance
decreased. These ecosystem responses resulted from envi-
ronmental changes were induced by not only climate change,
but also anthropogenic activities. After 1998, phosphorus de-
pletion in upper layer may be associated with a shift from
diatoms to dinoflagellates and cyanophytes domination. Acknowledgements. This study was supported by the National
Science Foundation of China (NSFC) key projects under the
contracts No. 90211021 and No. 90711006. The authors would
like to thank the Information Center of SOA for providing the en-
vironmental data, and to Paul Harrison for his significant comments. Edited by: M. Dai Edited by: M. Dai Biogeosciences, 6, 2227–2243, 2009 X. Ning et al.: Long term changes in the northern South China Sea X. Ning et al.: Long term changes in the northern South China Sea 2241 Chen, L., Zhu, W., Wang, W., Zhou, X., and Li, W.: Studies on cli-
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Environmental and Molecular
Modulation of Motor Individuality in
Larval Zebrafish
John Hageter1, Matthew Waalkes1, Jacob Starkey1, Haylee Copeland1, Heather Price1,
Logan Bays1, Casey Showman1, Sean Laverty2, Sadie A. Bergeron1,3 and
Eric J. Horstick1,3* Yet, genetic and environmental factors modulating
behavioral variation remain poorly understood, especially in vertebrates. To identify
genetic and environmental factors that influence behavioral variation, we take advantage
of larval zebrafish light-search behavior. During light-search, individuals preferentially
turn in leftward or rightward loops, in which directional bias is sustained and non-
heritable. Our previous work has shown that bias is maintained by a habenula-rostral
PT circuit and genes associated with Notch signaling. Here we use a medium-
throughput recording strategy and unbiased analysis to show that significant individual
to individual variation exists in wildtype larval zebrafish turning preference. We classify
stable left, right, and unbiased turning types, with most individuals exhibiting a
directional preference. We show unbiased behavior is not due to a loss of photo-
responsiveness but reduced persistence in same-direction turning. Raising larvae at
elevated temperature selectively reduces the leftward turning type and impacts rostral
PT neurons, specifically. Exposure to conspecifics, variable salinity, environmental
enrichment, and physical disturbance does not significantly impact inter-individual
turning bias. Pharmacological manipulation of Notch signaling disrupts habenula
development and turn bias individuality in a dose dependent manner, establishing a
direct role of Notch signaling. Last, a mutant allele of a known Notch pathway affecter
gene, gsx2, disrupts turn bias individuality, implicating that brain regions independent
of the previously established habenula-rostral PT likely contribute to inter-individual
variation. These results establish that larval zebrafish is a powerful vertebrate model
for inter-individual variation with established neural targets showing sensitivity to specific
environmental and gene signaling disruptions. Our results provide new insight into how
variation is generated in the vertebrate nervous system. Keywords:
zebrafish,
inter-individual
variation,
individuality,
environment,
Notch,
Gsx,
modulation,
thermoregulation Edited by: Edited by:
Benjamin L. De Bivort,
Harvard University, United States Reviewed by:
Maxim Nikitchenko,
Duke University, United States
Michael Brian Orger,
Champalimaud Foundation, Portugal *Correspondence:
Eric J. Horstick
eric.horstick@mail.wvu.edu Specialty section:
This article was submitted to
Individual and Social Behaviors,
a section of the journal
Frontiers in Behavioral Neuroscience
Received: 15 September 2021
Accepted: 17 November 2021
Published: 06 December 2021 Specialty section:
This article was submitted to
Individual and Social Behaviors,
a section of the journal
Frontiers in Behavioral Neuroscience Specialty section:
This article was submitted to
Individual and Social Behaviors,
a section of the journal
Frontiers in Behavioral Neuroscience Received: 15 September 2021
Accepted: 17 November 2021
Published: 06 December 2021 ORIGINAL RESEARCH
published: 06 December 2021
doi: 10.3389/fnbeh.2021.777778 Environmental and Molecular
Modulation of Motor Individuality in
Larval Zebrafish
John Hageter1, Matthew Waalkes1, Jacob Starkey1, Haylee Copeland1, Heather Price1,
Logan Bays1, Casey Showman1, Sean Laverty2, Sadie A. Bergeron1,3 and
Eric J. Horstick1,3* John Hageter1, Matthew Waalkes1, Jacob Starkey1, Haylee Copeland1, Heather Price1,
Logan Bays1, Casey Showman1, Sean Laverty2, Sadie A. Bergeron1,3 and
Eric J. Horstick1,3* 1 Department of Biology, West Virginia University, Morgantown, WV, United States, 2 Department of Mathematics
and Statistics, University of Central Oklahoma, Edmond, OK, United States, 3 Department of Neuroscience, West Virginia
University, Morgantown, WV, United States Innate behavioral biases such as human handedness are a ubiquitous form of inter-
individual variation that are not strictly hardwired into the genome and are influenced by
diverse internal and external cues. Yet, genetic and environmental factors modulating
behavioral variation remain poorly understood, especially in vertebrates. To identify
genetic and environmental factors that influence behavioral variation, we take advantage
of larval zebrafish light-search behavior. During light-search, individuals preferentially
turn in leftward or rightward loops, in which directional bias is sustained and non-
heritable. Our previous work has shown that bias is maintained by a habenula-rostral
PT circuit and genes associated with Notch signaling. Here we use a medium-
throughput recording strategy and unbiased analysis to show that significant individual
to individual variation exists in wildtype larval zebrafish turning preference. We classify
stable left, right, and unbiased turning types, with most individuals exhibiting a
directional preference. We show unbiased behavior is not due to a loss of photo-
responsiveness but reduced persistence in same-direction turning. Raising larvae at
elevated temperature selectively reduces the leftward turning type and impacts rostral
PT neurons, specifically. Exposure to conspecifics, variable salinity, environmental
enrichment, and physical disturbance does not significantly impact inter-individual
turning bias. Pharmacological manipulation of Notch signaling disrupts habenula
development and turn bias individuality in a dose dependent manner, establishing a
direct role of Notch signaling. Last, a mutant allele of a known Notch pathway affecter
gene, gsx2, disrupts turn bias individuality, implicating that brain regions independent
of the previously established habenula-rostral PT likely contribute to inter-individual
variation. These results establish that larval zebrafish is a powerful vertebrate model
for inter-individual variation with established neural targets showing sensitivity to specific
environmental and gene signaling disruptions. Our results provide new insight into how
variation is generated in the vertebrate nervous system. Innate behavioral biases such as human handedness are a ubiquitous form of inter-
individual variation that are not strictly hardwired into the genome and are influenced by
diverse internal and external cues. INTRODUCTION However, this behavioral bias is primarily fixed in the
population and offers little insight into inter-individual variation. Larval zebrafish also perform a light-search behavior that is onset
by the loss of visual navigating cues, which drives a period of
stereotypic leftward or rightward circling (Horstick et al., 2017),
consistent with search patterns observed in other species (Bell
et al., 1985; Hills et al., 2004, 2013; Gray et al., 2005). An
individual’s leftward or rightward circling direction is persistent
over at least multiple days, observed at equal proportions in
the population, and is not heritable (Horstick et al., 2020). The features of light-search share many of the hallmark traits
observed in well-established invertebrate models of turn bias
that have been instrumental for characterizing mechanisms that
regulate inter-individual variation (Ayroles et al., 2015; Buchanan
et al., 2015; Akhund-Zade et al., 2019). Moreover, our work
has shown that neurons in the habenula and rostral posterior
tuberculum (PT) are essential for maintaining zebrafish turn
bias (Horstick et al., 2020). Therefore, larval zebrafish is a
potentially powerful vertebrate model to determine how inter-
individual variation is imposed in the vertebrate brain. What
remains lacking is a rigorous analysis of turn bias variation in the
population and the identification of external and internal factors
modulating inter-individual turn bias differences. Research to date shows that binary handed-like behavioral
variation is observed in isogenic Drosophila (Kain et al.,
2012; Buchanan et al., 2015; Linneweber et al., 2020) and
clonal fish (Izvekov et al., 2012; Bierbach et al., 2017). Even
more complex behavioral modalities in isogenic mouse strains
(Freund et al., 2013, 2015; Hager et al., 2014), Caenorhabditis
elegans (Stern et al., 2017), Drosophila (Linneweber et al.,
2020), and clonal crayfish (Vogt et al., 2008) species display
stable individual phenotypes with significant inter-individual
variation at the population level, suggesting external events
contribute to behavioral diversity across individuals. Even in
humans, external or stochastic factors are likely important as
discordant handedness is frequently observed in monozygotic
twins (Jäncke and Steinmetz, 1995). These examples suggest that
environmental, chemical, or physical events during development,
even at early developmental stages, could produce inter-
individual differences. In Drosophila, the availability of numerous
isogenic strains and the ability to assay large numbers
of individuals have been instrumental in elucidating key
components generating inter-individual variation (Buchanan
et al., 2015). INTRODUCTION by gene and environment interaction. In murine models,
exploratory behavior is a thoroughly investigated example
of inter-individual variation, where phenotype variation is
enhanced by environmental enrichment and correlated changes
in hippocampal neurogenesis (Freund et al., 2013; Körholz et al.,
2018; Zocher et al., 2020). Despite this well-studied mammalian
model and other known handed behaviors that suggest changes
in neuron number or activity patterns may regulate inter-
individual variation, the mechanisms instructing inter-individual
differences remain poorly understood. Therefore, two prevailing
questions are what neural substrates generate biases and what
mechanisms instruct specific bias types, i.e., left versus right-
handed or consistent versus inconsistent handedness. Inter-individual variation, or individuality, is a hallmark of nearly
all animal species and contributes to the population’s fitness and
ability to adapt when confronted with environmental change
(Dingemanse et al., 2004; Klein et al., 2017; Horváth et al., 2020). One form of inter-individual variation is sensory and motor
biases. Handedness in humans is a familiar example, expressed
as a sustained preference for left- or right-hand use, which
the expression of a specific hand preference is only partially
explained by genetics, suggesting complex interactions contribute
to imposing handed phenotypes (Cuellar-Partida et al., 2020). Indeed, significant complexity underlies human handedness. The
preferred hand usage is context-dependent, largely independent
of other behavioral biases, and shows variable consistency –
observed as consistent or inconsistent hand dominance in a task
dependent manner (Watson and Kimura, 1989; Souman et al.,
2009; Chu et al., 2012). Handed biases are also a conserved
form of individual behavioral variation with species as diverse
as hagfish (Miyashita and Palmer, 2014), Drosophila (Kain et al.,
2012; Buchanan et al., 2015), chicken (Rogers, 1982; Casey
and Karpinski, 1999), and various vertebrate paw/foot biases
(Bulman-Fleming et al., 1997; Brown and Magat, 2011; Giljov
et al., 2013; Schiffner and Srinivasan, 2013; Manns et al., 2021)
showing sustained individual motor preferences. Despite the
prevalence of handed behaviors, mechanisms instructing these
behaviors and the variation observed across individuals are still
poorly understood. Zebrafish have emerged as a powerful model for elucidating
mechanisms that instruct visceral and neural differences between
individuals (Gamse et al., 2003, 2005; Dreosti et al., 2014). Moreover, similar to other teleost species, zebrafish have a visual
bias, preferentially using the left eye to assess novelty (Bisazza
et al., 1997; De Santi et al., 2001; Sovrano, 2004; Andrew et al.,
2009). Citation: Hageter J, Waalkes M, Starkey J,
Copeland H, Price H, Bays L,
Showman C, Laverty S, Bergeron SA
and Horstick EJ (2021) Environmental
and Molecular Modulation of Motor
Individuality in Larval Zebrafish. Front. Behav. Neurosci. 15:777778. doi: 10.3389/fnbeh.2021.777778 December 2021 | Volume 15 | Article 777778 1 Frontiers in Behavioral Neuroscience | www.frontiersin.org Modulation of Zebrafish Individuality Hageter et al. INTRODUCTION When navigating in their environment, Drosophila
display a turn bias, where individuals preferentially use same-
direction turns, and the magnitude of this bias is modulated by
genetic background, activity in the central complex, and exposure
to environmental enrichment as well as social experiences
(Ayroles et al., 2015; Buchanan et al., 2015; Akhund-Zade
et al., 2019; Versace et al., 2020). These findings demonstrate
that functional variation in the invertebrate nervous system is
maintained by specific neural substrates and further modified Here, we capitalize on the larval zebrafish turning bias to
characterize environmental factors and signaling pathways that
modulate inter-individual variation. Previous work identified
a persistent left/right turn bias maintained by a habenula-
rostral PT circuit and Notch associated signaling pathways
(Horstick et al., 2020). However, locomotor features or factors
instructing turn direction phenotypes was unexplored. Here
we develop a multiplex recording pipeline and a new metric,
bias ratio, which permit turn bias recording in a medium-
throughput manner and rigorous unbiased analysis of inter-
individual variation. Previous work used metrics that weighted
behavior on a single trial to categorize turning type (Horstick
et al., 2020), which these metrics are potent indicators of bias,
yet can easily compound error over serial testing that is typically
required to study probabilistic behavior like turning bias. Using December 2021 | Volume 15 | Article 777778 Frontiers in Behavioral Neuroscience | www.frontiersin.org 2 Modulation of Zebrafish Individuality Hageter et al. of a 10 cm petri dish. Previously, LEGO
R⃝blocks have been
used to stimulate novel object recognition and interaction in
larval zebrafish (Bruzzone et al., 2020). In addition, 5–8 plastic
aquarium leaves were included to float on the surface. Last, dishes
were positioned on platforms with mixed white and black shape
substrates. A total of four enriched environments were created
with variable LEGO
R⃝colors and sized blocks, and larvae were
rotated daily between enriched environments. As controls, larvae
were raised in plain 10 cm dishes placed on either a solid white
substrate. For experiments, larvae were maintained in enriched
or control dishes from 1 dpf until behavior testing. Shaking:
We tested the impact of environmental instability on motor
bias by continuously shaking larvae from 1 to 4 dpf. At 1 dpf,
embryos were placed in a 75 cm2 cell culture flask (Sigma) with
approximately 80 mL E3h. Flasks were propped at 30 degrees on a
Stovall Belly Dancer orbital rotator, set to 70 rpm. Behavior Tracking and Analysis Behavior Tracking and Analysis
Behavioral experiments were performed on 6–7 dpf larvae,
except as noted. All experiments were recorded using infrared
illumination (940 nm, CMVision Supplies), a µEye IDS1545LM-
M CMOS camera (1st Vision) fitted with a 12 mm lens, and
a long-pass 780 nm filter (Thorlabs, MVL12WA and FGL780,
respectively). Visible illumination was provided by a white
light LED (Thorlabs) positioned above the larvae, adjusted
to 40–50 µW/cm2 (International Light Technologies, ILT2400
Radiometer with SED033 detector). Testing conditions were
maintained between 26 and 28◦C for all behavioral recording,
and all larvae adapted to the recording room conditions
for 20 min before recording under matched illumination to
recording rigs. Custom DAQtimer software was used to control
lighting, camera recording parameters, and real-time tracking
as previously described (Yokogawa et al., 2012; Horstick et al.,
2017). The camera field of view was set to record four 10 cm
dishes simultaneously with one larva per dish for multiplex
recordings. A total of four recording rigs were used. Path
trajectories of individual larvae are recorded over 30-s recording
intervals at 10 fps and analyzed using five measures: net turn
angle (NTA), total turning angle (TTA), match index (MI),
bias ratio (BR), and performance index (PI) (see Table 1 for
metric reference). A minimum of 100 points were required to
be included in the analysis. NTA is the summation of leftward
and rightward angular displacement (−leftward, +rightward)
over the recording interval, whereas TTA is the sum of absolute
values of all angular displacement. MI measures the proportion
of events in a series going in the same direction. Leftward and
rightward trials are scored as 0 or 1, and MI is the percent of
events matching the direction of the first trial in a testing series. For example, a MI = 1 is all events are in the same direction as
the first trial, whereas 0.33 is a third of the events matching the
first trial. For MI analysis, individuals missing the first dark trial
were excluded from analysis. BR is a proportion of directional INTRODUCTION At 4 dpf, larvae
were removed from culture dishes and raised under standard
conditions prior to testing at 6–7 dpf. our new testing pipeline, we first characterized turning types,
finding previously described left and right turning types and
a previously undescribed unbiased turning type in a wildtype
strain. We further establish that turning types are distinguishable
by unique path trajectory features. Second, we determined that
temperature selectively impacts inter-individual variation and
rostral PT neurons, establishing a tentative mechanism for
temperature dependent regulation of inter-individual variation. Last, we investigate molecular pathways, demonstrating a direct
role for Notch signaling using pharmacological inhibition. We
establish levels of Notch inhibition that disrupts habenula
development and bias, yet well-established Notch mechanisms
such as neuronal proliferation or morphological development
are unaffected. By testing a mutant associated with Notch
signaling, gsx2, we implicate that brain regions beyond that
previously described circuit could be important for developing
variation in a vertebrate. This work develops zebrafish search
behavior as a model for inter-individual variation and reveals
how environmental and molecular cues impact specific neural
substrates to generate distinct behavior types in a vertebrate. Transgenic lines used were enhancer trap Tg(y279-Gal4)
(Marquart et al., 2015) and Tg(UAS:Kaede)s1999t (Davison et al.,
2007). Mutant line used was gsx2113a (Coltogirone et al., 2021). Frontiers in Behavioral Neuroscience | www.frontiersin.org Animal Husbandry y
All experiments were approved by the West Virginia University
Institutional Animal Care and Use Committee. Zebrafish (Danio
rerio) Tübingen long-fin (TL) wildtype strain was used in all
experiments and used as the genetic background to maintain
transgenic and mutant lines. Experiments were conducted during
the first 7 days post fertilization (dpf), which is before sex
determination. Larval rearing conditions were 28◦C, 14/10 h
light-dark cycle, in E3h media (5 mM NaCl, 0.17 mM KCl,
0.33 mM CaCl2, 0.33 mM MgSO4, and 1 mM HEPES, pH
7.3), and at a stocking density of 40 embryos per 30 mL E3h,
unless stated otherwise. Social environment: To test the effect
of social interaction, we raised larvae under two different social
conditions: 20 larvae in a 6 cm petri dish or a single larva per 6cm
dish. Social or isolation rearing started at 5–8 h post fertilization
(hpf) and continued until testing at 6 dpf. Temperature: To
test the impact of temperature on the development of turn
bias, larvae were raised 1–4 dpf at either 24, 28, or 32◦C. At
4 dpf, all groups were moved to 28◦C until testing at 6 dpf. To determine if a specific development period was sensitive to
elevated temperature, separate groups of larvae were raised at
32◦C from either 31–55 hpf or 55–79 hpf, after which they
were returned to standard rearing temperature and tested at
6 dpf. Salinity: The impact of increased salinity was tested over
4 salt concentrations (1, 2, and 5 ppt – parts per thousand)
and standard E3h (∼0.5 ppt) as a control. Larvae were reared
in variable salinities from 1 to 4 dpf, and behavior tested at
6 dpf. An elevated salinity stock of E3h was made by adding
9.5g NaCl (Sigma) to standard E3h, creating a 10 ppt stock,
which was diluted for working concentrations with standard E3h
media. Environmental enrichment: Enriched environments were
created by adhering mixed size and color (predominately red,
blue, gray, and white colors) LEGO
R⃝blocks onto the bottom December 2021 | Volume 15 | Article 777778 Frontiers in Behavioral Neuroscience | www.frontiersin.org Frontiers in Behavioral Neuroscience | www.frontiersin.org 3 Modulation of Zebrafish Individuality Hageter et al. TABLE 1 | Reference for metrics and assays. Behavior
metrics
Name
Measure
NTA
Net turn angle
Net sum of leftward and rightward angular
movement
TTA
Total turn angle
Absolute sum of all angular movement
BR
Bias ratio
NTA/TTA ratio. Pharmacology Notch signaling was inhibited using the ϒ-secretase inhibitor
LY411575 (Sigma, SML050). A 10 mM stock of LY411575 was
prepared in DMSO and diluted to working concentrations with
a final volume of 0.08–0.1% DMSO for all trials. To test Notch
inhibition on turn bias, mid-gastrulation (6–8 hpf) groups
of embryos were treated with 0.05, 0.1, 0.15, 0.2, 0.25, 1 or
10 µM LY411575 until 4 dpf; the drug was replaced daily. At 4 dpf, LY411575 was removed and larvae placed in fresh
E3h until behavior testing at 6 dpf. Phenotypic categorization
was performed at 3 dpf. Individuals were scored as normal
(visually no abnormal tail curvature, edema, reduced/decreased
swim bladder size, necrosis, or overt abnormal swimming), mild
(abnormal touch responsiveness), moderate (tail curvature), or
severe (gross developmental defects, necrosis). Only normal
larvae were used for behavioral testing. Vehicle controls were
0.08–0.1% DMSO treated. turning compared to total turning, calculated by dividing NTA
by TTA, e.g., −1 represents that all directional movement in a
single trial occurred in a leftward direction, while −0.5 indicates
that 50% of all turning was in a net leftward direction (e.g., −200
degrees NTA out of 400 TTA). PI was calculated by averaging
binary bias ratios, with leftward trials scored as 0 and rightward
1. Where noted on figures, bias ratios were weighted by the
proportion of larvae within a PI group in order to demonstrate
changes in the number of larvae within a performance group. In all analyses that required a PI for categorizing larvae,
all individuals that had missing trials were excluded. This
criteria was necessary to ensure rigorous categorization. For
gsx2 experiments, larvae were housed individually following
behavior testing for post hoc genotyping. Genotyping was
performed as previously described (Coltogirone et al., 2021). In brief, genotypes were confirmed using PCR spanning
the deletion: gsx2 (primers: 5′TGCGTATCCTCACACATCCA,
5′TGTCCAGGGTGCGCTAAC; 134 bp wildtype, 121 bp mutant,
and 134/121 bp heterozygous). Previous reports describe that
gsx2 mutants have reduced swim bladder inflation (Coltogirone
et al., 2021), which was minimized by raising larvae in shallow
water dishes. Only larvae with normal swim bladder inflation and
balance were used for experiments. Animal Husbandry Proportion of directional
movement
MI
Match index
Proportion of trials matching direction of
first trial
PI
Performance index Average of binarized turn directions
(0 = left; 1 = right)
Behavior
assays
Name
Measure
4×
NA
Paired 30 s Light ON and OFF recordings,
repeated 4×
8×
NA
Paired 30 s Light ON and OFF recordings,
repeated 8×
q4×
Quad 4×
Four repeated ‘4×’ recordings separated
by 10 min TABLE 1 | Reference for metrics and assays. turn bias. Individual larvae were first tested at 3 dpf, and were
separately raised in 6-well plates and retested daily through 6 dpf. For analysis, larvae were grouped as left or right biased based
on BR (average BR+, right bias; −, left bias) at 6 dpf when turn
bias is well-established (Horstick et al., 2020). To ensure rigorous
categorization, larvae with ambiguous responses at 6 dpf (BR
between −0.1 and 0.1) were removed. Frontiers in Behavioral Neuroscience | www.frontiersin.org Labeling and Imaging
Immunohistochemistry To assay neuronal proliferation, we labeled with anti-HuC/D
(Elav protein) (Invitrogen A21271). Control (0.08% DMSO)
and LY411575 groups (100 nM and 8 µM) were prepared
as described above. At 24 hpf, embryos were fixed overnight
using 4% paraformaldehyde in 1× PBS at 4◦C. Washes were
performed with 1× PBS containing 0.1% TritonX-100. We used
primary antibody mouse anti-HuC/D (1:500, Invitrogen, 16A11). Secondary detection was performed with goat anti-mouse IgG1
Alexa 488 (1:500, Invitrogen, A32723). To analyze images, signal
intensity of a 56 µm × 6 µm (W × H) region spanning a lateral
to midline hemi-section of the anterior spinal cord was recorded
using ImageJ. Three sections were measured per larva, averaged
and standardized for comparison between groups. Fluorescent in situ Hybridization y
To determine the levels of Notch signaling we examined
transcript
levels
of
her12
(Jacobs
and
Huang,
2019). Hybridization
chain
reaction
(Molecular
Instruments)
probes and labeling technology was used to detect her12
transcripts. Her12 mRNA sequence (NM_205619) was provided
to Molecular Instruments to design a custom gene-specific
her12 probe detection set. LY411575 and control larvae were
treated as described above. At 30 hpf, larvae were fixed overnight
using 4% paraformaldehyde in 1× PBS at 4◦C. Fixed larvae
were washed in 1× PBS containing 0.1% Tween20 and labeled
following Molecular Instruments HCR RNA-Fish protocol for
whole-mount zebrafish embryos (Schwarzkopf et al., 2021). All
images were collected using the same parameters. For analysis, The 4× recording assay was performed by recording larval
path trajectories over four recording intervals, each composed of
30 s baseline recordings, immediately followed by 30 s recording
following the loss of visible illumination. Each recording interval
was separated by 3 min of baseline illumination. The 8×
recording was performed in a similar format, including four
additional light ON/OFF recording intervals performed in series. The quad 4× (q4×) assay is identical to the 4×, except that the
4× recording interval is repeated four times, separated by 10 min
baseline illumination (see Table 1 for assay reference). A 4×
recording strategy was used to test the developmental onset of December 2021 | Volume 15 | Article 777778 4 Modulation of Zebrafish Individuality Hageter et al. Boxplots show median and quartiles with outliers identified
beyond 2.7 standard deviations from the mean. the percent area of her12 expression was quantified within the
spinal cord using ImageJ. Permutation
and
bootstrapping
was
performed
using
“sample” R function without and with replacement, respectively. For permutation experiments, bias ratios values were randomized
across all individuals in a dataset. Randomization was performed
only within the same trial, e.g., reshuffling of bias ratios within
the first light offtrial. Permutations were simulated 1,000 times
and average bias ratios and MAD values calculated using custom
R code, and used to plot permutated probability density curves
and MAD values. Probability density plots and area under the
curve measurements were performed using custom R code. For area under the curve analysis, ±0.3 tails were chosen for
comparison, which are approximately two standard deviations
from the population average. To generate error bars for MAD
analysis, average bias ratios were bootstrapped (1,000 bootstrap
replicates) with resampling. Imaging All imaging was performed on an Olympus Fluoview FV1000. For live imaging, larvae were anesthetized in a low dose of MS222
(Sigma) and embedded in 2% low melting temp agar. Fixed
samples were transferred into 70% glycerol/30% 1× PBS and
slide-mounted for imaging. Shows High Inter Individual Variation
We developed a multiplexed strategy to record path trajectories
to assess inter-individual variation during larval zebrafish light
search behavior (Figure 1A). Previously, the stereotypic turning
was described using a large recording arena (14,400 mm2) to
record single larva (Horstick et al., 2017). Larvae are recorded
in 100 mm diameter dishes (7,854 mm2) for our multiplexed
strategy, and robust circling is observed following light extinction
(Figure 1B). To characterize individual motor biases, we initially
recorded larval path trajectories over a series of four intervals
of paired 30-s baseline illumination and 30 s following the loss
of illumination, with each of these recording pairs separated by
3 min of illumination to restore baseline behavior (Horstick et al.,
2020), which we refer to as 4× recording (Figure 1C). This
recording yields four paired light on and offevents per individual. We recorded responses from 374 individuals, representing 1,496
paired baseline and dark responses. The presence of motor
bias was previously described using a match index (MI) – the
percent of turning trials in which turning direction was the
same as the first dark trial (Horstick et al., 2020). Here we Fluorescent in situ Hybridization For each resampled dataset a MAD
was calculated and MAD values across all resampled datasets
used to calculate a 95% confidence interval applied as an error
bar. A 1-way comparison was used to calculate significance for
all simulated dataset comparisons. To generate a p-value, the
number of resampled dataset MAD values were totaled that fall
within or exceed the 95% confidence interval of the comparison
group, and this total was divided by 1,000 to produce a final
p-value. This represents the fraction of simulated experimental
groups that fall within a range that supports a null hypothesis of
no difference between groups. For example, 600 bootstrapped
datasets from a simulated control that fall within or exceed the
confidence interval of an experimental group yields p = 0.60,
implicating that 60% of simulated datasets do not support the
statistical difference between compared groups. Direction of
comparison is noted in the legend for each dataset. Neuron Sensitivity to Notch Inhibition Neuron Sensitivity to Notch Inhibition
Using
Tg(y279-Gal4)/Tg(UAS:kaede)
carrier
in-crosses
we
performed LY411575 as described above, except treatments
ended at 3 dpf when both the habenula and PT could be
observed, while attempting to minimize severe morphological
phenotypes and death at higher concentrations. We treated
embryos at concentrations of 0.1 and 1 µM with a vehicle
control. All treatments had a final DMSO concentration of
0.01%. Imaging the habenula and PT was performed as above
and neuron counts performed using max projections in ImageJ. Counts were only performed on groups were habenula and
PT neurons could be reliable identified. For counting neuron
numbers, the larger habenula was classified as the ‘left’ habenula
regardless of hemisphere. Habenula were classified as symmetric
if the left to right neuron ratio was less than 2. Neuron Temperature Sensitivity Rostral PT and habenula image stacks were captured and
neurons counted in max projections using ImageJ. All imaging
was performed using larvae from Tg(y279-Gal4)/Tg(UAS:kaede)
carrier in-crosses. At 1 dpf, larvae were screened for Kaede and
reared at elevated temperatures as described above. Larvae were
moved to standard raising conditions at 4 dpf, and live-imaged
at 6 dpf. Larvae were anesthetized using MS-222 (Sigma) and
mounted in 2% low melting temp agar. To determine if a specific
developmental time period was crucial, larvae were similarly
prepared and analyzed, yet only raised at elevated temperature
during either 31–55 hpf or 55–79 hpf intervals. Controls were
raised at standard rearing temperatures. Statistical Analysis y
Analysis was performed in R R Core Team (2020). R: A
language and environment for statistical computing, 2020), R
ggplot2 package (Wickham, 2016) (R Core Team) and Prism
(GraphPad). All statistical comparisons were two-sided, unless
noted otherwise. Standard error of the mean (±SEM) was
used for all experiments, except MAD analysis which display
95% confidence intervals. Cohen D was calculated in R using
package effsize. For all experiments, data was collected from a
minimum of three independent clutches. Normality was tested
using the Shapiro–Wilks test. Normally distributed data was
compared using either 1 or 2-way t-tests. Non-normal data was
analyzed using a Mann–Whitney U test or Wilcoxon signed-
rank test for 2 or 1-way tests, respectively. To perform multiple
comparisons, ANOVAs were performed in GraphPad and
multiple comparisons adjusted using a Bonferroni correction. December 2021 | Volume 15 | Article 777778 Frontiers in Behavioral Neuroscience | www.frontiersin.org Frontiers in Behavioral Neuroscience | www.frontiersin.org 5 Modulation of Zebrafish Individuality Hageter et al. FIGURE 1 | Turning behavior following the loss of light exhibits high
inter-individual variation. (A) Schematic of multiplex recording rig. (B) Illustrative path trajectories following loss of light from multiplex recording. Color scale is time (seconds). (C) Diagram of 4× recording. Outlined regions
denote recording intervals. (D–F) 4× recordings strategy to show high
inter-individual variation in turn bias. (D) Average bias ratio probability density
curve for dark responses (solid blue line, N = 374) and paired curve following
reshuffling (dotted blue line, average of 1,000 resampled datasets). (E) Same
as (D) except the paired baseline responses (solid gray line, N = 374) and
random permutation density curve (dotted gray line). Cyan fill shows the area
in each tail corresponding to the probability of observing a result more extreme
or equal to ±0.3 average bias ratio. (F) MAD for baseline (yellow), and dark
(gray) responses. N = 374. Error bars are 95% confidence intervals generated
by bootstrap resampling. Asterisk in circle, p < 0.05 to MAD permutation
value shown with an X. (G) Dark responses (gray outline) and baseline (H)
(yellow outline) from 4× recording showing frequency of direction change
between first (circles) and second (arrows) pairs of responses. Initial N shown
in circles. Bold, solid, and dotted arrows delineate responses that produce
perfectly matched bias, partial bias, and unbiased responses, respectively. Statistical Analysis We calculated a bias ratio by dividing net turning angle
(NTA) by total turning angle (TTA – absolute sum of all
angular displacement) for each baseline, and dark trial recorded
to examine the spectrum of wildtype larvae inter-individual
variation during search behavior (Supplementary Figure 1B). This metric provides the proportion of same-direction turning
within a continuous numerical range bounded by −1 and
1, representing all directional movement in a leftward or
rightward direction, respectively. The average bias ratio across
the entire population during baseline illumination and light-
search did not significantly vary from zero showing no population
bias [one-sample t-test against 0, baseline: t(373) = 0.007842,
p = 0.9937; dark: t(373) = 0.1696, p = 0.89] (Supplementary
Figure 1C). Despite similar population-level bias ratios between
baseline and dark, significant variation is observed in the dark
that is not observed during baseline (Figures 1D–F). Using
a probability density curve, where the area under the curve
represents the proportion of individuals in the population,
we find that during dark turning, 12.38% of the population
displayed a robust sustained turning bias over 4 trials (bias
ratio <−0.7 = 6.41%, left bias; >0.7 = 5.97%, right bias)
(Figure 1D). Conversely, 1.72% of baseline events displayed
sustained directional turning (Figure 1E). The distribution of
bias ratios shows that, following light extinction, a significantly
greater number of individuals utilize sustained same-direction
turning [χ2(1) = 51.02, p < 0.0001]. To determine whether
these distributions were the product of chance, we simulated
‘randomized’ baseline and dark datasets by resampling bias ratios
(1,000 resamples) within each trial (Figures D,E, dotted line). Following randomizing, 2.35% of the simulated dark responses
maintained strong directional turning, similar to that observed
during baseline. A previous study used mean absolute deviation
(MAD) as a metric to quantify variation in a population; a higher
MAD represents increased variation across individuals in the
population (Buchanan et al., 2015). Here, MAD was calculated
for baseline, dark, and simulated datasets. As MAD was generated
from the whole population, average bias ratios were bootstrapped
(1,000 boots) to generate 95% confidence intervals for statistical
comparison. MAD is 44.10% (p < 0.001) and 15.79% (p < 0.001)
reduced in baseline or in randomized dark groups compared to
light-search dark responses, respectively (Figure 1F), whereas no
difference was observed between baseline MAD and randomized
baseline responses (Figure 1F, yellow bar). Statistical Analysis These findings show
that turn bias during light search behavior shows significant
variation beyond what is expected by chance or while larvae
navigate in an illuminated environment. O
l
i
l
ith fidi
f
i
t FIGURE 1 | Turning behavior following the loss of light exhibits high FIGURE 1 | Turning behavior following the loss of light exhibits high
inter-individual variation. (A) Schematic of multiplex recording rig. (B) Illustrative path trajectories following loss of light from multiplex recording. Color scale is time (seconds). (C) Diagram of 4× recording. Outlined regions
denote recording intervals. (D–F) 4× recordings strategy to show high
inter-individual variation in turn bias. (D) Average bias ratio probability density
curve for dark responses (solid blue line, N = 374) and paired curve following
reshuffling (dotted blue line, average of 1,000 resampled datasets). (E) Same
as (D) except the paired baseline responses (solid gray line, N = 374) and
random permutation density curve (dotted gray line). Cyan fill shows the area
in each tail corresponding to the probability of observing a result more extreme
or equal to ±0.3 average bias ratio. (F) MAD for baseline (yellow), and dark
(gray) responses. N = 374. Error bars are 95% confidence intervals generated
by bootstrap resampling. Asterisk in circle, p < 0.05 to MAD permutation
value shown with an X. (G) Dark responses (gray outline) and baseline (H)
(yellow outline) from 4× recording showing frequency of direction change
between first (circles) and second (arrows) pairs of responses. Initial N shown
in circles. Bold, solid, and dotted arrows delineate responses that produce
perfectly matched bias, partial bias, and unbiased responses, respectively. FIGURE 1 | Turning behavior following the loss of light exhibits high
inter-individual variation. (A) Schematic of multiplex recording rig. (B) Illustrative path trajectories following loss of light from multiplex recording. Color scale is time (seconds). (C) Diagram of 4× recording. Outlined regions
denote recording intervals. (D–F) 4× recordings strategy to show high
inter-individual variation in turn bias. (D) Average bias ratio probability density
curve for dark responses (solid blue line, N = 374) and paired curve following
reshuffling (dotted blue line, average of 1,000 resampled datasets). (E) Same
as (D) except the paired baseline responses (solid gray line, N = 374) and
random permutation density curve (dotted gray line). Frontiers in Behavioral Neuroscience | www.frontiersin.org Statistical Analysis As all PI groups
exhibited normal levels of total turning, this ruled out variable
photo responsiveness as the basis of different turning types. Unexpectedly, partially biased populations (0.25, 0.75 PI) showed
a similar average bias ratio as unbiased larvae (Figure 2A). To
explain this observation, we reasoned that bias ratio magnitude
might vary depending on whether an individual trial matches
or opposes the overall larva turning type. For example, for 0.25
PI larvae, leftward matched direction bias ratios compared to
rightward opposed direction trials. We analyzed all individual
trials between all performance groups to explore this idea, sorting
trials into matched or opposing based on the average PI for
each individual. Perfect performance trials (0,1) were categorized
as all matched, whereas unbiased trials (0.5) as all unmatched. Left and right direction bias ratios did not vary in these groups;
therefore, we combined these groups to simplify comparison
(Supplementary Figure 2C). A significant effect was observed
across groups [1-way ANOVA F(3,1408) = 27.93, p < 0.0001],
with trials opposed to overall PI direction showing lower overall
bias ratio strength (Figure 2B, magenta lines). These data suggest
that the basis of unbiased motor types is due to a lower bias
ratio or less persistent same-direction turning, yet not a loss of
overall turning. Interestingly, we noted that matched bias ratios
were reduced in partially matched trials compared to events in
the fully matched group [match 0.594 ± 0.013: partial match
0.514 ± 0.015: t(1408) = 4.046 adjusted p = 0.003] (black line),
implicating that the underlying differences between biased and
unbiased larvae may be graded. overall leftward or rightward preference per trial, respectively. From these binary values, we created a transition index for the
first and second set of responses from the 4× dataset, i.e., left
(LL = 0), right (RR = 1), or random (LR; RL = 0.5) responses
that can be compared between the first and last response pairs. Using the transition pair PI, we assessed the frequency of turn
direction change or conservation (Figures 1G,H). During dark
trials, 36% of all transitions showed sustained turn direction
(left = 17%, right = 19%; average PI = 0 or 1), whereas during
baseline illumination 12% of larvae sustained turn direction
[χ2(1) = 54.545, p < 0.0001]. Statistical Analysis Conversely, 21 and 35% of
transitions yielded sustained random behavior between dark
and baseline recording conditions, respectively (for example,
LR to RL or RR to LL; average PI = 0.5) [χ2(1) = 8.615,
p = 0.0033] (Figures 1G,H). Interestingly, during light-search
initially random response pairs transitioned to directional (RR or
LL) responses 22% of the time, yielding partial turn bias (average
PI 0.75 or 0.25). To confirm our observations persisted over longer timescales,
we ran an additional 8× experiment, testing 189 larvae as before,
with four additional light ON/OFF intervals in series. From
this extended testing condition, we observed conserved trends
demonstrating significant inter-individual variation in turning
bias during light-search, yet not during baseline illumination
(Supplementary Figures 1D–F). Neither 4× or 8× recording
showed a change in TTA over time, establishing overall behavior
is not disrupted by our assays (Supplementary Figures 1G,H). As 4× and 8× experiments were broadly consistent, we focused
on the 4× recording strategy for ongoing investigations. Our
data show that wildtype larvae exhibit significant inter-individual
variation in turn bias during light-search, greater than that
expected by chance, with a subset of individuals potentially
exhibiting a previously unexplored unbiased turning type. Statistical Analysis Cyan fill shows the area
in each tail corresponding to the probability of observing a result more extreme
or equal to ±0.3 average bias ratio. (F) MAD for baseline (yellow), and dark
(gray) responses. N = 374. Error bars are 95% confidence intervals generated
by bootstrap resampling. Asterisk in circle, p < 0.05 to MAD permutation
value shown with an X. (G) Dark responses (gray outline) and baseline (H)
(yellow outline) from 4× recording showing frequency of direction change
between first (circles) and second (arrows) pairs of responses. Initial N shown
in circles. Bold, solid, and dotted arrows delineate responses that produce
perfectly matched bias, partial bias, and unbiased responses, respectively. FIGURE 1 | Turning behavior following the loss of light exhibits high
inter individual variation (A) Schematic of multiplex recording rig Our analysis, along with findings from previous reports,
illustrates robust left and right turners, or turning types, within
the population. However, the distribution of bias ratios from 4×
recordings shows that over 14% of the population exhibits an
average bias ratio consistent with no sustained turn direction
(−0.1 < BR < 0.1) (see Figure 1D). These individuals could
represent either a stable unbiased population or endogenous
behavioral fluctuation. To evaluate whether unbiased individuals
are a sustained turning type in the population, in addition
to left/right biased turners, we created a performance index
(PI) by transforming all individual trials to either 0 or 1 for confirm previous findings showing a significant MI increase
following the loss of illumination (Wilcoxon matched-pairs test,
p < 0.001), showing the number of individuals recorded can be
upscaled via multiplexing (Supplementary Figure 1A). Overall,
our current approach for multiplexed recording recapitulates
previous findings. These data show that our multiplexed strategy
provides medium-throughput recording, allowing a rigorous
analysis of larval zebrafish inter-individual variation. Frontiers in Behavioral Neuroscience | www.frontiersin.org December 2021 | Volume 15 | Article 777778 6 Modulation of Zebrafish Individuality Hageter et al. [PI = 0, 0.603 ± 0.022; PI = 1, 0.58 ± 0.021: t(352) = 0.7811
adjusted p > 0.9999]. Consistent with earlier observations, no
differences were observed across PI groups during baseline [1-
way ANOVA F(4,352) = 2.087, p = 0.082], consistent with
an absence of turn individuality (Supplementary Figure 2A). Moreover, there was no significant change in TTA during dark
turning [1-way ANOVA F(4,352) = 1.263, p = 0.28] across
all PI groups (Supplementary Figure 2B). Development of Inter-Individual Variation
Is Sensitive to Specific Environmental
Factors (C) Cumulative
summation of bias ratio over the q4× from strong right (magenta, N = 34),
strong left (cyan, N = 34), and unbiased (yellow, N = 18) individuals. Individuals
were selected based on the first 4× average PI (strong right PI = 1, strong left
PI = 0, unbiased PI = 0.5). (D) Average q4× PI based on initial 4× PI (PI = 0,
N = 66; PI = 0.25, N = 74; PI = 0.5, N = 75; PI = 0.75, N = 75; PI = 1, N = 67). Black bars represent sample mean. Left, right, and unbiased performance
categorized within equal thirds of the PI scale. Cyan background highlights
average PI with left or right turning type behavior. (E,F) Characterization of
path trajectory features during baseline (yellow boxes) and dark (gray boxes)
responses from individuals tested in the q4× assay. Turning type determined
by 16× average PI distribution in (D). (E) Fractal dimension and
(F) Displacement (L, left, N = 36; U, unbiased, N = 40; R, right, N = 38). Asterisk p < 0.05. Many instances of motor and behavioral biases show limited
heritability (Collins, 1969; Buchanan et al., 2015; Linneweber
et al., 2020). This observation suggests that inter-individual
variation is, at least in part, modulated through individual
experience with environmental factors. Indeed, previous studies
show that social experience and environmental enrichment
modify inter-individual variation of specific behaviors (Freund
et al., 2015; Akhund-Zade et al., 2019; Versace et al., 2020; Zocher
et al., 2020). As larval zebrafish turning bias is not heritable
(Horstick et al., 2020), we reasoned that the environment
might contribute to overall inter-individual variation or the
generation of specific turning types. To test this hypothesis, we
first established that turn bias appears at 4 dpf (Supplementary
Figures 3A,B). Therefore, larvae were exposed to changes in the
environment from 1 through either 4 or 7 dpf, dependent on
the tested factor. The four parameters we screened were social
experience, environmental enrichment, temperature, and salinity
(Figure 3A). Social interaction and environmental enrichment
were selected because each has been shown to modulate inter-
individual variation (Akhund-Zade et al., 2019; Versace et al.,
2020). For social interaction, larvae are raised in isolation or
groups. Multiple Stable Turning Types Exist With
Distinct Locomotor Features No difference
was observed between strong left and right biased turners December 2021 | Volume 15 | Article 777778 Frontiers in Behavioral Neuroscience | www.frontiersin.org 7 Modulation of Zebrafish Individuality Hageter et al. FIGURE 2 | Biased and unbiased motor types present during light-search. (A) Absolute bias ratio from 4× recording per PI (PI 0, N = 66; PI 0.25,
N = 74; PI 0.5, N = 75; PI 0.75, N = 75; PI 1, N = 67). (B) Absolute bias ratio
magnitude for single 30 s recordings occurring in a direction matching (blue)
overall average PI direction or occurring in the opposed (beige) direction
shown for perfect bias (0,1 PI, N = 517), partially biased (0.25, 0.75 PI,
N = 596), and unbiased (0.5 PI, N = 301) populations. (C) Cumulative
summation of bias ratio over the q4× from strong right (magenta, N = 34),
strong left (cyan, N = 34), and unbiased (yellow, N = 18) individuals. Individuals
were selected based on the first 4× average PI (strong right PI = 1, strong left
PI = 0, unbiased PI = 0.5). (D) Average q4× PI based on initial 4× PI (PI = 0,
N = 66; PI = 0.25, N = 74; PI = 0.5, N = 75; PI = 0.75, N = 75; PI = 1, N = 67). Black bars represent sample mean. Left, right, and unbiased performance
categorized within equal thirds of the PI scale. Cyan background highlights
average PI with left or right turning type behavior. (E,F) Characterization of
path trajectory features during baseline (yellow boxes) and dark (gray boxes)
responses from individuals tested in the q4× assay. Turning type determined
by 16× average PI distribution in (D). (E) Fractal dimension and
(F) Displacement (L, left, N = 36; U, unbiased, N = 40; R, right, N = 38). Asterisk p < 0.05. F(2,222) = 0.42, p = 0.66; F.D: F(2,222) = 2.12, p = 0.12], yet
the expected changes in behavior following light extinction were
observed [main effect due to illumination 2-way ANOVA displ:
F(1,222) = 604, p < 0.0001; F.D: F(1,222) = 643, p < 0.0001]
(Figures 2E,F). Interestingly, upon closer inspection, we did
notice a small yet significant change in F.D. between left and
right turning groups during dark trials [left 1.240 ± 0.012; right
1.200 ± 0.014: t(222) = 2.974, adjusted p = 0.0489, effect size
d = 0.63]. Multiple Stable Turning Types Exist With
Distinct Locomotor Features This effect was specific, and not observed during
baseline [left F.D. 1.021 ± 0.003; right F.D. 1.021 ± 0.004:
t(222) = 0.059, adjusted p > 0.9999] or for displacement. These
results show that the difference of left and right turning type also
generate mild changes to search pattern behavior, yet not motor
trajectories during baseline movement. FIGURE 2 | Biased and unbiased motor types present during light search Multiple Stable Turning Types Exist With
Distinct Locomotor Features In order to confirm rigorously the three motor types, we
performed a quad 4× assay (q4×), using the standard 4× assay
repeated four times, with each recording sequence separated by
10 min of baseline illumination (Supplementary Figure 2D). We recorded 114 larvae, and consistent with our previous
measures, individuals showed significant inter-individual turn
bias variation during light-search (±0.3 probability density
tails: 7.34% dark; 0.00037%, randomized dark), and sustained
left, right, or unbiased locomotor preferences (Supplementary
Figure 2E). The cumulative summation of bias ratios provided
a qualitative measure of turn performance over time (Figure 2C). From this analysis, we noted that some larvae initially categorized
as strong or unbiased turners, seemingly switched over time. Therefore, we next aimed to utilize the q4× analysis to quantify
bias determination accuracy by comparing the first 4× PI to
overall q4× performance. We equally divided the 0 to 1 PI
scale for classifying left, right, or unbiased behavior (left ≤0.33;
unbiased 0.33 < 0.66; right ≥0.66) (Figure 2D). Of the larvae
that show an initial strong or partial bias during the first 4×
interval, 2/96 (2.08%) reverse bias direction during the q4× assay,
and 27/96 (28.13%) of these individuals ultimately switch to an
unbiased response after serial q4× testing. However, switching is Characterizing changes in locomotor parameters in zebrafish
has been a powerful strategy to develop etiological and
mechanistic models (Burgess and Granato, 2007; Horstick
et al., 2013; Chen and Engert, 2014; Dunn et al., 2016). Therefore, we next aimed to establish what underlying locomotor
changes account for unbiased and biased motor types. We
hypothesized that three possible modes could generate unbiased
behavior: (1) normal turning with high rates of direction
switching across trials, (2) reduced same-direction turning
within single trials, or (3) weak photo-responsiveness and,
therefore, low total turning. To differentiate between these
hypotheses, we categorized all larvae based on average PI across
all four trials, generating five categories. Across PI groups,
we compared the absolute average bias ratio to determine if
the magnitude of directional turning changed based on PI. During light search the average bias ratio magnitude significantly
changed based on PI [1-way ANOVA F(4,352) = 10.43,
p < 0.0001], where partial and unbiased PI groups showed
less overall directional turning (Figure 2A). Frontiers in Behavioral Neuroscience | www.frontiersin.org Development of Inter-Individual Variation
Is Sensitive to Specific Environmental
Factors FIGURE 2 | Biased and unbiased motor types present during light-search. (A) Absolute bias ratio from 4× recording per PI (PI 0, N = 66; PI 0.25,
N = 74; PI 0.5, N = 75; PI 0.75, N = 75; PI 1, N = 67). (B) Absolute bias ratio
magnitude for single 30 s recordings occurring in a direction matching (blue)
overall average PI direction or occurring in the opposed (beige) direction
shown for perfect bias (0,1 PI, N = 517), partially biased (0.25, 0.75 PI,
N = 596), and unbiased (0.5 PI, N = 301) populations. (C) Cumulative
summation of bias ratio over the q4× from strong right (magenta, N = 34),
strong left (cyan, N = 34), and unbiased (yellow, N = 18) individuals. Individuals
were selected based on the first 4× average PI (strong right PI = 1, strong left
PI = 0, unbiased PI = 0.5). (D) Average q4× PI based on initial 4× PI (PI = 0,
N = 66; PI = 0.25, N = 74; PI = 0.5, N = 75; PI = 0.75, N = 75; PI = 1, N = 67). Black bars represent sample mean. Left, right, and unbiased performance
categorized within equal thirds of the PI scale. Cyan background highlights
average PI with left or right turning type behavior. (E,F) Characterization of
path trajectory features during baseline (yellow boxes) and dark (gray boxes)
responses from individuals tested in the q4× assay. Turning type determined
by 16× average PI distribution in (D). (E) Fractal dimension and
(F) Displacement (L, left, N = 36; U, unbiased, N = 40; R, right, N = 38). Asterisk p < 0.05. FIGURE 2 | Biased and unbiased motor types present during light-search. (A) Absolute bias ratio from 4× recording per PI (PI 0, N = 66; PI 0.25, FIGURE 2 | Biased and unbiased motor types present during light-search. (A) Absolute bias ratio from 4× recording per PI (PI 0, N = 66; PI 0.25,
N = 74; PI 0.5, N = 75; PI 0.75, N = 75; PI 1, N = 67). (B) Absolute bias ratio
magnitude for single 30 s recordings occurring in a direction matching (blue)
overall average PI direction or occurring in the opposed (beige) direction
shown for perfect bias (0,1 PI, N = 517), partially biased (0.25, 0.75 PI,
N = 596), and unbiased (0.5 PI, N = 301) populations. Development of Inter-Individual Variation
Is Sensitive to Specific Environmental
Factors For enrichment, we generated two environments: (1) an
enriched environment where a petri dish was fitted with internal
surfaces, diverse color, hiding spots, water surface cover, and
dynamic substrate pattern, and (2) an empty petri dish with a
uniform white bottom as a control. In addition, we also tested
the impact of etiologically relevant temperature (24 or 32◦C) and
salinity [0.5–5 parts per thousand (ppt)] variations during early
development compared to standard rearing parameters (Engeszer
et al., 2007; Sundin et al., 2019). Thus, our parameters test factors
that generated inter-individual variation in other models and
abiotic environmental fluctuations that larvae could encounter in
a native habitat. primarily observed in larvae showing an initially partial bias, as
the larvae that displayed an initially strong bias (0, 1 PI) in the
q4× assay, 50/59 (84.75%) maintained a left or rightward turning
type. Interestingly, at the population level, the 9/114 (7.89%) of
unbiased individuals initially categorized with a strong bias was
comparable to that expected by random chance, i.e., the same
6.25% likelihood of flipping 4 heads with a coin [χ2(1) = 0.609,
p = 0.435]. As expected, classifying unbiased larvae was less
accurate, yet a single 4× trial accurately represented 10/18
(55.56%) of individuals. Altogether, the q4× testing strategy
confirms our earlier findings and demonstrates the veracity of our
recording strategies to detect specific turning types. As the q4× assay allowed for a rigorous confirmation of
turning type, we next wanted to determine whether left, right,
or unbiased turning types exhibited unique path trajectory
characteristics. A PI was calculated from all 16 trials in the q4×
assay for each individual and categorized as left, unbiased, or right
type. For each turning type, we examined fractal dimension (F.D.)
and displacement (displ) as measures of local search behavior
(Tremblay et al., 2007; Horstick et al., 2017). Comparison across
all three motor types yielded no differences in the tested motor
parameters [main effect due to turn type 2-way ANOVA displ: To determine if any of the tested parameters altered
turning type development or magnitude of inter-individual
variation, we looked at the average population bias ratio and
MAD, respectively (Figures 3B,C) (Supplementary Figure 3C). Interestingly, the elevated temperature during early development December 2021 | Volume 15 | Article 777778 8 Modulation of Zebrafish Individuality Hageter et al. increase in rightward turning [0.75 PI t(364) = 2.904, adjusted
p = 0.012; red line]. Elevated Temperature Impacts Rostral
Posterior Tuberculum Specification Posterior Tuberculum Specification
A basic circuit involving the rostral posterior tuberculum
(PT) and dorsal habenula (dHb) neurons has previously been
described for zebrafish turn bias (Horstick et al., 2020). However, in wildtype larvae, no hemispheric differences in
these neurons were found to account for left or right turning
preference (Horstick et al., 2020). Because we found that
elevated temperature disrupted left and right turning balance,
we next wanted to determine if elevated temperature caused
changes in neurons necessary for turn bias. We reasoned our
environmental variables could alter neuronal development, as
bias maintaining PT neurons are present as early as 2 dpf
(Horstick et al., 2020), and dHb differentiation begins on 1 dpf
(Gamse et al., 2003; Amo et al., 2010). First, we wanted to
identify if a specific period during early development was
sensitive to increased temperature. We found that elevated
temperature during either 31–55 hpf or 55–79 hpf intervals did
not recapitulate the population shift observed during the 1–
4 dpf exposure (Supplementary Figure 4); therefore, we selected
the full testing duration for further investigation. To visualize
key dHb and PT neurons, we used the enhancer trap line
y279:Gal4, which labels both populations of neurons (Horstick
et al., 2020) (Figure 4A). In zebrafish, the left dHb is considerably
larger than the right dHb (Gamse et al., 2003; Roussigné et al.,
2009). We found that elevated temperature did not alter the
habenula, and typical left/right asymmetry was observed [2-
way ANOVA: interaction between temperature and hemisphere
F(1,56) = 0.070, p = 0.79; effect of hemisphere F(1,56) = 101.2,
p < 0.0001) (Figures 4B,E]. No hemispheric differences [main
effect of hemisphere 2-way ANOVA F(1,56) = 0.493, p = 0.49]
were observed in the number of y279 positive PT neurons
(Figure 4C). Therefore, we combined PT measures from both
hemispheres. Interestingly, from these combined pools, y279 FIGURE 3 | Temperature selectively changes inter-individual variation in turn
bias. (A) Schematic of environmental manipulations (Social Isolated N = 87,
Social Group N = 87; Enriched N = 172, Plain N = 134; Temp. 32◦C N = 112,
Temp. 28◦C N = 136, Temp. 24◦C N = 144; Standard salinity N = 102, 1 ppt
N = 107, 2 ppt N = 107). Each condition has an independent control denoted
by superscript Cntrl. (B) Average bias ratios across the entire tested
population per condition. Statistical comparison performed to 0, denoting no
population level bias. Development of Inter-Individual Variation
Is Sensitive to Specific Environmental
Factors Conversely, low temperature depressed turn
bias performance in the population (Figure 3D, blue lines). These results suggest a specific temperature-mediated change. However, in larval zebrafish, fluctuating temperature is a stressor,
and elevation of stress signaling has been shown to attenuate
visual bias in chickens (Rogers and Deng, 2005; Long et al.,
2012; Haesemeyer et al., 2018). Therefore, we tested the effect
of shaking on turn bias which is a potent stressor for larval
zebrafish (Eto et al., 2014; Castillo-Ramírez et al., 2019; Apaydin
et al., 2020). Sustained shaking during early development resulted
in no population or turn bias magnitude changes (Figures 3E–
G). Moreover, external temperature impacts the rate of zebrafish
development, and based on previous studies, our conditions
would lead to an estimated ±13 h shift in development (Kimmel
et al., 1995). We show that our temperature assay results in a
change in hatching, a developmental marker, yet no gross changes
in morphology or survival (Supplementary Figures 3D–F). These data illustrate that etiologically relevant temperature
fluctuations differentially and specifically affect inter-individual
turn bias variation. FIGURE 3 | Temperature selectively changes inter-individual variation in turn
bias. (A) Schematic of environmental manipulations (Social Isolated N = 87,
Social Group N = 87; Enriched N = 172, Plain N = 134; Temp. 32◦C N = 112,
Temp. 28◦C N = 136, Temp. 24◦C N = 144; Standard salinity N = 102, 1 ppt
N = 107, 2 ppt N = 107). Each condition has an independent control denoted
by superscript Cntrl. (B) Average bias ratios across the entire tested
population per condition. Statistical comparison performed to 0, denoting no
population level bias. (C) MAD for dark responses, error bars are 95%
confidence levels generated from 1,000 bootstraps. Individual p-values shown
above bars, calculated by comparing experimental groups to controls. (D) Average bias ratio per PI weighted by the percent of individuals. N’s
indicated within bars. (E) Representative diagram of the setup used to shake
larvae during early development. Average population bias ratio (F) and MAD
(G) between shake experiments. Number above bar represents p-value
compared to the control group. Asterisk p < 0.05. Motor Individuality Is Sensitive to Gene
Signaling Associated With Neuronal
Proliferation Studies from C. elegans (Bertrand et al., 2011) and Drosophila
(Linneweber et al., 2020) demonstrate that Notch signaling
can generate functional asymmetries in the brain that drive
unique individual behavioral responses. Established zebrafish
mutant lines mindbomb (mib) and mosaic eyes (moe), E3
ubiquitin ligase and Epb41l5 adapter, respectively, do not directly
disrupt the Notch cascade, yet impair Notch signaling (Itoh
et al., 2003; Ohata et al., 2011; Matsuda et al., 2016). Indeed,
haploinsufficiency in these lines abrogates zebrafish turn bias,
suggesting sensitivity to the levels of Notch signaling (Horstick
et al., 2020). One of the canonical roles of Notch during early
development is the regulation of neuronal proliferation (Appel
et al., 2001; Mizutani et al., 2007; Yoon et al., 2008). Therefore,
we next aimed to elucidate if turn bias is (1) sensitive to direct
Notch antagonism in a dose-dependent manner and (2) if partial
Notch inhibition impairs neuronal proliferation. To confirm that LY411575 exposure impaired Notch signaling,
we examined her12 expression, a downstream target of the
Notch signaling cascade that is robustly expressed in the spinal
cord, providing an unambiguous region to quantify expression
changes (Jacobs and Huang, 2019). Exposure to micromolar
inhibitor concentrations resulted in a near-total absence of her12
expression, consistent with previous reports (Jacobs and Huang,
2019). The her12 expression was, however, observed in the spinal
cord of the 100 nM group at an intensity indistinguishable from
controls (Figures 5E,L). A canonical and conserved role for Notch during early
development
is
regulating
neuronal
proliferation
and
maintaining progenitor pools, and the loss of Notch leads
to increased proliferation (Appel et al., 2001; Cheng et al., 2004;
Sharma et al., 2019). Therefore, we next wanted to determine
whether the level of Notch inhibition that impairs turn bias
individuality
also
disrupts
proliferation. During
zebrafish
embryonic development, proliferative neurons are readily
visualized in the anterior hindbrain using Elav (HuC/D) protein
expression as a marker (Kim et al., 1996; Sharma et al., 2019). These proliferative neuron pools expand following high levels of
Notch inhibition or in the mindbomb mutant background (Itoh
et al., 2003; Sharma et al., 2019). Consistent to our observation
with her12 and PT neuron counts, partial pharmacological
Notch inhibition (100 nM drug) induced no change in actively
proliferating neurons, yet positive controls (8 µM) displayed
robust expansion of proliferating neurons (Figures 5K,M). Motor Individuality Is Sensitive to Gene
Signaling Associated With Neuronal
Proliferation p
p
To disrupt Notch signaling, we used the specific ϒ-secretase
inhibitor LY411575, which blocks the activation of the Notch
signaling cascade (Geling et al., 2002; Fauq et al., 2007). Previous
reports show that treatment with micromolar concentrations
of LY411575 starting at mid-gastrulation results in a near-
total loss of Notch signaling, which largely recapitulates the
mindbomb mutant (Jacobs and Huang, 2019; Sharma et al.,
2019). Therefore, we used 10 µM as a maximum dose and
positive control for inhibitor efficacy across trials. To identify
a level of Notch inhibition that could impair turning bias, we
LY411575-treated larvae from mid-gastrulation to 4 dpf over
7 concentrations ranging from 50 nM to 10 µM and scored
phenotypes at 3 dpf (Figure 5A). Developmental exposure
of LY411575 up to 100 nM left most larvae morphologically
normal, which we used as a maximum dose to test the
impact on turn bias. Notch inhibition resulted in a significant
change in TTA following the loss of illumination [1-way
ANOVA F(2,152) = 4.614, p = 0.011], causing an increase in
overall turning at 100 nM inhibitor treatment compared to
controls [vehicle 1175.95 ± 53.34, 100 nM 1411.39 ± 66.50:
t(152) = 2.786, adjusted p = 0.018] (Supplementary Figure 5). Whereas turn bias performance was reduced by Notch inhibition
[main effect due to treatment 2-way ANOVA F(2,144) = 8.995,
p = 0.0002], with 100 nM inhibitor nullifying bias ratio
strength differences due to PI, which was not observed at
lower inhibitor concentrations (Figures 5B,C). In addition,
100 nM but not 50 nM treatment reduced overall inter-
individual turn bias variation in the population (Figure 5D). This data suggests that a critical threshold of Notch signaling Notch
signaling
is
ubiquitous
in
the
larval
zebrafish
nervous system (Tallafuss et al., 2009; Banote et al., 2016;
Kumar et al., 2017), and pharmacological inhibition is not
specific. Consequentially, we next aimed to determine whether
proliferative pathways in restricted areas of the brain may also
contribute to turn bias. Genomic screen homeobox transcription
factors (Gsx1 and 2, formerly Gsh1 and 2) are affecters of
the Notch signaling pathway in mouse, and Gsx2 maintains
neural
progenitor
pools
in
the
developing
telencephalon
(Wang et al., 2009; Pei et al., 2011; Roychoudhury et al.,
2020). In larval zebrafish, gsx2 is predominantly expressed
in the pallium, preoptic area, hypothalamus, and hindbrain,
with an established putative null TALEN deletion mutant line
(Coltogirone et al., 2021). Elevated Temperature Impacts Rostral
Posterior Tuberculum Specification (C) MAD for dark responses, error bars are 95%
confidence levels generated from 1,000 bootstraps. Individual p-values shown
above bars, calculated by comparing experimental groups to controls. (D) Average bias ratio per PI weighted by the percent of individuals. N’s
indicated within bars. (E) Representative diagram of the setup used to shake
larvae during early development. Average population bias ratio (F) and MAD
(G) between shake experiments. Number above bar represents p-value
compared to the control group. Asterisk p < 0.05. FIGURE 3 | Temperature selectively changes inter-individual variation in turn
bias. (A) Schematic of environmental manipulations (Social Isolated N = 87, caused a significant population shift from random [high temp
0.094 ± 0.044: one-sample t-test against 0, t(112) = 2.157,
p = 0.033], implicating a population-level rightward bias, whereas
no significant changes were observed in other temperature
conditions
or
any
other
tested
environmental
parameter
(Figure 3B). Conversely, the magnitude of turn bias variation
during light-search was only reduced by low-temperature
rearing, yet unaffected by other testing conditions (Figure 3C). To confirm the observed temperature-dependent changes, we
examined the bias ratio per PI, weighted for the number of
individuals per PI group. We observed that temperature imposed
a significant effect on turn bias persistence [main effect of
temperature 2-way ANOVA F(2,364) = 9.275, p = 0.0001]
(Figure 3D). Indeed, the tested high temperature resulted in
a significant depression of leftward turning [within PI group
comparison t(364) = 3.031, adjusted p = 0.0078; red line] and December 2021 | Volume 15 | Article 777778 Frontiers in Behavioral Neuroscience | www.frontiersin.org 9 Modulation of Zebrafish Individuality Hageter et al. is required for generating variation in turn bias and overall
performance, which is lower than levels necessary for normal
gross morphological development. To identify a potential
neuronal basis for the loss of bias following Notch inhibition,
we LY411575-treated y279:Gal4 embryos to quantify transgene
positive
dHb
and
rostral
PT
neurons,
focusing
on
the
inhibitor concentration that specifically impairs behavior yet
not morphological development. Interestingly, we found that
the levels of inhibition that abrogate bias also disrupts typical
dHb hemispheric asymmetry, producing an increase in reversed
and symmetric habenular phenotypes (Figure 5E). Similarly, we
observed an increase in the smaller ‘right’ dHb neuron number
[vehicle 4.25 ± 1.21, 100 nM 10.22 ± 1.64: t(15) = 2.870,
p = 0.012], consistent with increased habenular symmetry
(Figures 5F,H,I). Conversely, the rostral PT was unaffected
(Figures 5G–I). Elevated Temperature Impacts Rostral
Posterior Tuberculum Specification positive neurons in the PT were reduced after exposure to
elevated etiological temperature during early development [high
temperature 17.64 ± 0.885; normal temperature 27.59 ± 1.172:
2-tail t-test t(58) = 6.625, p < 0.0001] (Figures 4D,F and
Supplementary Figures 4C–F), establishing a potential neuronal
basis for how high temperature during development modifies
turn bias inter-individual variation. Frontiers in Behavioral Neuroscience | www.frontiersin.org Motor Individuality Is Sensitive to Gene
Signaling Associated With Neuronal
Proliferation As gsx2 mutants show no gross December 2021 | Volume 15 | Article 777778 Frontiers in Behavioral Neuroscience | www.frontiersin.org 10 Modulation of Zebrafish Individuality Hageter et al. FIGURE 4 | Temperature impacts y279 specified expression in the PT. (A) Single sagittal slice of larval zebrafish brain showing expression of enhancer trap
Tg(y279:Gal4) obtained from the ‘Zebrafish Brain Browser’ atlas. Circled regions highlight the habenula (Hb) and rostral posterior tuberculum (PT) and white lines
show the telencephalon (Tel), hypothalamus (Hyp), and cerebellum (Cb). (B–F) Effect of elevated temperature during early development on the expression of y279 in
the habenula and PT. (B) Expression of y279 in the left and right hemisphere Hb nuclei (28◦C purple, N = 16; 32◦C orange, N = 14). (C) y279 positive PT neurons
(28◦C purple, N = 16; 32◦C orange, N = 14). (D) Combined left and right hemisphere PT neuron counts (28◦C purple, N = 16; 32◦C orange, N = 14). (E,F)
Representative images showing maximum intensity projections for y279 positive Hb (left habenula, LHb; right habenula, RHb) (E) and PT (left PT, LPT; right PT, RPT)
(F) neurons for larvae raised at 28 or 32◦C. Scale bar 20 µm. Asterisk p < 0.05. FIGURE 4 | Temperature impacts y279 specified expression in the PT. (A) Single sagittal slice of larval zebrafish brain showing expression of enhancer trap
Tg(y279:Gal4) obtained from the ‘Zebrafish Brain Browser’ atlas. Circled regions highlight the habenula (Hb) and rostral posterior tuberculum (PT) and white lines
show the telencephalon (Tel), hypothalamus (Hyp), and cerebellum (Cb). (B–F) Effect of elevated temperature during early development on the expression of y279 in
the habenula and PT. (B) Expression of y279 in the left and right hemisphere Hb nuclei (28◦C purple, N = 16; 32◦C orange, N = 14). (C) y279 positive PT neurons
(28◦C purple, N = 16; 32◦C orange, N = 14). (D) Combined left and right hemisphere PT neuron counts (28◦C purple, N = 16; 32◦C orange, N = 14). (E,F)
Representative images showing maximum intensity projections for y279 positive Hb (left habenula, LHb; right habenula, RHb) (E) and PT (left PT, LPT; right PT, RPT)
(F) neurons for larvae raised at 28 or 32◦C. Scale bar 20 µm. Asterisk p < 0.05. morphological abnormalities during larval stages, we used these
lines to test turn bias. Motor Individuality Is Sensitive to Gene
Signaling Associated With Neuronal
Proliferation Heterozygous and mutant gsx2 larvae
displayed reduced inter-individual variation and a shift toward
less persistent turn bias (Figure 6A). The loss of persistent
same-direction turning was similarly observed using match
index (MI), an analogous metric (Figure 6B). Yet, TTA during
light-search was not significantly changed across genotypes
[1-way ANOVA F(2,187) = 2.730, p = 0.068], suggesting the loss
of same-direction turning is not due to reduced light-driven
behavior (Figure 6C). Thus, our analysis implies that broad
and local haploinsufficient changes in Notch signaling and Gsx2
contribute to inter-individual variation in turn bias behavior,
independent of canonical roles in proliferation. pathways associated with neuronal proliferation affected turn
bias development, using either pharmacological inhibition of
Notch signaling or a presumable null Gsx2 mutant. Notch
and Gsx2 represent canonical broad and regional regulators of
proliferation, respectively. Interestingly, turn bias attenuation is
observed with partial Notch inhibition and in gsx2 heterozygotes,
suggesting dose-dependent sensitivity. Despite a well-established
role for Notch in cell proliferation, the inhibitor concentrations
that selectively impairs turn bias did not result in observable
changes in proliferation, at least early in development (see
Figure 5). Our findings confirm that three turning types can
be categorically defined, are modulated by specific etiological
relevant environmental cues, and are sensitive to internal
proliferative associated signaling pathways. One potential caveat
is
that
zebrafish
strains
are
not
isogenic
and
maintain
some genetic heterogeneity (Butler et al., 2015), potentially
contributing to inter-individual differences. Nevertheless, our
work develops larval zebrafish as a powerful model to identify
mechanisms generating inter-individual variation in vertebrates. DISCUSSION Here we reveal that during light-search initiated by the loss of
illumination, larval zebrafish exhibit significant inter-individual
variation in turn bias, a handed-like behavior. Based on our
newly developed assays, we were further able to show mild
changes in search behavior correlated with left and right turning
types. However, the impact of turning on search motor patterns
was specific, as we found no evidence of individual motor
changes during baseline illumination, consistent with previous
studies (Horstick et al., 2020). We demonstrated a turn bias
spectrum across the population which shows the previously
described left/right turning types (Horstick et al., 2020). In
addition, our analysis revealed a consistently unbiased turning
type, supported by multiple independent recording strategies
(4×, 8×, and q4×). Furthermore, we show that temperature
changes during early development result in sustained changes
in inter-individual variation. Finally, we tested how signaling Frontiers in Behavioral Neuroscience | www.frontiersin.org Determination of Bias Our findings suggest a ‘hemispheric noise’ model where turn
bias and inter-individual variation is modulated by conflicting
brain hemisphere signals in turn bias driving neurons (Figure 7). We elucidated that change in bias ratios strength distinguishes
unbiased versus biased larvae. Moreover, we establish that
this change is not a result of a loss of photo-responsiveness
in unbiased individuals (total turning, see Supplementary
Figure 1); rather a failure to navigate in a single direction
during light-search consistently. This observation supports
the conclusion that unbiased individuals are not a subset
with impaired photo-responsiveness, but a distinct behavioral
motor profile during search behavior. Supporting this model, December 2021 | Volume 15 | Article 777778 Frontiers in Behavioral Neuroscience | www.frontiersin.org 11 Modulation of Zebrafish Individuality Hageter et al. FIGURE 5 | Turn bias is sensitive to levels of Notch signaling. (A) Phenotypic counts following Notch inhibitor treatment (Vehicle, N = 125; 50 nM, N = 88; 100 nM,
N = 114; 150 nM, N = 123; 200 nM, N = 120; 250 nM, N = 101; 500 nM, N = 48; 1 µM, N = 43; 10 µM, N = 100). (B) Weighted absolute bias ratio averages
(matched PI = 0,1 blue bar; Partial match PI = 0.25, 0.75 gray bar; unbiased PI = 0.50 cyan bar. (C) Illustrative traces for treatment groups. Scale bar color
represents time in seconds. (D) Effect of Notch inhibition on MAD. p-values shown in bar, 1-way comparison of treatment groups to control. (E) Habenula symmetry
for vehicle controls (N = 8) and 100 nM Notch inhibitor treated (N = 9) larvae showing proportion with WT, symmetric (Sym), or reversed (Rev) phenotypes. WT larvae
have the larger habenula in the left hemisphere. y279 neuron counts in the habenula (F: vehicle N = 8; 100 nM N = 9) and PT (G: vehicle N = 8; 100 nM N = 9). For
neuron counts, regardless of hemisphere the larger habenula was classified as the ‘left’ habenula. (H,I) Representative maximum projection images showing y279
labeled neurons in vehicle and control. Scale bar 20 µm. (J) Area of her12 expression in the spinal cord following LY411575 treatment (Vehicle, N = 12; 100 nM,
N = 13; 8 µM, N = 11). (K) Normalized distribution of HuC/D positive neurons following notch inhibition (Vehicle: Blue, N = 18; 100 nM: Yellow, N = 13; 8 µM:
Magenta, N = 13). Determination of Bias X-axis distance spans half the spinal cord (0 micron = lateral spinal cord; 55 micron = spinal cord midline). Comparison shown is between vehicle
and 8 µM along the whole length of black bar between matched positions. Ribbons ± SEM. (L) Representative images of her12 expression in 27 hpf embryos. Lateral view of spinal cord (dotted outline). Scale bar 20 µm. (M) Representative HuC labeling in 24 hpf embryos showing dorsal view. Dotted line denotes spinal
cord midline. Scale Bar 40 µm. Asterisk p < 0.05. FIGURE 5 | Turn bias is sensitive to levels of Notch signaling. (A) Phenotypic counts following Notch inhibitor treatment (Vehicle, N = 125; 50 nM, N = 88; 100 nM,
N = 114; 150 nM, N = 123; 200 nM, N = 120; 250 nM, N = 101; 500 nM, N = 48; 1 µM, N = 43; 10 µM, N = 100). (B) Weighted absolute bias ratio averages
(matched PI = 0,1 blue bar; Partial match PI = 0.25, 0.75 gray bar; unbiased PI = 0.50 cyan bar. (C) Illustrative traces for treatment groups. Scale bar color
represents time in seconds. (D) Effect of Notch inhibition on MAD. p-values shown in bar, 1-way comparison of treatment groups to control. (E) Habenula symmetry
for vehicle controls (N = 8) and 100 nM Notch inhibitor treated (N = 9) larvae showing proportion with WT, symmetric (Sym), or reversed (Rev) phenotypes. WT larvae
have the larger habenula in the left hemisphere. y279 neuron counts in the habenula (F: vehicle N = 8; 100 nM N = 9) and PT (G: vehicle N = 8; 100 nM N = 9). For
neuron counts, regardless of hemisphere the larger habenula was classified as the ‘left’ habenula. (H,I) Representative maximum projection images showing y279
labeled neurons in vehicle and control. Scale bar 20 µm. (J) Area of her12 expression in the spinal cord following LY411575 treatment (Vehicle, N = 12; 100 nM,
N = 13; 8 µM, N = 11). (K) Normalized distribution of HuC/D positive neurons following notch inhibition (Vehicle: Blue, N = 18; 100 nM: Yellow, N = 13; 8 µM:
Magenta, N = 13). X-axis distance spans half the spinal cord (0 micron = lateral spinal cord; 55 micron = spinal cord midline). Comparison shown is between vehicle
and 8 µM along the whole length of black bar between matched positions. Ribbons ± SEM. Determination of Bias (L) Representative images of her12 expression in 27 hpf embryos. Lateral view of spinal cord (dotted outline). Scale bar 20 µm. (M) Representative HuC labeling in 24 hpf embryos showing dorsal view. Dotted line denotes spinal
cord midline. Scale Bar 40 µm. Asterisk p < 0.05. visual task latency (Manns and Römling, 2012). Therefore,
variable balance in hemispheric signaling may be a conserved
mechanism in generating inter-individual variation (Chen-Bee
and Frostig, 1996; Linneweber et al., 2020). Inter-hemispheric
communication is vital for the function of the visual system (Bui
Quoc et al., 2012; Chaumillon et al., 2018), including photo-
driven behavior in larval zebrafish (Gebhardt et al., 2019). The
counter hypothesis is a ’switching model’ where unbiased larvae
would display vigorous directional turning, yet in randomly
selected directions over sequential trials. This model is consistent
with a ‘winner take all’ circuit function (Fernandes et al., 2021). Indeed, within the primary visual processing center in zebrafish, when we quantify the strength of individual trials, the bias
ratios exhibit a step-wise decrease, i.e., PI 1 < 0.75 < 0.5,
suggesting accumulating inter-hemispheric noise that degrades
overall individual bias persistence. Corroborating this model,
previous studies showing that unilateral ablation of rostral PT
neurons, which are required for turn bias in larval zebrafish,
increases turning strength in the direction ipsilateral to the
intact neurons, indicating ablation removes conflicting input
from the contralateral hemisphere (Horstick et al., 2020). In
pigeons, a classic model for hemispheric specialization and
individual variation (Güntürkün et al., 1998; Freund et al.,
2016), increased conflict between hemispheres exacerbates December 2021 | Volume 15 | Article 777778 Frontiers in Behavioral Neuroscience | www.frontiersin.org 12 Modulation of Zebrafish Individuality Hageter et al. FIGURE 6 | Loss of gsx2 reduces inter-individual variation. (A) Effect of gsx2 genotype on weighted average bias ratio per performance groups (Matched: PI = 0,1;
Partial PI = 0.25,0.75; and Unbiased PI = 0.5). Numbers on bars indicate N. Weighing was based on the percent of larvae within each PI per genotype. (B) MI shows
that only gsx2+/+larvae maintain persistent same-direction turning following the loss of illumination. Dotted line at 0.5 indicates random movement. (C) TTA between
genotypes is not affected (gsx2+/+, N = 45; gsx2±, N = 113; gsx2–/–, N = 32). Baseline (yellow) and dark (gray) responses in (B,C). Asterisk p < 0.05. FIGURE 6 | Loss of gsx2 reduces inter-individual variation. Determination of Bias In
individuals with similar turn bias drive from both hemispheres, no single turn
direction persists due to conflict between hemispheres, resulting in lower bias
ratios and unbiased turning types. Determination of Bias (A) Effect of gsx2 genotype on weighted average bias ratio per performance groups (Matched: PI = 0,1;
Partial PI = 0.25,0.75; and Unbiased PI = 0.5). Numbers on bars indicate N. Weighing was based on the percent of larvae within each PI per genotype. (B) MI shows
that only gsx2+/+larvae maintain persistent same-direction turning following the loss of illumination. Dotted line at 0.5 indicates random movement. (C) TTA between
genotypes is not affected (gsx2+/+, N = 45; gsx2±, N = 113; gsx2–/–, N = 32). Baseline (yellow) and dark (gray) responses in (B,C). Asterisk p < 0.05. longer be malleable to environmental experiences beyond this
developmental interval. the optic tectum, neurons operate in a winner take all style during
visually guided behavior (Fernandes et al., 2021). However, turn
bias is driven by the loss of visual cues that activate rostral PT
neurons, which do not project to the tectum (Horstick et al.,
2020), implicating that even though turn bias is visually evoked,
the mechanism is likely independent of a tectal winner take
all mechanism. Despite the neurons maintaining zebrafish turn
bias being identified, the underlying mechanism imposing a
specific turning type remains unknown (Horstick et al., 2020). Our analysis suggests a model of competitive inter-hemispheric
communication modulating the magnitude of inter-individual
turn bias variation that is further adjusted by fluctuating and
specific variables in the internal and external environment. We also tested temperature and salinity, emphasizing
etiological ranges that zebrafish could experience in their native
environments (Engeszer et al., 2007; Sundin et al., 2019). Salinity
and temperature are critical environmental determinants and
have been shown to drive evolutionary changes in stickleback
populations (Gibbons et al., 2017). However, we found that only
raising larvae at varying temperatures resulted in modifications to
inter-individual variation. We show that temperature-dependent FIGURE 7 | Model for generating different turning types. Interhemispheric
differences in turn bias driving motor signals are a potential mechanism for
establishing turning types. Left (cyan) and right (magenta) hemispheres shown
for left, right, and unbiased motor types, with corresponding motor drive
shown by scale of descending arrow. For individuals with robust left or right
turning types, a strong ipsilateral turn bias signal persists in a single
hemisphere, with limited conflicting input from the contralateral hemisphere. Regulation of Individuality The mechanisms driving unique individual behavioral responses
based on sex or sensory context are well described (Asahina
et al., 2014; Lewis et al., 2015; Yapici et al., 2016; Marquart
et al., 2019; Ishii et al., 2020; Nelson et al., 2020). However,
why individuals in a population will show variable response
types to a consistent stimulus is poorly understood, especially
in vertebrates. One of our goals was to determine what internal
and external elements may modulate turn bias variation as
a basis to explain how different response types potentially
arise. To test external environmental factors, we selected
environmental enrichment, social experience, temperature, and
salinity. One hypothesis for how factors like enrichment or
social experience influence inter-individual variation is through
micro-environmental interactions that create unique individual
experiences (Kain et al., 2015). However, our data indicate
that these interactions do not influence turn bias variation
in zebrafish. One possible explanation is that during early
development, 1–3 days post-fertilization, larvae are primarily
inactive and only begin actively exploring around 4 days post
fertilization (Colwill and Creton, 2011; Lambert et al., 2012). Conversely, responsiveness to conspecifics is not observed until
3 weeks (Dreosti et al., 2015; Larsch and Baier, 2018). As bias
is established by 4 dpf, the underlying mechanisms may no FIGURE 7 | Model for generating different turning types. Interhemispheric
differences in turn bias driving motor signals are a potential mechanism for
establishing turning types. Left (cyan) and right (magenta) hemispheres shown
for left, right, and unbiased motor types, with corresponding motor drive
shown by scale of descending arrow. For individuals with robust left or right
turning types, a strong ipsilateral turn bias signal persists in a single
hemisphere, with limited conflicting input from the contralateral hemisphere. In
individuals with similar turn bias drive from both hemispheres, no single turn
direction persists due to conflict between hemispheres, resulting in lower bias
ratios and unbiased turning types. December 2021 | Volume 15 | Article 777778 Frontiers in Behavioral Neuroscience | www.frontiersin.org 13 Modulation of Zebrafish Individuality Hageter et al. are highly sensitive to the strength of Notch signaling (De
Smedt et al., 2005; Shen et al., 2021), the low inhibitor
concentrations used may be sub-threshold for disturbing the
spatial-temporal patterns of her12 and HuC/D tested here. Regulation of Individuality In
addition, the downstream effects of Notch are dependent on
the cellular micro-environments, determined by the local co-
expression of Notch receptors, ligands, and auxiliary proteins
(Demehri et al., 2009; Bertrand et al., 2011; LaFoya et al.,
2016). Therefore, the levels of Notch reduction that impair turn
bias, but not morphology, may not be sufficient to alter Notch
associated mechanisms impacting proliferation. Nevertheless,
subtle changes in Notch could lead to changes in cellular micro-
environments, thereby altering downstream signaling cascades,
and ultimately impacting turn bias maintaining neurons. Notch
haploinsufficiency is known to generate a myriad of defects
and disease states, including vasculature defects, seizure, autism,
and brain malformations, demonstrating that reduced Notch
signaling can disrupt biological functions (Krebs et al., 2004;
Connor et al., 2016; Fischer-Zirnsak et al., 2019; Blackwood
et al., 2020). However, the pharmacological inhibition used
in our current study is not regionally specific. Therefore, we
also tested an established zebrafish gsx2 mutant line, and
gsx2 is predominately expressed in subsets of hypothalamic,
preoptic area, pallium, and hindbrain neurons (Coltogirone et al.,
2021). The reduction in turn bias in gsx2 heterozygotes and
mutants suggests that turn bias variation is sensitive to local
changes in brain regions where gsx2 is expressed, independent
of the previously described rostral PT and habenula (Horstick
et al., 2020). As the levels of Notch that reduce turn bias
do not impact proliferation, it seems possible that Notch and
Gsx2 modulate turn bias by independent mechanisms. Our
current analysis identifies two conserved molecular signaling
and transcriptional control mechanisms, Notch and Gsx2, and
novel neuroanatomical substrates as important for generating
variation in turn bias. effects are not a generic thermal stress response. Etiological
increases in temperature have been shown to attenuate turn
bias in adult reef fish, implicating a potentially broader thermal
sensitivity in bias establishing mechanisms (Domenici et al.,
2014). Our analysis establishes that early developmental exposure
to etiological temperature fluctuation results in sustained and
specific turn bias changes. g
Intriguingly, the specification of habenular hemispheric
asymmetry is sensitive to the rate of development (Aizawa
et al., 2007), and developmental rate is temperature sensitive
(Kimmel et al., 1995). This observation could provide a potential
mechanism for thermal driven changes in turn bias. However,
our testing conditions produced no gross change in the habenula
nuclei morphology. This observation, however, does not exclude
functional or subcellular changes. Regulation of Individuality Surprisingly, we observe a
bilateral reduction in Tg(y279) positive PT neurons, which are
essential for maintaining turn bias, in the elevated temperature
experiments. A primary function of the PT is to integrate diverse
sensory inputs (Striedter, 1991; Derjean et al., 2010; Yaeger
et al., 2014). However, thermosensitivity of the PT neurons
has not been previously described, and we believe this is a
novel observation. Future studies identifying the genetic basis
of the Tg(y279) enhancer trap, which is currently unknown,
will be instrumental in elucidating how temperature impacts PT
neuron specification and inter-individual variation. The specific
abrogation of leftward turning types in increased temperature
conditions provides a powerful model to interrogate underlying
neural changes in a vertebrate brain associated with individual
behavioral patterns. p
Last, we wanted to identify molecular signaling pathways
regulating turn bias. Biased turning in larvae is largely lost in
heterozygotes of mutant lines associated with Notch signaling, yet
the impact of direct Notch inhibition was unexplored (Horstick
et al., 2020). In Drosophila and C. elegans, Notch signaling is
essential for establishing individual visual navigational strategies
and asymmetric chemosensory neuron identities, respectively
(Bertrand et al., 2011; Linneweber et al., 2020). Thus, work
from several species implicates Notch as a driver of variation
at behavioral and neuronal levels. Indeed, we show that partial
Notch disruption, using a specific pharmacological inhibitor,
disrupts biased turning in larval zebrafish, yet not the ability
to respond to illumination changes, establishing a direct role
of Notch signaling for turn bias, which is independent of
gross morphological development. Despite the established role
of Notch in neural proliferation, we found no significant
change in proliferative neurons, her12 expression, or number
of PT neurons at the dosages used for behavioral studies. However, we did observe disruption to the typical left/right
hemispheric asymmetry of the habenula, observing an increase in
reversed or symmetric habenula. Interestingly, similar disruption
to habenula symmetry is observed in mib zebrafish which
have severely reduced Notch signaling (Aizawa et al., 2007). Our results, show a novel Notch dose-sensitivity for habenula
asymmetry development, which may be a potential neural basis
for the absence of biased behavior following low levels of Notch
inhibition. Since Notch signaling is essential for diverse cellular
functions, and the precise downstream signaling mechanisms Frontiers in Behavioral Neuroscience | www.frontiersin.org ACKNOWLEDGMENTS We thank Alexandra Schmidt and Rebecca Coltogirone from
the Bergeron lab for help with imaging and fluorescent in situ
and Erik Duboué and Andrew Dacks for helpful comments
on the manuscript. FUNDING This work was supported by West Virginia University and
Department of Biology startup funds to EH and SB, Research
and Scholarship Advancement award to EH, and Program to
Stimulate Competitive Research funds provided to EH. MW
was supported by the Ruby Distinguished Doctoral Fellowship. HP was supported by NICHD R15HD101974 award to SB. The content is solely the responsibility of the authors and
does not necessarily represent the official views of the National
Institutes of Health. Function of Turn Bias and Inter-Individual
Variation Behavioral
variation
is
observed
in
diverse
species
and
behavioral
modalities
(Byrne
et
al.,
2004;
Elnitsky
and
Claussen, 2006; Cauchard et al., 2013; Horváth et al., 2020). In zebrafish, even complex neuromodulatory processes such
as startle habituation display inter-individual variation with
distinguishable ’habituation types’ (Pantoja et al., 2016, 2020). Yet, the general question remains, “why do specific behavioral
modalities manifest inter-individual differences?” Considering
a simple form of inter-individual variation, such as turn bias,
may offer insights to these questions. Zebrafish are active
hunters during larval stages and predatory success depends
on visual input, thus establishing a potent drive to remain
in illuminated areas (Gahtan et al., 2005; Filosa et al., 2016;
Muto et al., 2017). Following the loss of light and of overt
navigation cues, larvae initiate a local light-search, where
individual turn bias is triggered, causing looping trajectories
(Horstick et al., 2017). Looping search trajectories are observed
in various species in the absence of clear navigational cues, December 2021 | Volume 15 | Article 777778 Frontiers in Behavioral Neuroscience | www.frontiersin.org 14 Modulation of Zebrafish Individuality Hageter et al. DATA AVAILABILITY STATEMENT Inquiries can be directed to the corresponding author for
R scripts and datasets generated in this study. ETHICS STATEMENT The Supplementary Material for this article can be found
online
at:
https://www.frontiersin.org/articles/10.3389/fnbeh. 2021.777778/full#supplementary-material The animal study was reviewed and approved by West Virginia
University Institutional Animal Care and Use Committee. AUTHOR CONTRIBUTIONS suggesting an efficient systematic strategy (Collins et al., 1994;
Conradt et al., 2000; Zadicario et al., 2005). However, even
seemingly optimal behaviors may not be advantageous in all
contexts (Simons, 2011). Variation in turning types may ensure
individuals across the population possess strategies to mitigate
erratic environmental challenges, a form of bet-hedging (Simons,
2011; Kain et al., 2015). Similarly, behavioral variation adds
unpredictability to a population. Predictable behavioral patterns
can be exploited by predators (Catania, 2009, 2010). For aquatic
species, this may be advantageous as some heron species, a
predator of small fish, use a canopy hunting strategy, covering the
water surface with their wings and blocking light (Kushlan, 1976). Prey populations with unpredictable responses would potentially
provide a more challenging target (Humphries and Driver, 1970). Even though larval fish may not be the intended target of
heron canopy hunting, larval behavioral patterns may persist
over their lifespan. Indeed, adult zebrafish display a persistent
turn direction preference (Fontana et al., 2019), although
the correlation to larval turn bias is currently unexplored. Ultimately, the etiological purpose for turn bias variation is most
likely a combination of multiple explanations, including bet-
hedging, generating unpredictability, and genetically encoded
sources of variation. EH conceived the experiments. EH and JH wrote the manuscript
and analyzed data. SL wrote custom R scripts for data analysis. SB provided gsx2 lines and associated analysis. JH, MW, JS, HC,
HP, LB, and CS performed experiments. All authors contributed
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Age-Differential Effects of Job Characteristics on Job Attraction: A Policy-Capturing Study
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University of Groningen University of Groningen Age-Differential Effects of Job Characteristics on Job Attraction
Zacher, Hannes; Dirkers, Bodil T.; Korek, Sabine; Hughes, Brenda Age-Differential Effects of Job Characteristics on Job Attraction
Zacher, Hannes; Dirkers, Bodil T.; Korek, Sabine; Hughes, Brenda Age-Differential Effects of Job Characteristics on Job Attraction
Zacher, Hannes; Dirkers, Bodil T.; Korek, Sabine; Hughes, Brenda Published in:
Frontiers in Psychology DOI:
10.3389/fpsyg.2017.01124 IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from
it. Please check the document version below. Document Version
Publisher's PDF, also known as Version of record IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from
it. Please check the document version below. Document Version Publication date:
2017 Link to publication in University of Groningen/UMCG research database Citation for published version (APA):
Zacher, H., Dirkers, B. T., Korek, S., & Hughes, B. (2017). Age-Differential Effects of Job Characteristics on
Job Attraction: A Policy-Capturing Study. Frontiers in Psychology, 8, 1-11. Article 1124. https://doi.org/10.3389/fpsyg.2017.01124 Copyright
Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the
author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Age-Differential Effects of Job
Characteristics on Job Attraction: A
Policy-Capturing Study Based on an integration of job design and lifespan developmental theories, Truxillo
et al. (2012) proposed that job characteristics interact with employee age in
predicting important work outcomes. Using an experimental policy-capturing design, we
investigated age-differential effects of four core job characteristics (i.e., job autonomy,
task variety, task significance, and feedback from the job) on job attraction (i.e.,
individuals’ rating of job attractiveness). Eighty-two employees between 19 and 65 years
(Mage = 41, SD = 14) indicated their job attraction for each of 40 hypothetical job
descriptions in which the four job characteristics were systematically manipulated (in
total, participants provided 3,280 ratings). Results of multilevel analyses showed that
the positive effects of task variety, task significance, and feedback from the job were
stronger for younger compared to older employees, whereas we did not find significant
age-differential effects of job autonomy on job attraction. These findings are only partially
consistent with propositions of Truxillo et al.’s (2012) lifespan perspective on job design. Copyright strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of th
or copyright holder(s), unless the work is under an open content license (like Creative Commons). The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license. More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-
amendment. Take-down policy
If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately
and investigate your claim. Take-down policy
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believe that this document breaches copyright please contact us providing details, and we will remove access to the wo
vestigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the
number of authors shown on this cover page is limited to 10 maximum. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the
number of authors shown on this cover page is limited to 10 maximum. Download date: 24-10-2024 ORIGINAL RESEARCH
published: 30 June 2017
doi: 10.3389/fpsyg.2017.01124 Edited by: Edited by:
Ilias Kapoutsis,
Athens University of Economics and
Business, Greece Business, Greece
Reviewed by:
Sara Zaniboni,
Università di Bologna, Italy
Johannes Wendsche,
Bundesanstalt für Arbeitsschutz und
Arbeitsmedizin, Germany
*Correspondence:
Hannes Zacher
hannes.zacher@uni-leipzig.de Reviewed by:
Sara Zaniboni,
Università di Bologna, Italy
Johannes Wendsche,
Bundesanstalt für Arbeitsschutz und
Arbeitsmedizin, Germany Keywords: age, job design, job characteristics, job attraction, policy-capturing INTRODUCTION Populations and workforces around the globe are aging and becoming increasingly age diverse
(Hedge and Borman, 2012; Truxillo et al., 2015). This implies that organizations have to identify
effective ways to attract highly qualified younger and older job applicants. So far, however,
only a small number of survey studies have examined age-differential associations between job
characteristics and work outcomes (e.g., Zaniboni et al., 2013, 2014). Using an experimental
policy-capturing design (Aguinis and Bradley, 2014), the goal of the present study was to investigate
which jobs are most attractive to younger and older workers, respectively. Adopting a lifespan
perspective on job design, Truxillo et al. (2012) suggested that young and older workers have
different preferences with regard to job characteristics. They offered a model, based on an
integration of job design and lifespan developmental theories, that outlines possible moderating
effects of age on relationships between various job characteristics and work outcomes. *Correspondence:
Hannes Zacher
hannes.zacher@uni-leipzig.de Specialty section:
This article was submitted to
Organizational Psychology,
a section of the journal
Frontiers in Psychology Specialty section:
This article was submitted to
Organizational Psychology,
a section of the journal
Frontiers in Psychology
Received: 26 April 2017
Accepted: 19 June 2017
Published: 30 June 2017 With this article, we aim to contribute to the literature on age and job design in three ways: First,
using an experimental vignette methodology design, we conduct a rigorous empirical investigation
of core propositions of Truxillo et al.’s (2012) lifespan perspective on job design. In particular, we
investigated how four core job characteristics influence workers’ job attraction, and whether these
influences vary depending on age (see also Griffiths, 1999; Truxillo and Zaniboni, 2017). Keywords: age, job design, job characteristics, job attraction, policy-capturing Citation: Zacher H, Dirkers BT, Korek S and
Hughes B (2017) Age-Differential
Effects of Job Characteristics on Job
Attraction: A Policy-Capturing Study. Front. Psychol. 8:1124. doi: 10.3389/fpsyg.2017.01124 Second, Truxillo et al. (2012) included job satisfaction, work engagement, and performance as
outcome variables in their model. We extend research on this model by focusing on job attraction, June 2017 | Volume 8 | Article 1124 1 Frontiers in Psychology | www.frontiersin.org Zacher et al. Age and Job Characteristics pay, type of work) and organizational characteristics (i.e., work
environment, organizational image, location, size, familiarity,
work hours). However, these researchers did not include
motivational job characteristics in their meta-analysis. or individuals’ ratings of job attractiveness, as an outcome
variable. Job attraction describes the extent to which applicants
would like to carry out a given job (Singh, 1975; Rynes and
Lawler, 1983; Rynes and Miller, 1983). In the context of an aging
workforce and a “war for talent,” it is important to investigate
predictors of job attraction, as organizations are interested in
attracting and hiring highly qualified or qualifiable younger and
older job applicants (Zacher et al., in press). Morgeson and Humphrey (2006) reviewed an extensive
array of important job characteristics and combined several
characteristics in a comprehensive measurement tool, the Work
Design Questionnaire (WDQ). In our study, we focus on four
job characteristics included in both job characteristics theory
(Hackman and Oldham, 1976) and the WDQ, which have been
identified as having age-differential effects on work outcomes
by Truxillo et al. (2012). Specifically, we included four job
characteristics (i.e., job autonomy, task variety, task significance,
feedback from the job) that belong to the broader category
of task characteristics. According to Morgeson and Humphrey
(2006), task characteristics include those features of the job that
describe how the work itself is done and the nature and breadth
of tasks in a job (in addition to task characteristics, Morgeson
and Humphrey (2006) include knowledge characteristics, social
characteristics, and contextual characteristics). Job attraction is a popular criterion in the recruitment
literature. It differs from other recruitment-related constructs
such as job pursuit intentions, acceptance intentions, and
job choice (Chapman et al., 2005). Job attraction is typically
measured by asking applicants to provide an overall evaluation
of the attractiveness of the job they are applying for (e.g., “How
attractive is the job to you?”; Saks et al., 1994). Job Autonomy Job autonomy refers to the extent to which workers are able to
independently make decisions, and have autonomy in planning
and carrying out their work tasks (Hackman and Oldham, 1976;
Morgeson and Humphrey, 2006). Job autonomy has been shown
to be positively related to job satisfaction and work motivation
(Humphrey et al., 2007). In their model, Truxillo et al. (2012)
suggest that job autonomy has a stronger positive effect on job
satisfaction and performance among older compared to younger
workers. Older workers typically have been working in their jobs
for longer and therefore are more interested in autonomy to make
use of their experiential knowledge and skills (see also Zacher and
Frese, 2009). They further proposed that younger workers are still
gaining work experience and have a higher need for supervision
and thus expect less autonomy. Furthermore, Truxillo et al. (2012) suggested that older workers value job autonomy more
than younger workers because it allows them to adapt to job
demands and possibly compensate for age-related limitations,
such as decreases in physical strength and fast information
processing abilities (Kanfer and Ackerman, 2004). Therefore,
job autonomy should be more attractive to older compared to
younger workers. Consistent with these assumptions, a study by
Zaniboni et al. (2016) showed that job autonomy was stronger
positively related to the job satisfaction of older compared to
younger construction workers. Job satisfaction, in turn, was
positively related to mental health. According to Chapman et al. (2005), the influence of job
characteristics on job attraction can be explained by objective
factor theory (Behling et al., 1968), which states that applicants
form their job-related attitudes based on evaluations of objective
job or position characteristics. Surprisingly, however, research on
the effects of motivational job characteristics, particularly those
proposed by the job characteristics model (i.e., job autonomy,
task variety, task identity, task significance, feedback from the
job; Hackman and Oldham, 1976), is very sparse. We identified
only one early study by Farh and Scott (1983), which showed
that three of these job characteristics (i.e., job autonomy, task
variety, and feedback from the job) are positively and moderately
related to job attraction. Thus, our study also contributes to the
literature by investigating general effects of job characteristics on
job attraction. Job Autonomy Finally, the results of our study could inform how jobs are
designed and advertised, to increase the likelihood that younger
and older people apply for open job positions in the first place. In addition, our results may provide advice to companies on how
to (re-)structure jobs so that younger and older workers are more
likely to stay with the organization, are more satisfied with their
job conditions, and potentially work more efficiently (Zacher and
Schmitt, 2016). Hypothesis 1: The positive effect of job autonomy on job
attraction is moderated by age, such that the effect is stronger for
older compared to younger workers. JOB CHARACTERISTICS AND AGE Organizational researchers have argued that job characteristics
influence employees’ psychological states and, in turn, work
outcomes such as job satisfaction, strain, absenteeism, and
turnover (Hackman and Oldham, 1976; Fried and Ferris, 1987;
Steyn and Vawda, 2014). Chapman et al. (2005) showed in their
meta-analysis that job attraction is influenced directly by job and
position characteristics (i.e., compensation and advancement, Citation: In contrast,
job pursuit intentions include “a person’s desire to submit an
application, attend a site visit or second interview, or otherwise
indicate a willingness to enter or stay in the applicant pool
without committing to a job choice” (Chapman et al., 2005,
p. 929). Acceptance intentions describe “the likelihood that an
applicant would accept a job offer if one were forthcoming”
(Chapman et al., 2005, p. 929). Finally, job choice is an action
that entails “choosing whether to accept a real job offer involving
an actual job” (Chapman et al., 2005, p. 929). Frontiers in Psychology | www.frontiersin.org METHODS We used a policy-capturing design, a specific design that
is part of the broader category of experimental vignette
methodology designs, to test our hypotheses. Experimental
vignette methodology designs can be used to assess behaviors,
attitudes,
and
intentions
in
experimental
settings
while
improving experimental realism through the construction
of realistic scenarios (Aguinis and Bradley, 2014). Aguinis
and Bradley (2014) suggest that policy-capturing designs are a
particularly useful method for assessing implicit decision-making
processes. Hypothesis 2: The positive effect of task variety on job attraction
is moderated by age, such that the effect is stronger for younger
compared to older workers. In the present study, we created hypothetical scenarios in
which each of the four job characteristics (i.e., job autonomy,
task variety, task significance, and feedback from the job) was
manipulated (Karren and Barringer, 2002). Specifically, a number
of scenarios was created using statements from the German
version of the WDQ (Morgeson and Humphrey, 2006; Stegmann
et al., 2010). For each scenario, participants were asked to rate the
attractiveness of the job described in the scenario. In line with
best practices (Aiman-Smith et al., 2002; Rotundo and Sackett,
2002; Ohme and Zacher, 2015), we conducted a pilot study
before the main study to validate the statements used in the
scenarios. Task Variety Task variety describes the diversity of the job requirements, that
is, how many different tasks a worker is expected to perform
as part of the job (Hackman and Oldham, 1976; Morgeson
and Humphrey, 2006). Jobs that have higher levels of task
variety are generally assumed to be more pleasant to perform June 2017 | Volume 8 | Article 1124 Frontiers in Psychology | www.frontiersin.org 2 Zacher et al. Age and Job Characteristics (Humphrey et al., 2007). Lifespan theories (e.g., Carstensen
et al., 1999) propose that younger workers will find high task
variety more useful than older workers, as they have yet to
gain experience in different tasks, whereas older workers already
have acquired skills necessary for the job (see also Truxillo
et al., 2012). Thus, older workers might see task variety as a
burden in that they have to fulfill tasks that do not focus on
their existing experience and specialized expertise. Task variety
should therefore be more attractive for younger compared to
older workers. This proposition has been supported by survey
research which found that task variety has a stronger influence
on younger workers’ job satisfaction (Zaniboni et al., 2013,
2014). (Humphrey et al., 2007). Lifespan theories (e.g., Carstensen
et al., 1999) propose that younger workers will find high task
variety more useful than older workers, as they have yet to
gain experience in different tasks, whereas older workers already
have acquired skills necessary for the job (see also Truxillo
et al., 2012). Thus, older workers might see task variety as a
burden in that they have to fulfill tasks that do not focus on
their existing experience and specialized expertise. Task variety
should therefore be more attractive for younger compared to
older workers. This proposition has been supported by survey
research which found that task variety has a stronger influence
on younger workers’ job satisfaction (Zaniboni et al., 2013,
2014). Hypothesis 4: The positive effect of feedback from the job on job
attraction is moderated by age, such that the effect is stronger for
younger compared to older employees. Participants and Procedure In total, 20 participants completed the pilot study after they
were provided with a link to an online survey. Participants
were recruited through personal and professional contacts in
Germany. No demographic data were collected. Feedback from the Job Feedback from the job reflects the extent to which workers
receive direct and explicit feedback on how effectively they
are performing the required tasks (Hackman and Oldham,
1976; Morgeson and Humphrey, 2006). Feedback from the
job refers to feedback that is obtained through the results
of a worker’s performance rather than feedback given by
other people (Morgeson and Humphrey, 2006). Feedback has
a positive influence on job satisfaction, work motivation, as
well as job performance (Humphrey et al., 2007). Truxillo
et al. (2012) suggested in their model that feedback from the
job will be particularly valued by younger workers as they
still lack work experience and seek feedback to improve their
performance to further their careers. In contrast, older workers
are more experienced and have more confidence regarding their
performance and therefore need less feedback (Wang et al.,
2015). Feedback should therefore be more attractive for younger
compared to older workers. Task Significance Task significance refers to the influence and impact that people’s
jobs have on other people’s lives or work (Hackman and Oldham,
1976; Morgeson and Humphrey, 2006). Perceptions of task
significance are thought to enhance workers’ experience of
meaningfulness, which is believed to mediate the relationship
between task significance and work outcomes (Humphrey et al.,
2007). Furthermore, task significance is positively related to
job satisfaction, work motivation, and performance (Humphrey
et al., 2007; Grant, 2008). Based on the lifespan theory of
socioemotional selectivity (Carstensen et al., 1999), Truxillo
et al. (2012) argued that older workers are more likely to
value task significance in a job than younger workers. Workers
are increasingly looking for meaning in their jobs as they get
older (and their future time perspective becomes more limited),
whereas younger workers (who typically have higher levels of
future time perspective) are more focused on acquiring new and
useful skills and various job-related experiences. Both the pilot study and the main study were reviewed
and approved by the Ethical Committee Psychology at the
University of Groningen (Netherlands; see http://www.rug.nl/
research/heymans-institute/organization/ecp/?lang$=$en). All
participants gave written informed consent in accordance with
the Declaration of Helsinki. Pilot Study Hypothesis 3: The positive effect of task significance on job
attraction is moderated by age, such that the effect is stronger for
older compared to younger workers. Participants and Procedure Materials and Measures Statements describing different levels of each of the four job
characteristic were shown to the pilot study participants. For
each job characteristic, three statements were chosen randomly
from the respective items provided by the WDQ (Morgeson
and Humphrey, 2006). Each statement was shown with three
different levels of intensity (low, medium, high). Thus, there were
nine statements per job characteristic. Participants were asked,
“How much autonomy does this job offer?,” “How much task
variety does this job offer?,” “How significant or important is this
job?,” and “How much feedback does this job offer?,” respectively. Participants were asked to indicate their answers on 7-point
scales ranging from “very little” (1) to “very much” (7). Table 1
shows the wording of the items and descriptive statistics of the
pilot study. June 2017 | Volume 8 | Article 1124 Frontiers in Psychology | www.frontiersin.org 3 Age and Job Characteristics Zacher et al. TABLE 1 | Descriptive statistics from pilot study (N = 20). Job characteristics/Items
Low level
Medium level
High level
Difference
low-medium
Difference
medium-high
M
SD
M
SD
M
SD
t
t
JOB AUTONOMY
The job (very rarely/sometimes/very often) gives me
a chance to use my personal initiative or judgment
in carrying out the work. 1.53
0.77
3.68
0.75
6.16
0.77
−12.30***
−13.96***
The job allows me to make (very few/some/a lot of)
decisions on my own. 1.37
0.76
3.74
0.65
6.63
0.50
−12.43***
−22.25***
The job provides me with (very
little/moderate/significant) autonomy in making
decisions. 1.74
1.28
4.11
0.46
6.32
1.16
−8.52***
−7.84***
TASK VARIETY
The job involves performing a (low/moderate/great)
variety of tasks. 1.30
0.47
4.15
0.49
6.45
0.76
−26.05***
−14.04***
The job (very rarely/sometimes/very often) involves
doing a number of different things. 1.25
0.44
4.00
0.80
6.55
0.61
−15.64***
−12.07***
The job (very rarely/sometimes/very often) requires
the performance of a wide range of tasks. 1.30
0.57
4.00
0.46
6.35
0.75
−16.48***
−15.67***
TASK SIGNIFICANCE
The results of my work (very rarely/sometimes/very
often) significantly affect the lives of other people. 2.35
1.27
4.20
0.41
6.35
0.81
−6.75***
−10.30***
The work performed on the job has a (very
small/moderate/significant) impact on people
outside the organization. 2.40
1.43
4.35
0.75
6.25
0.79
−7.61***
−7.03***
The job has a (little/moderate/large) impact on
people outside the organization. TASK SIGNIFICANCE Items were adapted from the Work Design Questionnaire (Morgeson and Humphrey, 2006). Items were followed by the questions: “How much autonomy does this job offer?,” “How
much task variety does this job offer?,” “How significant is this job?,” and “How much feedback does this job offer?,” respectively. Responses were provided on 7-point scales ranging
from 1 (very little) to 7 (very much). ***p < 0.001. Participants were contacted by the second author and asked
whether they would be willing to take part in a research study
on job design. After a short introduction and explanation
of the study, participants were provided with a link to an
online survey. In total, 114 people started the online survey
and answered at least one question. However, only 82 workers
provided sufficient information on the study variables to be
included into the analyses. All participants were employed
or self-employed. There were 46 female participants and 36
male participants. Ages ranged from 19 to 65 years (M =
41.41, SD = 14.08). Participants had been working in their
current jobs for an average of 14.44 years (SD = 12.57). The
sample was highly educated with 42 of the participants (51.2%)
holding a university degree, and another 20 (24.4%) of the
participants having acquired the German general qualification
for entering university. Participants’ professions were very Results of a series of t-tests showed that the differences
between participants’ ratings of low intensity statements vs. medium intensity statements, as well as the differences between
participants’ rating of medium intensity statements vs. high
intensity statements for each of the job characteristic statements
were significant and consistent with expectations, in that low
intensity statements were rated lower than medium intensity
statements and medium intensity statements were rated lower
than high intensity statements (see Table 1). Thus, we concluded
that it was acceptable to use the statements to create the scenarios
for our main study. Materials and Measures 2.37
1.17
4.11
0.81
6.47
0.84
−5.90***
−10.21***
FEEDBACK FROM THE JOB
The work activities themselves provide (very
rarely/sometimes/very often) direct and clear
information about the effectiveness (e.g., quality and
quantity) of my job performance. 1.50
0.76
3.95
0.69
6.45
0.76
−10.97***
−9.38***
The job itself (very rarely/sometimes/very often)
provides feedback on my performance. 2.45
1.57
3.90
0.64
6.20
1.06
−3.81***
−9.52***
The job itself provides me (very
rarely/sometimes/very often) with information about
my performance. 1.90
0.91
3.90
0.85
6.55
0.76
−7.65***
−11.40***
Items were adapted from the Work Design Questionnaire (Morgeson and Humphrey, 2006). Items were followed by the questions: “How much autonomy does this job offer?,” “How
much task variety does this job offer?,” “How significant is this job?,” and “How much feedback does this job offer?,” respectively. Responses were provided on 7-point scales ranging
from 1 (very little) to 7 (very much). ***p < 0.001. TABLE 1 | Descriptive statistics from pilot study (N = 20). y
Participants and Procedure Participants and Procedure We recruited a convenience sample for the main study, again
relying on personal and professional contacts in Germany. June 2017 | Volume 8 | Article 1124 Frontiers in Psychology | www.frontiersin.org 4 Age and Job Characteristics Zacher et al. Information Network (O∗Net) database (Peterson et al., 2001; see
https://www.onetonline.org/) and a coding scheme developed
by Gonzalez-Mulé (2015). Specifically, as outlined by Gonzalez-
Mulé (2015), we used values of items from the work activities
and work context inventories of O∗NET that correspond to the
four job characteristics of interest. Specifically, job autonomy was
measured with the item “freedom to make decisions” (i.e., how
much decision making freedom, without supervision, does the
job offer?); task variety was measured with the item “importance
of repeating the same task” (reverse coded; i.e., how important
is repeating the same physical activities... or mental activities... over and over, without stopping, to performing this job?); task
significance was measured with the item “impact of decisions
on co-workers or company results” (i.e., what results do your
decisions usually have on other people or the image or reputation
or financial resources of your employer?); and feedback from the
job was measured with the item “making decisions and solving
problems” (i.e., how often workers receive feedback on their
performance and act upon it; see Table 5 in Gonzalez-Mulé,
2015). The values of the O∗Net items were obtained from a large
random sample of job incumbents who rated the extent to which
their jobs are characterized by the different descriptors. Previous
research has demonstrated the high reliability of the O∗Net
inventories (Childs et al., 1999; Strong et al., 1999; Peterson et al.,
2001). diverse. For example, job descriptions included teachers, lawyers,
administrators, and biologists. diverse. For example, job descriptions included teachers, lawyers,
administrators, and biologists. Materials and Measures We set up an online survey in which participants were first asked
to answer some general demographic questions, including age,
gender, education, job tenure, and job description. Subsequently,
participants were instructed to read a series of descriptions of
hypothetical job descriptions and rate how much they would
like to carry out these jobs. Each job description included four
statements validated in the pilot study; the statements presented
different levels of job autonomy, task variety, task significance,
and feedback from the job. As in the pilot study, for each job
characteristic there were three different statements based on the
WDQ items (Morgeson and Humphrey, 2006), and for each
statement there were three different levels of intensity: low,
medium, and high. The statements for each scenario were chosen
randomly with regard to the statement itself, as well as with
regard to the intensity level of each job characteristic. In total,
we created 41 scenarios, including one duplicate scenario to
assess reliability (Rotundo and Sackett, 2002; Ohme and Zacher,
2015). Each scenario contained four randomly selected and
randomly ordered statements. This was done to avoid possible
primacy or recency effects (Rotundo and Sackett, 2002). The
order in which the scenarios were presented to participants
was also randomized. Participants provided their ratings of
job attraction on a 7-point Likert scale ranging from “very
strongly disagree” (1) to “very strongly agree” (7). An example
scenario is shown in Figure 1. Overall, 82 participants provided
3,280 ratings, suggesting that all participants rated all 40 job
descriptions. Statistical Analyses Data were analyzed with random coefficient (i.e., multilevel)
models
using
the
hierarchical
linear
modeling
software
(Hofmann et al., 2000; Raudenbush et al., 2011), because
scenario ratings were nested within participants and the software
supports the analysis of both within- as well as between-person
variance (Kristof-Brown et al., 2002; Rotundo and Sackett,
2002). The within-person predictors of job attraction (i.e.,
the independent variables of job autonomy, task variety, task
significance, and feedback from the job) were group-mean
centered. Age as a between-person predictor and moderator
variable was centered at the grand mean (the control variables As
participants’
demographic
characteristics
and
characteristics of their current jobs may influence job attraction
(Chapman et al., 2005), we controlled in the analyses for gender
(1 = male, 2 = female), education (ranging from 1 = no school
degree to 5 = university degree), job tenure (in years), and
the characteristics of participants current jobs. We coded job
descriptions provided by participants using the Occupational FIGURE 1 | Example Scenario. The scenario describes a job with high levels of job autonomy and task variety, as well as low levels of task significance and medium
levels of feedback from the job. FIGURE 1 | Example Scenario. The scenario describes a job with high levels of job autonomy and task variety, as well as low levels of task significance and medium
levels of feedback from the job. June 2017 | Volume 8 | Article 1124 5 Frontiers in Psychology | www.frontiersin.org Zacher et al. Age and Job Characteristics characteristics and control variables significantly predicted job
attraction (see Table 3). In contrast, at the within-person level,
all four job characteristics positively predicted job attraction were also grand mean centered). To probe significant interaction
effects, we created plots of the regions of significance, which
show the simple slopes (i.e., effect of job characteristic on job
attraction) for different values of the moderator variable (i.e.,
age; Bauer and Curran, 2005; Preacher et al., 2006). In addition,
this plotting technique may help detect potential curvilinear
moderating effects of age (Rauschenbach et al., 2013; Zacher and
Schmitt, 2016). TABLE 3 | Results of multilevel analysis predicting job attraction. Preliminary Analyses Descriptive statistics and correlations of the study variables are
shown in Table 2. Of note, at the bivariate and between-person
level, aggregated job attraction ratings were negatively related
to age, job tenure, and job autonomy (O∗Net). Thus, older
workers, as well as workers with higher job tenure and higher job
autonomy generally rated the hypothetical job descriptions less
favorably. y
We first ran a null (or intercept-only) model to test whether
the use of multilevel modeling was appropriate. The chi-square
test for the intercept (r0) was significant, χ2
(81) = 473.90 with
p < 0.001, and the intraclass correlation coefficient was 0.12
(see Table 3). This value indicates that approximately 12% of
the variance in job attraction can potentially be explained
by between-person factors (e.g., participants’ age), leaving
approximately 88% of the variance that could potentially be
explained by within-person factors (i.e., the job characteristics in
our study). Thus, the use of multilevel modeling in our study was
appropriate. To assess test-retest reliability, we included a duplicate
scenario in our study (see also Rotundo and Sackett, 2002; Ohme
and Zacher, 2015). The duplicate scenario was not included in
subsequent analyses. Results showed that Cronbach’s alpha was
α = 0.65, indicating acceptable test-retest reliability of the job
attraction ratings. Statistical Analyses Predictor
Null model
Predictor model
γ
SE
t
γ
SE
t
Intercept
3.55
0.06
56.36***
3.55
0.06
59.54***
BETWEEN-PERSON PREDICTOR AND CONTROL VARIABLES
Age
−0.01
0.01
−1.39
Gender
0.09
0.14
0.63
Education
0.00
0.08
0.04
Job tenure
−0.00
0.01
−0.03
Job autonomy (O*Net)
−0.01
0.01
−1.65
Task variety (O*Net)
−0.00
0.00
−0.54
Task significance (O*Net)
0.00
0.01
0.52
Feedback from the job (O*Net)
−0.00
0.01
−0.13
WITHIN-PERSON PREDICTORS
Job autonomy
0.73
0.03
27.74***
Task variety
0.58
0.03
19.87***
Task significance
0.36
0.03
13.64***
Feedback from the job
0.37
0.03
14.47***
CROSS-LEVEL INTERACTIONS
Job autonomy × Age
−0.00
0.00
−1.89
Task variety × Age
−0.01
0.00
−3.69***
Task significance × Age
−0.01
0.00
−4.99***
Feedback from the job × Age
−0.01
0.00
−3.12***
VARIANCE COMPONENTS
Level 1 (σ2)
1.95
1.30
Level 2 Intercept (τ00)
0.26
0.25
ADDITIONAL INFORMATION
ICC
0.12
Pseudo R2
0.30
N = 82 participants provided 3,280 job attraction. Unstandardized coefficients (γ )
and standard errors (SE) are shown. ICC, intraclass correlation coefficient. The ICC is
calculated by dividing the between-person variance component (τ00) of the null model (i.e.,
the model with no predictors) by the sum of τ00 and the within-person variance component
(σ 2) of the null model. Pseudo R2, proportion of variance explained in dependent variable
by predictors at the between-person and within-person levels. ***p < 0.001. TABLE 3 | Results of multilevel analysis predicting job attraction. June 2017 | Volume 8 | Article 1124 Main Effects of Age and Job
Characteristics Results
of
the
multilevel
analysis
showed
that,
at
the
between-person level, neither age nor the other demographic TABLE 2 | Descriptive statistics and correlations. Variable
M
SD
1
2
3
4
5
6
7
8
9
1. Job attraction
3.57
0.58
–
2. Age
41.41
14.08
−0.37**
–
3. Gendera
1.56
0.50
0.16
−0.34**
–
4. Education
4.24
0.88
0.03
−0.07
0.19
–
5. Job tenure
14.44
12.57
−0.28*
0.82**
−0.30**
−0.34**
–
6. Job autonomy (O*Net)
81.49
9.79
−0.25*
0.19
0.10
0.08
0.06
–
7. Task variety (O*Net)
40.65
16.75
−0.10
0.08
−0.05
0.26*
−0.07
0.14
–
8. Task significance (O*Net)
71.84
12.86
−0.10
0.01
0.11
0.14
−0.09
0.70**
0.26*
–
9. Feedback from the job (O*Net)
63.17
11.40
−0.09
0.07
−0.18
0.11
−0.03
0.30**
0.38**
0.59**
–
N = 82. a1, male; 2, female. O*Net, Occupational Information Network (https://www.onetonline.org). **p < 0.01; *p < 0.05. Frontiers in Psychology | www.frontiersin.org 6 Age and Job Characteristics Zacher et al. (Table 3). Specifically, we found positive and significant main
effects of job autonomy (β1 = 0.73, p < 0.001), task variety (β2
= 0.58, p < 0.001), task significance (β3 = 0.36, p < 0.001), and
feedback from the job (β4 = 0.37, p < 0.001). These findings
suggest that job autonomy and task variety were somewhat more
important predictors of job attraction than task significance and
feedback from the job. for older workers (γ = 0.47, SE = 0.04, t = 11.47, p < 0.001). In
addition, the plot of the regions of significance suggests that the
simple slopes were significant across all age groups included in
our sample. Hypothesis 2 was, therefore, supported. Hypothesis 3 states that age moderates the positive effect of
task significance on job attraction, such that the effect is stronger
for older compared to younger workers. We found a significant
moderating effect of age (β33 = −0.01, p < 0.001; Table 3). However, the positive effect of task significance on job attraction
was stronger for younger workers (γ = 0.49, SE = 0.04, t = 59.54,
p < 0.001) than for middle-aged workers (γ = 0.36, SE = 0.03, t
= 13.64, p < 0.001) and for older workers (γ = 0.23, SE = 0.04, t
= 6.16, p < 0.001; see Figure 4). Moderating Role of Age Hypothesis 1 states that age moderates the positive effect of
job autonomy on job attraction, such that the effect is stronger
for older compared to younger workers. Results showed that
the moderating effect of age was not significant (β11 = −0.00,
p = 0.060; see Table 3). Thus, Hypothesis 1 did not receive
support. Nevertheless, we plotted the effect of job autonomy
on job attraction for different values of age, including younger
workers (i.e., −1SD of age), middle-aged workers (i.e., mean age),
and older workers (i.e., +1SD of age). Figure 2 shows that the
simple slope was positive and significant for younger (γ = 0.78,
SE = 0.04, t = 20.88, p < 0.001), middle-aged (γ = 0.73, SE =
0.03, t = 27.74, p < 0.001), and older workers (γ = 0.68, SE =
0.04, t = 18.37, p < 0.001). The non-significant interaction effect
and the plot of the regions of significance suggest that the simple
slopes did not differ for the various age groups included in our
sample. Finally, Hypothesis 4 proposes that age moderates the positive
effect of feedback from the job on job attraction, such that the
effect is stronger for younger compared to older workers. This
hypothesis was supported by a significant moderating effect of
age (β44 = −0.01, p < 0.001) and an interaction consistent
with the hypothesized pattern. The plot of the regions of
significance suggests that the simple slopes were significant across
all age groups included in our sample (Figure 5). Specifically, the
positive effect of feedback from the job on job attraction was
stronger for younger workers (γ = 0.46, SE = 0.04, t = 12.55,
p < 0.001) than for middle-aged workers (γ = 0.37, SE = 0.03, t
= 14.47, p < 0.001) and for older workers (γ = 0.29, SE = 0.04, t
= 7.93, p < 0.001). According to Hypothesis 2, age moderates the positive effect
of task variety on job attraction, such that the effect is stronger
for young compared to older workers. Consistent with this
hypothesis, we found a moderating effect of age (β22 = −0.01,
p < 0.001; Table 3). Main Effects of Age and Job
Characteristics Again, the plot of the regions of
significance suggests that the simple slopes were significant across
all age groups included in our sample. Thus, as we found the
opposite to what we proposed, Hypothesis 3 was not supported. Moderating Role of Age In their model, Truxillo et al. (2012)
suggested that older workers value job autonomy more than
younger workers because they are more experienced and need
less supervision compared to younger, less experienced workers. Consistently, Zaniboni et al. (2016) found that job autonomy was
more important for older construction workers in terms of job
satisfaction and mental health. However, meta-analytic research
on age-differential effects of job autonomy on work outcomes
has yielded mixed results (Ng and Feldman, 2015). For instance,
the association between job autonomy and job performance
was stronger for older workers, whereas the associations of
job autonomy with job satisfaction and affective commitment
were weaker for older workers. A potential explanation offered
by Ng and Feldman (2015) for these mixed results is that
the effects depend on the particular outcomes under study. It
may be possible that job autonomy is particularly important
for younger workers with regard to job attraction and other
attitudinal outcomes, because most younger workers apply for a
career job for the first time in their lives and, thus, motivational
job characteristics such as autonomy may be more important
to them than materialistic factors. In other words, it could
be that younger people pay more attention to what makes a
new job interesting, challenging, and important, as compared
to factors such as pay and the physical work environment. In contrast, job autonomy may be more important for older
workers with regard to job performance, because it allows them
to make use of their accumulated knowledge, experience, and
skills. FIGURE 4 | Effect of task significance on job attraction moderated by age
(with 95% confidence bands). FIGURE 5 | Effect of feedback from the job on job attraction moderated by
age (with 95% confidence bands). FIGURE 5 | Effect of feedback from the job on job attraction moderated by
age (with 95% confidence bands). Second, we hypothesized that the positive effect of task
variety on job attraction are stronger for younger compared to
older workers. This hypothesis was supported, providing further
support for the notion that task variety is more important
for younger than older workers. For instance, survey research
showed that younger workers with higher task variety are more
satisfied with their jobs (Zaniboni et al., 2013, 2014); we extend
this research by showing that younger workers are more attracted
to jobs that promise to provide them with high levels of task
variety. Moderating Role of Age As can be seen in Figure 3, the positive effect
of task variety on job attraction was stronger for younger workers
(γ = 0.69, SE = 0.04, t = 16.66, p < 0.001) than for middle-
aged workers (γ = 0.58, SE = 0.03, t = 19.87, p < 0.001) and Overall, according to the pseudo R2 statistic (LaHuis et al.,
2014), age, control variables, job characteristics, and interactions
between age and job characteristics explained 30% of the variance
in job attraction (see Table 3). FIGURE 2 | Effect of job autonomy on job attraction moderated by age (with
95% confidence bands). FIGURE 3 | Effect of task variety on job attraction moderated by age (with
95% confidence bands). FIGURE 3 | Effect of task variety on job attraction moderated by age (with
95% confidence bands). FIGURE 3 | Effect of task variety on job attraction moderated by age (with
95% confidence bands). FIGURE 2 | Effect of job autonomy on job attraction moderated by age (with
95% confidence bands). June 2017 | Volume 8 | Article 1124 Frontiers in Psychology | www.frontiersin.org 7 Zacher et al. Age and Job Characteristics FIGURE 4 | Effect of task significance on job attraction moderated by age
(with 95% confidence bands). FIGURE 5 | Effect of feedback from the job on job attraction moderated by
age (with 95% confidence bands). DISCUSSION FIGURE 4 | Effect of task significance on job attraction moderated by age
(with 95% confidence bands). (Fried and Ferris, 1987; Humphrey et al., 2007), we found that
job autonomy, task variety, task significance, and feedback from
the job had positive main effects on participants’ ratings of
job attraction. Furthermore, we found age-differential effects for
three out of the four job characteristics, of which two were
consistent with the hypothesized pattern (i.e., those including
task variety and feedback). y
We did not find support for our first hypothesis, which
stated that job autonomy has a stronger positive effect on
job attraction among older compared to younger workers. It
is important to note here that the interaction effect was not
significant according to conventional cut-offs (p = 0.06) and,
thus, statistical power may have been a problem. However, the
interaction plot suggested that job autonomy has somewhat
stronger effects among younger compared to older workers,
which is contrary to our assumption based on the lifespan
perspective on job design. Moderating Role of Age High task variety is particularly important for younger
workers, because it allows them to gain diverse work-related
experiences and develop new and useful skills (Truxillo et al.,
2012). DISCUSSION Summary and Interpretation of Findings June 2017 | Volume 8 | Article 1124 Zacher et al. Moreover, previous research
has used similar single item measures to assess job attraction
(Singh, 1975; Rynes and Lawler, 1983; Rynes and Miller, 1983). Also, due to the time-intensive nature of this approach, many
policy-capturing studies use single items and report test-retest
reliabilities across scenarios (Rotundo and Sackett, 2002; Ohme
and Zacher, 2015). The test-retest reliability was acceptable in our
study. Finally, our hypothesis on age-differential effects of feedback
from the job on job attraction was supported, suggesting that
feedback from the job is more important for job attraction of
younger compared to older applicants. This supports Truxillo
et al.’s (2012) assumption that younger workers are less
experienced and are interested in more feedback to improve their
performance, whereas older workers already have a great amount
of experience to rely on and hence need less feedback. Fourth, consistent with Truxillo et al.’s (2012) lifespan model
of job design, we did not include task identity (i.e., the extent to
which workers perform complete tasks, including goal setting,
planning, execution, and feedback processing; Hackman and
Oldham, 1976; Hacker, 1986; Morgeson and Humphrey, 2006) in
our study. Future research could focus on the importance of tasks
identity among younger and older workers (see Zacher et al.,
2016). p
y
Taken together, we could confirm only two out of four
hypotheses based on Truxillo et al.’s (2012) lifespan perspective
on job design. While job autonomy did not have an age-
differential effect on job attractiveness ratings, task variety,
feedback, and, unexpectedly, task significance had stronger
effects among younger compared to older workers. We extended
research based on Truxillo et al.’s (2012) model, and the
literature on recruitment more broadly, by focusing on job
attraction as an outcome of motivational job characteristics. Moreover, the use of the policy-capturing method has advantages
over survey designs, as it maximizes internal validity and
realism (Aguinis and Bradley, 2014). Despite these strengths,
our findings need to be interpreted in light of a number of
limitations. Finally, further research is needed that compares our current
findings with age-differential effects of job characteristics with
regard to different work outcomes, such as job satisfaction, work
engagement, and job performance. The difference between these
outcomes and job attraction is that the former outcomes can
only be answered by job incumbents, whereas job attraction
is mainly relevant among job seekers and job applicants who
are not yet in a concrete work role. Zacher et al. Zacher et al. Age and Job Characteristics Third, we expected that the effect of task significance on
job attraction is stronger for older compared to younger
workers. Our findings did not support this hypothesis, but
instead showed the opposite pattern: younger workers were more
attracted to jobs with higher task significance than older workers. This finding contradicts assumptions based on the lifespan
theory of socioemotional selectivity (Carstensen et al., 1999). Specifically, Truxillo et al. (2012) argued that older workers value
task significance more than younger workers as their limited
future time perspective renders meaningfulness and intrinsic
rewards more important than other job-related factors (e.g.,
pay). However, our findings are consistent with some recent
research that suggested that younger workers are more interested
in what influence their work has on other people and outside
of the company (Scroggins, 2008; Murray et al., 2011). As
noted above, it may also be the case that the motivational job
characteristics we studied are particularly relevant for younger
workers in terms of job attraction, because they focus more
on motivational job characteristics (i.e., interesting, important,
and challenging work) when evaluation a new career job as
compared to materialistic factors (e.g., pay, physical work
environment). Second, critics may question the external validity and
generalizability of studies using a policy-capturing design
(Karren and Barringer, 2002). We asked participants to form a
judgment about a hypothetical job description based on only four
variables, whereas in reality applicants and workers may have
access to more information about job openings. Furthermore,
it could be argued that our participants, who were all currently
working as employees or self-employed, may have answered
differently if they were actually searching for a job at the time
of answering the survey. Future research could replicate our
study with jobseekers, possibly with realistic job advertisements. Moreover, future research could assess different or additional
outcome variables, such as intentions to apply for a job opening
(Chapman et al., 2005). Third, we used a single item to assess job attraction, which may
raise concerns about reliability. Some researchers have suggested
that homogeneous constructs, such as global job satisfaction,
can be reliably assessed using single items (Wanous et al.,
1997; Fisher et al., 2016). We argue that job attraction is a
rather homogeneous attitudinal construct that is distinct from
other job-related attitudes and behavioral intentions (Highhouse
et al., 2003; Chapman et al., 2005). Zacher et al. Moreover, researchers
could investigate whether additional moderators of the job
characteristics-outcome relationships may play a role. For
instance, Truxillo et al. (2012) suggested that age-related
factors such as future time perspective, self-regulatory strategies,
and socioemotional selectivity processes (cf. Rudolph, 2016)
may mediate the moderating effect of age. Furthermore,
they proposed that additional individual differences (e.g.,
personality, health, cognitive abilities) and contextual factors
(e.g., organizational culture, climate), as well as interactions
among different job characteristics may act as boundary
conditions of the age-differential effects of job characteristics on
work outcomes. Summary and Interpretation of Findings Summary and Interpretation of Findings
The goal of this study was to test core propositions of Truxillo
et al. (2012) lifespan perspective on job design using a policy-
capturing design, which is one type of experimental vignette
methodology designs. Consistent with previous research on the
positive effects of job characteristics on other work outcomes
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O∗NET, eds N. G. Practical Implications and Conclusion on how to design jobs to motivate older employees. In
contrast, our findings provide practitioners with suggestions
on how to change job characteristics to make jobs more
attractive to younger job applicants, as well as workers of all
ages. Our study provides useful information for recruiters and human
resource managers interested in addressing the challenges of
an aging and increasingly age diverse workforce. Our findings
suggest that younger workers value certain job characteristics
more than older workers, including task variety and feedback
from the job. Thus, organizations aiming to recruit younger job
applicants could adjust their job advertisements by emphasizing
high levels of task variety and feedback from the job (i.e.,
targeted recruitment; Newman and Lyon, 2009). At the same
time, younger, middle-aged, and older applicants appear to value
job autonomy to a similar extent. Employers could use this
information in the recruitment process to gain more interest
from applicants by advertising the position as providing high
levels of job autonomy. FUNDING We acknowledge support from the German Research Foundation
(DFG) and Leipzig University within the program of Open
Access Publishing. In summary, our findings suggest that younger and older
workers differ with regard to their preferences for task
variety, task significance, and feedback from the job, but not
job autonomy. Specifically, younger workers rated jobs with
high levels of task variety, task significance, and feedback
from the job as more attractive than older workers. So far,
most research on age and job characteristics has focused ACKNOWLEDGMENTS We thank Karoline Hudl for her help with coding the O∗Net job
characteristics. AUTHOR CONTRIBUTIONS HZ and BD designed the study. BD collected the data. HZ
analyzed the data and wrote the first draft of the manuscript, and
SK and BH revised the manuscript. Limitations and Future Research First, participants had to read a large number of job descriptions. The hypothetical nature of these descriptions and increasing
fatigue while participating in policy-capturing studies have
frequently been mentioned as limitations of these designs
(Kristof-Brown et al., 2002; Rotundo and Sackett, 2002). For
example, Graham and Cable (2001) found that participants
experienced
more
stress
and
felt
more
exhausted
when
responding to 32 scenarios as compared to only 8 scenarios. We took a number of precautions while designing the study to
prevent fatigue from influencing our results, including relatively
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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) or licensor 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. Stegmann,
S.,
van
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et
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und
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Version
[The June 2017 | Volume 8 | Article 1124 Frontiers in Psychology | www.frontiersin.org 11
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Minimally invasive approach associated with lower resource utilization after aortic and mitral valve surgery
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JTCVS open
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From the aDivision of Cardiothoracic Surgery, Department of Surgery, Rutgers Rob-
ert Wood Johnson Medical School, New Brunswick, NJ; and bRobert Wood John-
son University Hospital, New Brunswick, NJ.
IRB: #Pro2021001533 approved on September 21, 2021.
Read at the 103rd Annual Meeting of The American Association for Thoracic Sur-
gery, Los Angeles, California, May 6-9, 2023.
Received for publication March 1, 2023; revisions received May 21, 2023; accepted
for publication June 5, 2023; available ahead of print July 25, 2023. ADULT: AORTIC VALVE ADULT: AORTIC VALVE ABSTRACT 0%
Total HRU
76
*P < .001
115
57
66
61
92
Direct Cost
75th Percentile
LOS 7 days OR
Readmission 30 days
80%
60%
40%
20%
*P < .001
*P < .001
High-Resource Utilization (%)
Full Sternotomy
Minimally Invasive
High-resource utilization overall and by definition
stratified by operative approach. Objective: To investigate the effect of minimally invasive cardiac surgery (MICS) on
resource utilization, cost, and postoperative outcomes in patients undergoing left-
heart valve operations. Methods: Data were retrospectively reviewed for patients undergoing single-valve
surgery (eg, aortic valve replacement, mitral valve replacement, or mitral valve
repair) at a single center from 2018 to 2021, stratified by surgical approach: MICS
vs full sternotomy (FS). Baseline characteristics and postoperative outcomes
were compared. Primary outcome was high resource utilization, defined as direct
procedure cost higher than the third quartile or either postoperative LOS
7 days or 30-day readmission. Secondary outcomes were direct cost, length of
stay, 30-day readmission, in-hospital and 30-day mortality, and major morbidity. Multiple regression analysis was conducted, controlling for baseline characteristics,
operative approach, valve operation, and lead surgeon to assess high resource
utilization. High-resource utilization overall and by definition
stratified by operative approach. Address for reprints: Leonard Y. Lee, MD, Division of Cardiothoracic Surgery,
Department of Surgery, Rutgers Robert Wood Johnson Medical School, 1 Robert
Wood Johnson Pl, New Brunswick, NJ 08901 (E-mail: leele@rwjms.rutgers.edu).
2666-2736
Copyright 2023 The Author(s). Published by Elsevier Inc. on behalf of The Amer-
ican Association for Thoracic Surgery. This is an open access article under the CC BY
license (http://creativecommons.org/licenses/by/4.0/).
https://doi.org/10.1016/j.xjon.2023.06.007 CENTRAL MESSAGE Minimally invasive approach in
left-heart valve surgery corre-
lates with lower resource utiliza-
tion relative to full sternotomy
with no concomitant increase in
postoperative complication
rates. Minimally invasive approach in
left-heart valve surgery corre-
lates with lower resource utiliza-
tion relative to full sternotomy
with no concomitant increase in
postoperative complication
rates. Results: MICS was correlated with a significantly lower rate of high resource utili-
zation (MICS, 31.25% [n ¼ 115] vs FS 61.29% [n ¼ 76]; P<.001). Median postop-
erative length of stay (MICS, 4 days [range, 3-6 days] vs FS, 6 days [range, 4 to 9
days]; P < .001) and direct cost (MICS, $22,900 [$19,500–$28,600] vs FS,
$31,900 [$25,900–$50,000]; P<.001) were lower in the MICS group. FS patients
were more likely to experience postoperative atrial fibrillation (P ¼ .040) and renal
failure (P ¼ .027). Other outcomes did not differ between groups. Controlling for
stratified Society of Thoracic Surgeons predicted risk of mortality, cardiac valve
operation, and lead surgeon, FS demonstrated increased likelihood of high resource
utilization (P<.001). Minimally invasive approach associated with lower
resource utilization after aortic and mitral valve surgery NaYoung K. Yang, MPH,a Fady K. Soliman, BA,a Russell J. Pepe, MD, MSCTS,a,b Nadia K. Palte, BA,a
Jin Yoo, BS,a Sorasicha Nithikasem, BS,a Kayla N. Laraia, BS,a Abhishek Chakraborty, MS,a
Joshua C. Chao, MD, JD,a,b Gengo Sunagawa, MD,a,b Manabu Takebe, MD,a,b Anthony Lemaire, MD,a,b
Hirohisa Ikegami, MD,a,b Mark J. Russo, MD,a,b and Leonard Y. Lee, MDa,b Address for reprints: Leonard Y. Lee, MD, Division of Cardiothoracic Surgery,
Department of Surgery, Rutgers Robert Wood Johnson Medical School, 1 Robert
Wood Johnson Pl, New Brunswick, NJ 08901 (E-mail: leele@rwjms.rutgers.edu).
2666-2736 Statistical Analyses Baseline patient demographics, along with clinical and perioperative
characteristics, were evaluated and compared. Primary outcome was
HRU, defined as patients with either direct cost higher than the third quar-
tile5 and/or either postoperative LOS 7 days or 30-day readmission.6-10
Secondary outcomes investigated were total direct costs—defined as
expenses directly related to patient care, such as procedure and
periprocedural
costs19—postoperative
LOS,
30-day
readmission,
in-hospital and 30-day mortality, along with other postoperative complica-
tions including atrial fibrillation, acute kidney injury, bleeding requiring
reoperation, hospital readmission, reintubation, and stroke. Intraoperative
bypass and crossclamp times, along with intraoperative and postoperative
blood product utilization were also evaluated as secondary outcomes. complications, including sternal infections and postopera-
tive respiratory complications, and improved patient com-
fort with decreased postoperative pain.4 To quantify the benefits of MICS relative to traditional
approaches, clinicians have determined measures based
on procedure costs and outcomes. High-resource utilization
(HRU)—defined as direct procedure cost higher than the
third quartile5 or either postoperative LOS 7 days or 30-
day readmission6-10—is 1 such metric that can be used to
gauge
the
utility
of
MICS. Other
metrics
include
nonhome discharge location.10 Outcomes of continuous and categorical variables are reported as me-
dians and interquartile ranges (IQR) (25th–75th percentiles) or frequencies
and proportions (%), respectively. Wilcoxon rank sum test and Fisher exact
tests were used for comparison between cohorts. With thoughtful effort to curtail unnecessary surgical
injury, minimally invasive techniques have gained favor in
cardiac surgery, eventually propelling the popularization of
minimally invasive approaches for valve operations.11-13
Although studies on postoperative outcomes have been
conducted on isolated valve surgeries,14-18 minimally
invasive valve operations have not been compared to open
approaches comprehensively to include various valve types
within 1 investigation. Furthermore, minimally invasive
approaches to valve operations have not been compared to
their full sternotomy (FS) counterparts in the context of
patient resource utilization. Therefore, this study aims to
investigate the effect of MICS on resource utilization, cost,
and other postoperative outcomes in patients undergoing
left heart valve operations. For further analysis, a multivariable regression analysis was performed,
controlling for operative approach (either FS or MICS), valve operation,
lead surgeon, and STS predicted risk of mortality (STS PROM) score. Orig-
inally a continuous variable, “STS PROM score was stratified by low, me-
dium, and high risk defined as 1% to 4%, 4% to 8%, and 8% risk,
respectively, for this regression analysis. Patient Population Surgeries included patients who underwent isolated AVR, MVR, or MV
repair. Operative approach included either FS, partial sternotomy, or right
minithoracotomy, with the minimally invasive cohort comprising opera-
tions with partial sternotomy and right minithoracotomy approaches. Se-
lection for minimally invasive valve surgery vs conventional FS is based
on shared decision making between the surgeon and the patient. Patients
were stratified by operation approach (MICS vs FS). Patients who under-
went transcatheter operations, underwent concomitant operations, had
prior cardiac surgery of any kind, had indication for endocarditis, or who
underwent an emergency operation were excluded from the study. To view the AATS Annual Meeting Webcast, see the
URL next to the webcast thumbnail. PERSPECTIVE Abbreviations and Acronyms
AVR
¼ aortic valve replacement
ERAS
¼ Enhanced Recovery After Surgery
FS
¼ full sternotomy
HRU
¼ high-resource utilization
LOS
¼ length of stay
MICS
¼ minimally-invasive cardiac surgery
MVR
¼ mitral valve replacement
MV
¼ mitral valve
STS PROM ¼ Society of Thoracic Surgeons
predicted risk of mortality RESULTS The academic center has 28 operating rooms, with an
average annual surgical volume of 15,000 cases. Approx-
imately 10% of these are cardiac surgeries, with 1400 open
and 350 transcatheter valve operations performed annually. Notably, 650 of these are valve procedures. Of 492 patients
included in the study, 368 (74.80%) underwent MICS and
124 (25.20%) underwent FS. All patients, irrespective of
surgical approach, utilized a multimodal Enhanced Recov-
ery After Surgery (ERAS) protocol that included nutritional PERSPECTIVE Minimally invasive approaches are increasingly
common across surgical specialties, with postop-
erative benefits and noninferior outcomes. MICS
for left-heart valve pathology correlates with
lower resource utilization relative to full sternot-
omy with no increase in postoperative complica-
tions. MICS may optimize resource allocation and
provide higher value care. Conclusions: MICS for left-heart valve pathology demonstrated improved postop-
erative outcomes and resource utilization. (JTCVS Open 2023;15:72-80) with the case volume doubling in 2014 from 10 years prior.3
Within the field of cardiothoracic surgery, studies have
shown the benefits of minimally invasive cardiac surgery
(MICS) over traditional open techniques, including shorter
hospital lengths of stay (LOS), decreased postoperative First revolutionized by British surgeons in 1986 through
replacement of percutaneous nephrolithotomy with litho-
tripters,1,2 the minimally invasive approach has steadily
been incorporated into many surgical fields.2 Nationwide,
3 million operations use a minimally invasive approach, First revolutionized by British surgeons in 1986 through
replacement of percutaneous nephrolithotomy with litho-
tripters,1,2 the minimally invasive approach has steadily
been incorporated into many surgical fields.2 Nationwide,
3 million operations use a minimally invasive approach, JTCVS Open c September 2023 72 Yang et al Adult: Aortic Valve To view the AATS Annual Meeting Webcast, see the
URL next to the webcast thumbnail. Abbreviations and Acronyms
AVR
¼ aortic valve replacement
ERAS
¼ Enhanced Recovery After Surgery
FS
¼ full sternotomy
HRU
¼ high-resource utilization
LOS
¼ length of stay
MICS
¼ minimally-invasive cardiac surgery
MVR
¼ mitral valve replacement
MV
¼ mitral valve
STS PROM ¼ Society of Thoracic Surgeons
predicted risk of mortality To view the AATS Annual Meeting Webcast, see the
URL next to the webcast thumbnail. Abbreviations and Acronyms
AVR
¼ aortic valve replacement
ERAS
¼ Enhanced Recovery After Surgery
FS
¼ full sternotomy
HRU
¼ high-resource utilization
LOS
¼ length of stay
MICS
¼ minimally-invasive cardiac surgery
MVR
¼ mitral valve replacement
MV
¼ mitral valve
STS PROM ¼ Society of Thoracic Surgeons
predicted risk of mortality outcomes, and 30-day outcomes. This study was approved by the Institu-
tional Review Board of Robert Wood Johnson University Hospital under
protocol #Pro2021001533 on September 21, 2021. Patients provided
informed written consent for the publication of the study data. To view the AATS Annual Meeting Webcast, see the
URL next to the webcast thumbnail. Statistical Analyses Thresholds for stratification of
STS PROM scores were based on prior literature.20 Controlling for the
STS risk score ultimately controlled for baseline characteristics intrinsi-
cally included in calculating the patient’s score, including factors of age,
gender, race, ethnicity, preoperative atrial fibrillation, body mass index,
congestive heart failure, prior stroke or cerebrovascular disease, chronic
renal failure requiring dialysis, smoking history/status, diabetes, preopera-
tive ejection fraction, hypertension, hyperlipidemia, and New York Heart
Association (NYHA) functional class. Statistical analysis was conducted
using statistical program JMP Statistical Discovery Pro 16.2.0 (SAS Insti-
tute Inc). METHODS Baseline characteristics of minimally invasive cardiac su
Characteristic
Full sternotomy (n ¼ 12
Baseline demographics
Age (y)
63 (55-70)
Female
37 (29.84)
Hispanic ethnicity
18 (14.52)
Race
White
61 (49.19)
Black
11 (8.87)
Asian
5 (4.03)
Other (eg, Hispanic ethnicity)
44 (35.48)
Clinical characteristics
Atrial fibrillation
14 (11.48)
Body mass index
27.98 (24.28-33.23)
Congestive heart failure
39 (31.45)
Prior CVA
2 (1.63)
Cerebrovascular disease
17 (13.71)
Chronic lung disease moderate
21 (16.91)
Chronic renal failure
2 (1.61)
Cigarette smoking
History
41 (33.06)
Current at time of surgery
14 (11.29)
Diabetes mellitus
32 (25.81)
Ejection fraction (%)
58 (55-63)
Hypertension n (%)
98 (79.03) time (FS median, 106 minutes; interquartile range [IQR],
85-141.75 minutes vs MICS median, 65 minutes; IQR,
56-95 minutes; P<.001) and crossclamp time (FS median,
83 minutes; IQR, 54.25-112.25 minutes vs MICS median,
46 days; IQR, 39-65 minutes; P<.001) were significantly
longer with FS approach operation. Intensive care unit
(ICU) LOS (FS median, 28.5 days; IQR, 21-51.75 days vs
MICS median, 15; IQR, 8-27 days; P < .001) was also
longer in patients who underwent FS operative approach. Patients undergoing MICS were more likely to undergo
on-table extubation (P<.001) and less likely to receive in-
traoperative and postoperative transfusions (P<.001). supplementation, prehabilitation, goal-directed therapy,
multimodal
opioid-sparing
pain
management,
bowel
motility prophylaxis, early mobilization, and multimodal
analgesia. Of those who underwent MICS, 94.56%
(n ¼ 348) had right minithoracotomies (4 cm anterior
incision, just right lateral of the sternum in the second inter-
costal space) and the remaining 4.44% underwent partial
sternotomies (6 cm incision with a sternotomy “T’d”
into the third or fourth intercostal space). Baseline demographic and clinical characteristics of pa-
tients included are presented in Table 1. Patients who under-
went minimally invasive surgery were of older age
(P ¼ .014) and were more likely to be women (P ¼ .004). Whereas patients in the FS cohort had a greater proportion
of those identifying with Hispanic ethnicity (P ¼ .013), race
did not differ significantly between the 2 cohorts overall
(P ¼ .265). The proportion of patients with diabetes melli-
tus (P ¼ .182) or hypertension (P ¼ .395) did not differ be-
tween the two cohorts. NYHA functional class>III did not
differ between the two cohorts (P ¼ .258), along with STS
PROM score (P ¼ .733). METHODS Patients aged 18 years or older who had undergone aortic valve replace-
ment (AVR), mitral valve replacement (MVR), or mitral valve repair (MV
repair) at a single tertiary care academic medical center between 2018 and
2021 were identified. Data for this study was sourced from the cardiac surgery database of the
academic center (Robert Wood Johnson University Hospital), and was
developed according to The Society of Thoracic Surgeons (STS) Adult
Cardiac Database version 4.20.1 definitions to include patient demo-
graphics, baseline clinical and perioperative characteristics, in-hospital JTCVS Open c Volume 15, Number C 73 Adult: Aortic Valve Yang et al supplementation, prehabilitation, goal-directed therapy
multimodal
opioid-sparing
pain
management,
bowe
motility prophylaxis, early mobilization, and multimoda
analgesia. Of those who underwent MICS, 94.56%
(n ¼ 348) had right minithoracotomies (4 cm anterio
incision, just right lateral of the sternum in the second inter
costal space) and the remaining 4.44% underwent partia
sternotomies (6 cm incision with a sternotomy “T’d
into the third or fourth intercostal space). Baseline demographic and clinical characteristics of pa
tients included are presented in Table 1. Patients who under
went minimally invasive surgery were of older ag
(P ¼ .014) and were more likely to be women (P ¼ .004
Whereas patients in the FS cohort had a greater proportio
of those identifying with Hispanic ethnicity (P ¼ .013), rac
did not differ significantly between the 2 cohorts overa
(P ¼ .265). The proportion of patients with diabetes melli
tus (P ¼ .182) or hypertension (P ¼ .395) did not differ be
tween the two cohorts. NYHA functional class>III did no
differ between the two cohorts (P ¼ .258), along with ST
PROM score (P ¼ .733). Perioperative
characteristics
stratified
by
operativ
approach are described in Table 2. Cardiopulmonary bypas
TABLE 1. Values are presented as median (interquartile range) or n (%). CVA, Cerebrovascular accident; NYHA, New York Heart Association; STS PROM, Society of Thoracic Surgeons
predictive risk of mortality. *All bold font indicates statistically significant difference at a P-value of 0.05. METHODS The MICS group contained a significantly lower propor-
tion of patients in the HRU category: the MICS group un-
derwent fewer operations with a direct cost higher than
the 75th percentile ($33,000) compared with the FS group
(17.93% [n ¼ 66] vs 45.97% [n ¼ 57]; P <.001). The
MICS group contained fewer patients with either a postop-
erative LOS >7 days and/or readmission within 30 days,
compared with the FS group (25.0% [n ¼ 92] vs 49.19%
[n ¼ 61]; P<.001) (Figure 1). In the total population, there were 4 (0.81%) mortalities;
postoperative LOS ranged from 3 to 6.25 days (IQR) and In the total population, there were 4 (0.81%) mortalities;
postoperative LOS ranged from 3 to 6.25 days (IQR) and Perioperative
characteristics
stratified
by
operative
approach are described in Table 2. Cardiopulmonary bypass TABLE 1. Baseline characteristics of minimally invasive cardiac surgery and full sternotomy patients by procedure type*
Characteristic
Surgical approach
P value ⍺¼ 0.05
Full sternotomy (n ¼ 124)
Minimally-invasive surgery (n ¼ 368)
Baseline demographics
Age (y)
63 (55-70)
66 (58-73)
0.014
Female
37 (29.84)
165 (44.84)
0.004
Hispanic ethnicity
18 (14.52)
28 (7.61)
0.013
Race
0.265
White
61 (49.19)
211 (57.65)
Black
11 (8.87)
17 (4.64)
Asian
5 (4.03)
16 (4.37)
Other (eg, Hispanic ethnicity)
44 (35.48)
106 (28.96)
Clinical characteristics
Atrial fibrillation
14 (11.48)
77 (21.15)
0.018
Body mass index
27.98 (24.28-33.23)
27.68 (24.38-31.63)
0.452
Congestive heart failure
39 (31.45)
94 (25.54)
0.200
Prior CVA
2 (1.63)
14 (5.34)
0.088
Cerebrovascular disease
17 (13.71)
40 (10.87)
0.153
Chronic lung disease moderate
21 (16.91)
28 (7.61)
0.003
Chronic renal failure
2 (1.61)
7 (1.90)
0.835
Cigarette smoking
0.734
History
41 (33.06)
113 (30.71)
Current at time of surgery
14 (11.29)
32 (8.70)
Diabetes mellitus
32 (25.81)
74 (20.11)
0.182
Ejection fraction (%)
58 (55-63)
60 (55-63)
0.639
Hypertension n (%)
98 (79.03)
277 (75.27)
0.395
Hypercholesterolemia n (%)
38 (30.89)
179 (49.18)
<0.001
NYHA functional class III
25 (20.16)
58 (15.76)
0.258
STS PROM score (%)
1.36 (0.7-2.99)
1.32 (0.87-2.25)
0.733
Values are presented as median (interquartile range) or n (%). CVA, Cerebrovascular accident; NYHA, New York Heart Association; STS PROM, Society of Thoracic Surgeons
predictive risk of mortality. *All bold font indicates statistically significant difference at a P-value of 0.05. 74
JTCVS O
S
b
2023 minimally invasive cardiac surgery and full sternotomy patients by procedure type* TABLE 1. METHODS Baseline characteristics of minimally invasive cardiac surgery and full sternotomy patients by procedur Values are presented as median (interquartile range) or n (%). CVA, Cerebrovascular accident; NYHA, New York Heart Association; STS PROM, Society of Thoracic Surgeons
predictive risk of mortality. *All bold font indicates statistically significant difference at a P-value of 0.05. 74
JTCVS Open c September 2023 74 Yang et al Adult: Aortic Valve TABLE 2. Perioperative outcomes of patients undergoing minimally invasive cardiac surgery and full sternotomy by procedure type*
Characteristic
Surgical approach
P value ⍺¼ 0.05
Full sternotomy (n ¼ 124)
Minimally-invasive surgery (n ¼ 368)
Perioperative
Bypass time (min)
106 (85-141.75)
65 (56-95)
<0.001
Crossclamp time (min)
83 (54.25-112.25)
46 (39-67)
<0.001
On-table extubation
12 (9.68)
183 (49.73)
<0.001
ICU LOS (h)
28.5 (21-51.75)
15 (8-27)
<0.001
Operating surgeon
Surgeon A
39 (8.01)
258 (52.98)
<0.001
Surgeon B
27 (5.54)
52 (10.68)
Surgeon C
10 (2.05)
38 (7.80)
Surgeon D
36 (7.39)
0 (0.00)
Surgeon E
10 (2.05)
17 (3.49)
Valve operation
Aortic valve replacement
96 (19.51)
196 (39.84)
<0.001
Mitral valve replacement
25 (5.08)
54 (10.98)
Mitral valve repair
3 (0.61)
118 (23.98)
Blood products, 2 U
Intraoperative
Packed RBCs
20 (16.13)
7 (1.90)
<0.001
Fresh frozen plasma
28 (22.58)
11 (2.99)
<0.001
Cryoprecipitate
13 (10.48)
7 (1.90)
<0.001
Platelets
48 (38.71)
22 (5.98)
<0.001
Postoperative
Packed RBCs
35 (28.23)
25 (6.79)
<0.001
Fresh frozen plasma
26 (20.97)
37 (10.05)
0.002
Cryoprecipitate
16 (12.90)
7 (1.90)
<0.001
Platelets
19 (15.32)
12 (3.26)
<0.001
values are presented as median (interquartile range) or n (%). ICU LOS, Intensive care unit length of stay; RBCs, red blood cells. *All bold font indicates statistically significant
difference at a P-value of 0.05. ts undergoing minimally invasive cardiac surgery and full sternotomy by procedure type* tive outcomes of patients undergoing minimally invasive cardiac surgery and full sternotomy by procedure type* ABLE 2. Perioperative outcomes of patients undergoing minimally invasive cardiac surgery and full sternotomy by total direct cost from $20.3,000 to $32.9k (IQR). Postoper-
ative LOS (MICS median, 4 days; IQR, 3-6 days vs FS, 6
days; IQR, 4-9 days; P<.001) and direct costs (MICS me-
dian, $22,900; IQR, $19,500–$28,600] vs FS median,
$31,900; IQR, $25,900–$50,000; P <.001) were lower in
operations with a minimally-invasive approach (Figure 2). METHODS Postoperative 30-day mortality did not differ between pa-
tient cohorts even when contextualized by in-hospital mor-
tality, ICU readmission, and 30-day readmission rates,
which also did not show differences between the 2 cohorts. Other
postoperative
complications
such
as
bleeding
requiring reoperation, reintubation, stroke, and pneumonia
showed no significant difference between the MICS and
sternotomy groups. However, patients who underwent FS
approach were more likely to experience postoperative
atrial fibrillation (P ¼ .040) and renal failure (P ¼ .027)
(Figure 3). total direct cost from $20.3,000 to $32.9k (IQR). Postoper-
ative LOS (MICS median, 4 days; IQR, 3-6 days vs FS, 6
days; IQR, 4-9 days; P<.001) and direct costs (MICS me-
dian, $22,900; IQR, $19,500–$28,600] vs FS median,
$31,900; IQR, $25,900–$50,000; P <.001) were lower in
operations with a minimally-invasive approach (Figure 2). Postoperative 30-day mortality did not differ between pa-
tient cohorts even when contextualized by in-hospital mor-
tality, ICU readmission, and 30-day readmission rates,
which also did not show differences between the 2 cohorts. Other
postoperative
complications
such
as
bleeding
requiring reoperation, reintubation, stroke, and pneumonia
showed no significant difference between the MICS and
sternotomy groups. However, patients who underwent FS
approach were more likely to experience postoperative
atrial fibrillation (P ¼ .040) and renal failure (P ¼ .027)
(Figure 3). counterparts (FS odds ratio [OR], 3.28; 95% CI, 1.76-6.13
vs MICS OR, 0.30; 95% CI, 0.16-0.57; P<.001) (Figure 4). Furthermore, the lead surgeon had a significant effect on
odds of HRU, with more experienced surgeons showing
decreased odds of HRU compared with those with fewer
years of experience. DISCUSSION Comparison of high-resource utilization overall and separated by definition: direct procedure cost higher than the third quartile or either post-
operative length of stay (LOS) 7 days or 30-day readmission between patients who underwent valve operations stratified by operative approach (full ster-
notomy vs minimally invasive surgery). By both separate definitions and composite high resource utilization, we find that patients who underwent full
sternotomy cardiac valve operations experienced significantly higher rates of high resource utilization. HRU, High resource utilization; OR, odds ratio. DISCUSSION This study compared minimally invasive vs FS ap-
proaches for AVR, MVR, or MV repair at a single academic
medical center. Our results indicate that minimally invasive
approach is associated with shorter cardiopulmonary
bypass and crossclamp times, shorter ICU LOS, fewer
transfusion requirements, and lower postoperative rates of
acute kidney injury and atrial fibrillation. Additionally,
the MICS approach to left-heart valve surgery is associated
with shorter LOS, lower cost after valve surgery, and ulti-
mately lower rates of HRU, even when controlled for STS
PROM, valve operation, and lead surgeon. Minimally inva-
sive approaches did not compromise clinical outcomes as
evidenced by the lack of differences in postoperative
complications. Nominal logistic fit for HRU controlling for stratified
STS risk (low, medium, and high), type of cardiac valve
operation (AVR, MVR, MV repair), and lead surgeon
showed a significant model (P < .001), in with patients
who underwent FS significantly higher odds of high
resource utilization compared with their minimally invasive JTCVS Open c Volume 15, Number C 75 Yang et al Adult: Aortic Valve 0%
Total HRU
76
*P < .001
115
57
66
61
92
Direct Cost
75th Percentile
LOS 7 days OR
Readmission 30 days
80%
60%
40%
20%
*P < .001
*P < .001
High-Resource Utilization (%)
Full Sternotomy
Minimally Invasive
FIGURE 1. Comparison of high-resource utilization overall and separated by definition: direct procedure cost higher than the third quartile or either post-
operative length of stay (LOS) 7 days or 30-day readmission between patients who underwent valve operations stratified by operative approach (full ster-
notomy vs minimally invasive surgery). By both separate definitions and composite high resource utilization, we find that patients who underwent full
sternotomy cardiac valve operations experienced significantly higher rates of high resource utilization. HRU, High resource utilization; OR, odds ratio. LOS 7 days OR
Readmission 30 days Direct Cost
75th Percentile Full Sternotomy
Minimally Invasive FIGURE 1. Comparison of high-resource utilization overall and separated by definition: direct procedure cost higher than the third quartile or either post-
operative length of stay (LOS) 7 days or 30-day readmission between patients who underwent valve operations stratified by operative approach (full ster-
notomy vs minimally invasive surgery). By both separate definitions and composite high resource utilization, we find that patients who underwent full
sternotomy cardiac valve operations experienced significantly higher rates of high resource utilization. HRU, High resource utilization; OR, odds ratio. FIGURE 1. Clinical Outcomes of Minimally Invasive Valve colleagues21 reported no significant difference in mortality
(2.5% vs 1.0%; P ¼ .28), and postoperative complication
rates with shorter ICU LOS and transfusion requirements
after traditional FS vs right mini-thoracotomy AVR. Numerous other studies have replicated these outcomes at Surgery Postoperative complications, mortality, and readmission rates after left-heart valve surgery in patient cohorts stratified by operative approach. No significant difference was observed in mortality, readmission, and postoperative complications. Patients who underwent the full sternotomy approach
were more likely to experience postoperative atrial fibrillation and renal failure. ICU, Intensive care unit. Despite the evidence reporting benefits of MICS, barriers
remain precluding its wide-spread adoption. In a recent
retrospective report published by Nissen and Nguyen,25
the authors identified a number of factors essential to the
success of a nascent MICS program. In addition to the
logistical demands required to assemble and maintain a
high-complexity team, MICS programs require unique
administrative support and specialized education and
training for practitioners. Additionally, adequate referral
volume is required to allow for patient safety measures to
become part of routine care while maintaining appropriate
patient selection and increase surgeon chances of better out-
comes through increased practice and experience.26 The
marketing strategy for a MICS program, therefore, must
provide and advertise elements of patient experience,
recruitment of specialized health care professionals, and
availability of cutting-edge equipment. The academic cen-
ter in this study conducts 100 MICS prcedures per year,
with operations conducted under experienced surgeons
who, contrary to existing literature citing longer bypass
and crossclamp times,27 are able to achieve shorter times
in both areas. This is most likely possible through the exten-
sive experience by both the surgeon and operating room
team that over time have cultivated the expertise to achieve
these shorter bypass and crossclamp times. other high volume centers utilizing minimally invasive
approaches.22-24 These previous findings reported in the
literature are similar to the mortality effect observed in
our current study, demonstrating the safety of minimally
invasive techniques employed at high-volume centers
with experienced cardiac surgery teams. Full
Sternotomy
Minimally
Invasive
0
2
3.3
*P < .001
0.3
4
6
8
Odds ratio
FIGURE 4. Forest plot of nominal logistic regression depicting full ster-
notomy (FS) vs minimally invasive cardiac surgery (MICS) and their
respective odds of patients experiencing high resource utilization (HRU)
controlled for lead surgeon, operation (aortic valve replacement [AVR],
mitral valve replacement [MVR], or mitral valve [MV] repair), and Society
of Thoracic Surgeons predicted risk of mortality score stratified into low,
medium, and high risk defined as 1% to 4%, 4% to 8%, and 8% risk,
respectively. Surgery Findings in this study build on the growing literature in
favor of minimally invasive valve surgery. A propensity
score-matched cohort analysis conducted by Bowdish and 0
5
10
40
60
80
100
Surgical Approach
A
B
Postoperative Length of Stay (days)
Full Sternotomy
*P < .001
Minimally Invasive
$0k
$25k
$50k
$100k
$150k
$450k
$500k
Surgical Approach
Direct Cost (USD)
Full Sternotomy
*P < .001
Minimally Invasive
FIGURE 2. Comparison of (A) postoperative length of stay, and (B) total direct cost of patients who underwent valve operations stratified by operative
approach. Patients who underwent full sternotomy experienced both significantly longer postoperative length of stay and higher direct costs. 76
JTCVS Open c September 2023 B
$0k
$25k
$50k
$100k
$150k
$450k
$500k
Surgical Approach
Direct Cost (USD)
Full Sternotomy
*P < .001
Minimally Invasive 0
5
10
40
60
80
100
Surgical Approach
A
Postoperative Length of Stay (days)
Full Sternotomy
*P < .001
Minimally Invasive B A FIGURE 2. Comparison of (A) postoperative length of stay, and (B) total direct cost of patients who underwent valve operations stratified by operative
approach. Patients who underwent full sternotomy experienced both significantly longer postoperative length of stay and higher direct costs. FIGURE 2. Comparison of (A) postoperative length of stay, and (B) total direct cost of patients who underwent valv
approach. Patients who underwent full sternotomy experienced both significantly longer postoperative length of stay a 76
JTCVS Open c September 2023 6
JTCVS Open c September 2023 76 Yang et al Adult: Aortic Valve Postoperative Complication (%)
0%
5%
10%
15%
20%
25%
Atrial
Fibrillation
29
56
Acute Renal
Failure
30-Day
Mortality
30-Day
Readmission
ICU
Readmission
Reintubation
Reoperation
(Bleeding)
Stroke
In-Hospital
Mortality
Pneumonia
5
*P = .027
3
1
P = 1.000
1
1
P = .441
1
*P = .040
9
9
P = .192
13
29
P = .358
6
11
P = .392
5
6
P = .155
6
6
P = .083
2
2
P = .251
Full Sternotomy
Minimally Invasive
FIGURE 3. Postoperative complications, mortality, and readmission rates after left-heart valve surgery in patient cohorts stratified by operative approach. No significant difference was observed in mortality, readmission, and postoperative complications. Patients who underwent the full sternotomy approach
were more likely to experience postoperative atrial fibrillation and renal failure. ICU, Intensive care unit. FIGURE 3. Surgery The model showed that patients who underwent FS had
3.28 times the odds of HRU compared with their counterparts undergoing
MICS (FS OR, 3.28; 95% CI, 1.76-6.13 vs MICS OR, 0.30; 95% CI, 0.16-
0.57; P<.001). Full
Sternotomy
Minimally
Invasive
0
2
3.3
*P < .001
0.3
4
6
8
Odds ratio Minimally
Invasive FIGURE 4. Forest plot of nominal logistic regression depicting full ster-
notomy (FS) vs minimally invasive cardiac surgery (MICS) and their
respective odds of patients experiencing high resource utilization (HRU)
controlled for lead surgeon, operation (aortic valve replacement [AVR],
mitral valve replacement [MVR], or mitral valve [MV] repair), and Society
of Thoracic Surgeons predicted risk of mortality score stratified into low,
medium, and high risk defined as 1% to 4%, 4% to 8%, and 8% risk,
respectively. The model showed that patients who underwent FS had
3.28 times the odds of HRU compared with their counterparts undergoing
MICS (FS OR, 3.28; 95% CI, 1.76-6.13 vs MICS OR, 0.30; 95% CI, 0.16-
0.57; P<.001). JTCVS Open c Volume 15, Number C Resource Utilization and Cost of Minimally Invasive
Valve Surgery In addition to the clinical outcomes of MICS suggesting
substantial benefit over traditional FS, the effect on JTCVS Open c Volume 15, Number C 77 Adult: Aortic Valve Yang et al healthcare economics and resource utilization provide
another strong argument for its wide-spread adoption. This is especially important in the setting of a health care
system that is considered among the highest consumers of
gross domestic product expenditure per capita in the
world.28 Our study demonstrates a median direct cost differ-
ence of $9000 between full sternotomy ($31,900) vs mini-
mally
invasive
($22,900)
valve
surgery. More
than
80,000 aortic valve surgeries and 40,000 mitral sur-
geries are currently performed every year.29,30 Taken
together, these efforts to shift toward the widespread imple-
mentation of MICS may result in substantial reductions in
national health care expenditure. population increases the burden, especially financially, on
health care systems. Given the relationship between HRU
and increased costs, which are then passed onto the hospital,
there is benefit to the health care system when costs are
decreased through lower resource utilization. No difference
in postoperative complications was observed between the 2
groups in our study, thereby suggesting that the MICS
approach provides an avenue for safe, effective valve repair
or replacement without unnecessarily increasing financial
burden on health care payers. Beyond reducing the direct burden of cost on the health
care system, MICS has been shown to correlate with
decreased postoperative LOS. Prolonged hospital stay not
only carries the cost of facilities, staff, and providers needed
for patient care, but also increased risk of complications,
including avoidable nosocomial infections, iatrogenic
injury, and mortality.32-34 Patients undergoing MICS are
likely to have a shorter LOS in comparison to those
undergoing full FS, leading to reduced use of hospital
facilities and resources. Implications of HRU in Healthcare Expenditure In 2015, the Lancet Commission on Global Surgery pro-
posed 6 corresponding core surgical indicators to assess sur-
gical, anaesthetic, and obstetric health care systems in their
ability of preparedness, service delivery, and cost protec-
tion: geographic access to a Bellwether hospital within
2 hours, surgical, anaesthetic, and obstetric provider den-
sity, total operative volume, in-hospital postoperative mor-
tality, impoverishing, and catastrophic cost burden.31
Impoverishing and catastrophic costs can be assessed by
definition of HRU used in this study. Increased direct cost
and postoperative length of stay as seen in our FS Although costs of health care are often not directly trans-
lated directly to a patient’s hospital invoice, patients may
still inadvertently experience increased costs due to HRU. As an example, patients with a high deductible plan or large
required copayment, depending on insurance coverage,
may be required to pay additional costs that may present CONCLUSIONS Incorporation of minimally invasive surgery in various
surgical specialties has been shown to provide postoperative
benefits, including shorter hospital LOS, decreased postop-
erative complications, and greater patient comfort in the
path to recuperation. HRU is 1 metric to compare FS vs
minimally invasive approaches to cardiac surgery. Our
study of one academic institution shows that left-heart valve
surgery via MICS approach correlated with lower rates of
HRU and lower direct cost relative to full sternotomy
with no concomitant increase in postoperative complication
rate (Figure 5). MICS may optimize resource allocation and
provide higher value care for patients. 9. Fugar S, Okoh AK, Singh S, Garg A, Dave D, Chen C, et al. Healthcare and
health disparities and their impact on resource utilization and cost after transcath-
eter aortic valve replacement: a nationwide analysis of real-world data. Circula-
tion. 2019;140:A11233. 10. Dodoo SN, Okoh AK, Aggarwal T, Osman A, Nkansah E, Oseni A, et al. Dispar-
ities in health and healthcare: impact of race on resource utilization and costs
following transcatheter edge-to-edge repair. Cardiovasc Revascularization
Med. 2023;50:13-8. 11. Cohn LH, Adams DH, Couper GS, Bichell DP, Rosborough DM, Sears SP, et al. Minimally invasive cardiac valve surgery improves patient satisfaction while
reducing costs of cardiac valve replacement and repair. Ann Surg. 1997;226:421. 12. Cosgrove DM III, Sabik JF, Navia JL. Minimally invasive valve operations. Ann
Thorac Surg. 1998;65:1535-9. 13. Navia JL, Cosgrove DM. Minimally invasive mitral valve operations. Ann
Thorac Surg. 1996;62:1542-4. 14. Pojar M, Karalko M, Dergel M, Vojacek J. Minimally invasive or sternotomy
approach in mitral valve surgery: a propensity-matched comparison. J Cardio-
thorac Surg. 2021;16:228. Webcast
You can watch a Webcast of this AATS meeting presenta-
tion
by
going
to:
https://www.aats.org/resources/
minimally-invasive-approach-associated-with-lower-resource-
utilization-and-lower-cost-after-valve-surgery. 15. Ogami T, Yokoyama Y, Takagi H, Serna-Gallegos D, Ferdinand FD, Sultan I,
et al. Minimally invasive versus conventional aortic valve replacement: the
network meta-analysis. J Card Surg. 2022;37:4868-74. 16. Russo MJ, Thourani VH, Cohen DJ, Malaisrie SC, Szeto WY, George I, et al. Minimally invasive versus full sternotomy for isolated aortic valve replacement
in low-risk patients. Ann Thorac Surg. 2022;114:2124-30. 17. Iribarne A, Russo MJ, Easterwood R, Hong KN, Yang J, Cheema FH, et al. Mini-
mally invasive versus sternotomy approach for mitral valve surgery: a propensity
analysis. Ann Thorac Surg. 2010;90:1471-8. 18. Lange R, Voss B, Kehl V, Mazzitelli D, Tassani-Prell P, G€unther T. Conflict of Interest Statement an unexpected hardship. Accordingly, decreasing resource
utilization through limiting costs can decrease the financial
burden on health care systems, and by extension, to patients
and their families. Dr Russo discloses financial relationships with Edwards
Lifesciences and Abbott Laboratories. Dr Lee discloses a
financial relationship with Abbott Laboratories. All other
authors reported no conflicts of interest. The Journal policy requires editors and reviewers to
disclose conflicts of interest and to decline handling or re-
viewing manuscripts for which they may have a conflict
of interest. The editors and reviewers of this article have
no conflicts of interest. References
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invasive surgery in the United States, 2022: understanding its value using new
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ventional versus minimally invasive aortic valve replacement: pooled analysis of
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high resource utilization in pediatric skin and soft tissue infection hospitaliza-
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aortic valve replacement. Innovations. 2021;16:262-6. 7. Soliman FK, Volk L, Kenath R, Okoh AK, Chao JC, Baxi J, et al. Frailty, as
measured by the essential frailty toolset, is associated with higher resource utili-
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Auerbach AD, et al. Best definitions of multimorbidity to identify patients
with high health care resource utilization. Mayo Clin Proc Innov Qual Outcomes. 2020;4:40-9. Limitations Despite the benefits shown in this study, several limita-
tions exist. Given that this study is retrospective with an
observational design, bias is present due to the lack of
randomization and a priori data field selection. Moreover,
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tution, thereby subjecting results to differences in surgeon
technique and experience. Notably, majority of minimally
invasive operations at this institution were conducted by a
couple surgeons with extensive training and experience in
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troduces the possibility of a confounding variable into the
study, the variability of ERAS was deemed to not be 1
that would introduce significant bias. Surgeons with exten-
sive experience in MICS also conducted full sternotomies
regularly. Prospective
studies
in
larger
cohorts
are
warranted. Minimally Invasive Approach Associated with Lower Resource Utilization After Valve Surgery
Single-center retrospective review of adult patients who underwent AVR, MVR or MV repair from 2018-2021. Full sternotomy (n = 124) vs. MICS (n = 368) approach. Minimally invasive approach left-heart valve surgery correlates with lower rates of high-resource utilization relative to
full sternotomy with no concomitant increase in postoperative complication rates. reduction in median direct
cost associated with
MICS. Associated with MICS approach
High-Resource
Utilization
$9000
$ $ $
Median LOS
From 6 days in full
sternotomy to 4 days in
MICS. AVR aortic valve replacement, MVR mitral valve replacement, MV repair mitral
valve repair, MICS minimally invasive cardiac surgery, LOS length of stay
FIGURE 5. Outcomes of minimally invasive approach to left-heart valve surgery. Our results show that minimally invasive cardiac surgery (MICS)
approach correlated with lower rates of high resource utilization, shorter length of stay (LOS), and lower direct cost relative to full sternotomy with no
concomitant increase in postoperative complication rate. AVR, Aortic valve replacement; MVR, mitral valve replacement; MV, mitral valve. Minimally Invasive Approach Associated with Lower Resource Utilization After Valve Surgery Single-center retrospective review of adult patients who underwent AVR, MVR or MV repair from 2018-2021. Full sternotomy (n = 124) vs. MICS (n = 368) approach. AVR aortic valve replacement, MVR mitral valve replacement, MV repair mitral
valve repair, MICS minimally invasive cardiac surgery, LOS length of stay AVR aortic valve replacement, MVR mitral valve replacement, MV repair mitral
valve repair, MICS minimally invasive cardiac surgery, LOS length of stay FIGURE 5. Outcomes of minimally invasive approach to left-heart valve surgery. Our results show that minimally invasive cardiac surgery (MICS)
approach correlated with lower rates of high resource utilization, shorter length of stay (LOS), and lower direct cost relative to full sternotomy with no
concomitant increase in postoperative complication rate. AVR, Aortic valve replacement; MVR, mitral valve replacement; MV, mitral valve. FIGURE 5. Outcomes of minimally invasive approach to left-heart valve surgery. Our results show that minimally invasive cardiac surgery (MICS)
approach correlated with lower rates of high resource utilization, shorter length of stay (LOS), and lower direct cost relative to full sternotomy with no
concomitant increase in postoperative complication rate. AVR, Aortic valve replacement; MVR, mitral valve replacement; MV, mitral valve. 8
JTCVS Open c September 2023 78 Adult: Aortic Valve Yang et al CONCLUSIONS Right mini-
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academic medical centre. HPB (Oxford). 2015;17:311-7. 27. Cheng DC, Martin J, Lal A, Diegeler A, Folliguet TA, Nifong LW, et al. Mini-
mally invasive versus conventional open mitral valve surgery a meta-analysis
and systematic review. Innovations. 2011;6:84-103. 28. Papanicolas I, Woskie LR, Jha AK. Health care spending in the United States and
other high-income countries. JAMA. 2018;319:1024-39. 20. Badhwar V, Rankin JS, Jacobs JP, Shahian DM, Habib RH, D’Agostino RS, et al. The Society of Thoracic Surgeons Adult Cardiac Surgery database: 2016 update
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patterns, testing, and surgery in aortic valve disease: leaving women and elderly
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parison of aortic valve replacement via an anterior right minithoracotomy with
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Thorac Surg. 2016;49:456-63. 31. Meara JG, Leather AJ, Hagander L, Alkire BC, Alonso N, Ameh EA, et al. Global surgery 2030: evidence and solutions for achieving health, welfare, and
economic development. Lancet. 2015;386:569-624. 22. Hiraoka A, Totsugawa T, Kuinose M, Nakajima K, Chikazawa G, Tamura K,
et al. Propensity score-matched analysis of minimally invasive aortic valve
replacement. Circ J. 2014;78:2876-81. 32. Loke HY, Kyaw WM, Chen M, Lim JW, Ang B, Chow A. Length of stay and odds
of MRSA acquisition: a dose–response relationship? Epidemiol Infect. 2019;147:
e223. 23. Gilmanov D, Farneti PA, Ferrarini M, Santarelli F, Murzi M, Miceli A, et al. Full
sternotomy versus right anterior minithoracotomy for isolated aortic valve
replacement in octogenarians: a propensity-matched study. Interact Cardiovasc
Thorac Surg. 2015;20:732-41. 33. McCarthy BC Jr, Tuiskula KA, Driscoll TP, Davis AM. Medication errors result-
ing in harm: using chargemaster data to determine association with cost of hos-
pitalization and length of stay. CONCLUSIONS Am J Health Syst Pharm. 2017;74:S102-7. 24. Del Giglio M, Mikus E, Nerla R, Micari A, Calvi S, Tripodi A, et al. Right ante-
rior mini-thoracotomy versus conventional sternotomy for aortic valve replace-
ment: a propensity-matched comparison. J Thorac Dis. 2018;10:1588. 34. Rojas-Garcıa A, Turner S, Pizzo E, Hudson E, Thomas J, Raine R. Impact and
experiences of delayed discharge: a mixed-studies systematic review. Health
Expect. 2018;21:41-56. 25. Nissen AP, Nguyen S, Abreu J, Nguyen TC. The first 5 years: building a mini-
mally invasive valve program. J Thorac Cardiovasc Surg. 2019;157:1958-65. 26. Akmaz B, van Kuijk SM, Nia PS. Association between individual surgeon vol-
ume and outcome in mitral valve surgery: a systematic review. J Thorac Dis. 2021;13:4500. Key Words: minimally-invasive surgery, high-resource uti-
lization, left heart valve surgery JTCVS Open c September 2023 80
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Pengaruh Perubahan Pendapatan Yang Dimoderasi Oleh Siklus Kas Operasi Dalam Memprediksi Akrual
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Jurnal Eksplorasi Akuntansi
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JEA Jurnal Eksplorasi Akuntansi
Vol. 2, No 3, Seri C, Agustus 2020, Hal 3270-3285 JEA PENGARUH PERUBAHAN PENDAPATAN YANG DIMODERASI OLEH
SIKLUS KAS OPERASI DALAM MEMPREDIKSI AKRUAL
(Studi Empiris pada Perusahaan Manufaktur yang Terdaftar di Bursa Efek Indonesia
pada Tahun 2016-2018) Muhammad Nur Ihkwansyah1, Nurzi Sebrina2
1Alumni Jurusan Akuntansi Fakultas Ekonomi Universitas Negeri Padang
2Jurusan Akuntansi Fakultas Ekonomi Universitas Negeri Padang
*Korespondensi: muhammadnurihkwansyah@gmail.com Muhammad Nur Ihkwansyah1, Nurzi Sebrina2
1Alumni Jurusan Akuntansi Fakultas Ekonomi Universitas Negeri Padang
2Jurusan Akuntansi Fakultas Ekonomi Universitas Negeri Padang
*Korespondensi: muhammadnurihkwansyah@gmail.com Muhammad Nur Ihkwansyah1, Nurzi Sebrina2
1Alumni Jurusan Akuntansi Fakultas Ekonomi Universitas Negeri Padang
2Jurusan Akuntansi Fakultas Ekonomi Universitas Negeri Padang
*Korespondensi: muhammadnurihkwansyah@gmail.com Abstract: The purpose of this research is to examine the effect of operating cash cycle in
moderating the relationship of revenue changes and accruals. This research is classified as
causative research with a quantitative approach. The population are manufacturing companies
listed on the Indonesian Stock Exchange period of 2016 to 2018. By using purposive sampling
method, there were 43 companies as the research’s sample. The type of data used is secondary
data obtained from the official site of the Indonesia Stock Exchange and company’s official
website. The hypotheses were tests using panel regression analysis. The results indicate that
revenue changes has not significant effect on accrual, also operating cash cycle is not able to
strengthen or weaken the relationship between revenue changes and accruals. Keywords: Revenue Changes; Operating Cash Cycle; Accruals. How to cite (APA 6th style):
Ihkwansyah, M. N., & Nurzi, S. (2020). Pengaruh Perubahan Pendapatan yang Dimoderasi oleh
Siklus Kas Operasi Dalam Memprediksi Akrual (Studi Empiris pada Perusahaan
Manufaktur yang Terdaftar di Bursa Efek Indonesia Periode 2016-2018). Jurnal
Eksplorasi Akuntansi, 2(3), Seri C, 3270-3285. How to cite (APA 6th style): How to cite (APA 6th style):
Ihkwansyah, M. N., & Nurzi, S. (2020). Pengaruh Perubahan Pendapatan yang Dimoderasi oleh
Siklus Kas Operasi Dalam Memprediksi Akrual (Studi Empiris pada Perusahaan
Manufaktur yang Terdaftar di Bursa Efek Indonesia Periode 2016-2018). Jurnal
Eksplorasi Akuntansi, 2(3), Seri C, 3270-3285. ISSN : 2656-3649 (Online)
http://jea.ppj.unp.ac.id/index. ISSN : 2656-3649 (Online)
http://jea.ppj.unp.ac.id/index. ISSN : 2656-3649 (Online)
http://jea.ppj.unp.ac.id/index.php/jea/issue/view/27 PENDAHULUAN Perkembangan perekonomian pada masa sekarang ini yang begitu pesat membuat persaingan
dibidang ekonomi menjadi semakin ketat dan menuntut perusahaan agar dapat memberikan
informasi yang dapat digunakan sebagai dasar pengambilan keputusan ekonomi dan dapat
memberikan gambaran suatu keadaan perusahan di masa depan. Untuk mendapatkan informasi
tersebut, akuntansi ikut berperan dalam memberikan informasi yang diperlukan. Salah satu
informasi akuntansi yang memberikan informasi mengenai kinerja perusahaan adalah laporan
keuangan yang disusun oleh manajemen perusahaan. g
y
g
j
p
Manajemen (agent) dalam menyusun laporan keuangan perusahaan menggunakan asumsi-
asumsi dasar yang berlaku dalam akuntansi. Asumsi digunakan dalam penyusunan laporan
keuangan untuk menjadi konsep dasar yang melandasi penyusunan laporan keuangan. Asumsi
dasar yang digunakan dalam proses penyusunan laporan keuangan salah satunya adalah asumsi
akrual. Menurut PSAK No. 1 paragraf 27 dan 28 (IAI, 2018), entitas dalam menyusun laporan 3270 keuangan menggunakan dasar akrual, kecuali laporan arus kas. Ketika akuntansi berdasarkan
akrual digunakan, entitas mengakui pos-pos sebagai aset, liabilitas, ekuitas, penghasilan, dan
beban (unsur-unsur laporan keuangan) ketika pos-pos tersebut memenuhi defenisi dan kriteria
pengakuan untuk unsur-unsur tersebut dalam Kerangka Konseptual Pelaporan Keuangan. Asumsi akrual memberikan gambaran tentang pengaruh transaksi dan peristiwa lain, keadaan
sumber daya pelaporan entitas ekonomi, dan klaim pada periode dimana transaksi ekonomi
tersebut terjadi, walaupun penerimaan dan pembayaran kas terjadi dalam periode yang berbeda. Oleh karena itu akrual penting dalam penyusunan laporan keuangan (Lam, 2015). Akrual merupakan mekanisme perubahan arus kas saat ini untuk menghasilkan ukuran
kinerja yang lebih prediktif, yaitu laba. Perubahan arus kas tersebut dapat terlihat dari
penerimaan dan pembayaran kas bersih yang terjadi pada pendapatan. Namun, arus kas memiliki
sifat waktu (timing) dan pencocokan (matching) yang mengganggu kemampuan akrual untuk
mengukur kinerja tersebut (Dechow, 1994). Frankle (2018) menyatakan peran ekonomi dari
akrual
adalah
untuk
mengurangi
masalah
timing dan
masalah
matching
tersebut
dan
menghasilkan laba yang lebih baik mencerminkan peristiwa yang relevan. Masalah timing
mengacu pada penerimaan dan pembayaran kas bersih yang dapat terjadi pada periode yang
berbeda dari kejadian ekonomik. Sedangkan, masalah matching mengacu pada perbedaan persyaratan pelunasan antara
piutang dan utang yang dimiliki perusahaan yang menyebabkan arus kas masuk dan arus kas
keluar dari transaksi ekonomi terjadi dalam periode yang berbeda pula. PENDAHULUAN Frankle (2018)
menemukan bahwa menggabungkan perubahan arus kas, korelasi serial dalam perubahan arus
kas, dan panjang siklus kas operasi pada hubungan antara akrual dan perubahan pendapatan
dapat meningkatkan spesifikasi dan kekuatan penjelas dari akrual, membantu mendeteksi
manajemen laba di perusahaan, dan menghasilkan estimasi akrual nondiscretionary yang lebih
baik dalam memprediksi arus kas dan pendapatan masa depan. Hasil ini menunjukkan
pentingnya mempertimbangkan peran ekonomi akrual ketika memprediksi akrual. Prediksi akrual dapat dilakukan dengan memahami peran waktu arus kas dan properti
yang cocok, karena fungsi utama akrual adalah untuk mengimbangi komponen arus kas yang
tidak terkait dengan kinerja periode saat ini (Frankle, 2018). Komponen akrual yang
mengimbangi arus kas adalah akrual yang berasal dari perubahan modal kerja karena perubahan
penjualan periode berjalan yang diperkirakan akan kembali. Ini menunjukkan bahwa akrual
adalah fungsi dari perubahan penjualan saat ini dan sebelumnya. Koefisiennya adalah fungsi dari
kebijakan perusahaan atas persediaan dan persyaratan kredit dari piutang dan hutang, yang
merupakan siklus kas operasi perusahaan (Dechow, 1998). Dengan demikian, prediksi akrual
dapat didasarkan pada perubahan pendapatan yang menggabungkan siklus kas operasi karena
panjang siklus kas operasi dapat menangkap sifat timing dan matching arus kas yang diimbangi
oleh akrual (Frankle, 2018). Beberapa penelitian
terdahulu telah
membahas apakah
memprediksi akrual
dapat
bermanfaat dalam hal spesifikasi dan kekuatan penjelas terhadap akrual, kekuatan dalam
mendeteksi manajemen laba, dan prediksi arus kas dan pendapatan satu tahun ke depan. Jones
(1991) melakukan penelitian terkait dengan pengestimasi akrual diskresioner untuk mendeteksi
manajemen laba. Jones (1991) menunjukkan bahwa perubahan pendapatan mempengaruhi
perubahan akrual dari modal kerja (working capital) seperti piutang, persediaan, dan utang. Jones (1991) berusaha mengontrol pengaruh perubahan kondisi ekonomik pada akrual dengan
memasukkan variabel perubahan pendapatan dan gross property, plant, and equipment (PPE) ke
dalam model yang dibangunnya. Dechow et al. (1998) dalam penelitiannya juga memodelkan 3271 pendapatan, arus kas operasi, dan akrual untuk menghubungkan akrual modal kerja dengan arus
kas masa depan dan menjelaskan sifat rangkaian waktu dari pendapatan, arus kas, dan akrual. Model tersebut termasuk piutang, persediaan, dan hutang merupakan akrual modal kerja yang
paling penting dan menunjukkan bahwa akrual adalah fungsi dari perubahan penjualan saat ini
dan sebelumnya. Frankle (2018) memperluas Dechow (1998) dengan melakukan penelitian
bagaimana sifat arus kas dapat menjelaskan akrual. Frankle (2018) menemukan bahwa
menggabungkan panjang siklus kas operasi pada hubungan antara akrual dan perubahan
pendapatan meningkatkan kekuatan penjelas dari semua model akrual (yaitu, model Jones, Ball
dan Shivakumar, McNichols, dan Jeter dan Shivakumar). PENDAHULUAN ,
,
)
Penelitian ini mengacu pada penelitian Frankle (2018) tentang kemampuan properti arus
kas dalam memprediksi akrual pada perusahaan swasta di Amerika Serikat pada tahun 1989-
2013. Penelitian ini dilakukan oleh Frankel (2018) untuk memahami sejauhmana sifat arus kas
menjelaskan akrual, dengan menggunakan model Dechow et al. (1998). Motivasi dalam
melakukan penelitian ini adalah karena banyak peneliti yang menyoroti pemahaman yang tidak
memadai tentang proses yang menghasilkan akrual (Owens et al., 2013; Ball, 2013). Menurut
Ball (2013), adanya keterbatasan pengetahuan tentang faktor-faktor yang mempengaruhi akrual
akan membuat rancangan riset tidak memadai untuk memprediksi akrual. Peneliti juga
termotivasi untuk melakukan penelitian ini karena penelitian terkait memprediksi akrual dengan
perubahan pendapatan sebagai predictor di Indonesia masih sedikit dan juga peneliti ingin
mengetahui bagaimana implikasi penelitian tersebut pada perusahaan manufaktur yang ada di
Indonesia. Penelitian ini bertujuan untuk menemukan bukti empiris apakah perubahan pendapatan
memiliki pengaruh dalam memprediksi akrual dan peran siklus kas operasi dalam memoderasi
hubungan perubahan pendapatan dalam memprediksi akrual. Hasil penelitian ini diharapkan
dapat menambah wawasan dan pengetahuan dalam memprediksi akrual, memberikan masukan
dan pertimbangan untuk perusahaan dalam memprediksi akrual dengan memperhatikan
perubahan pendapatan dan siklus kas operasi yang terjadi dalam perusahaan, dan hasil penelitian
ini diharapkan dapat menambah literatur dan ilmu pengetahuan peneliti selanjutnya tentang
akrual. REVIU LITERATUR DAN HIPOTESIS
A
Th Agency theory (teori keagenan) adalah teori yang biasa digunakan dalam kontrak bisnis selama
ini. Bodroastuti (2009) menjelaskan bahwa teori keagenan merupakan teori yang menjelaskan
tentang adanya pemisahan kepentingan antara pemilik perusahaan (principals) dan pengelola
perusahaan (agent). Hal tersebut terjadi karena tidak tercapainya tujuan antara principal dan
agent dalam suatu perusahaan. Menurut Ellen dan Juniarti (2013), adanya konflik kepentingan
tersebut timbul karena pihak principals sebagai pemilik perusahaan menginginkan perusahaan
menghasilkan laba yang tinggi yang dapat memberikan manfaat kesejahteraan bagi principals,
disisi lain agent diberi wewenang dan tanggungjawab untuk mengelola perusahaan dan berupaya
untuk meningkatkan utilitasnya sendiri dan menyalahgunakan kepercayaan principals sebagai
pemilik perusahaan. Teori agensi ini banyak digunakan untuk menjelaskan fenomena yang
terjadi dalam pengambilan keputusan keuangan. Manajemen laba didasari oleh adanya theory agency yang menyatakan bahwa setiap
individu cenderung untuk memaksimalkan utilitasnya. Dalam hubungan antara principals dan
agent, akan timbul masalah jika terdapat informasi yang asimetri. Informasi yang asimetri terjadi 3272 apabila pihak yang terkait dalam transaksi bisnis lebih memiliki informasi dari pada pihak
lainnya (Scott, 1997). Informasi yang asimetri dapat berupa informasi yang terdistribusi dengan
tidak merata diantara principals dan agent, serta tidak mungkinnya principals untuk mengamati
secara langsung usaha yang dilakukan agent. Hal inilah yang cenderung menyebabkan agent
melakukan prilaku yang tidak semestinya (disfunctional behaviour). p
y
g
y (
f
)
Salah satu disfunctional beahviour yang dilakukan agent adalah pemanipulasian data
dalam laporan keuangan agar sesuai dengan harapan principals meskipun laporan tersebut tidak
menggambarkan kondisi perusahaan yang sebenarnya. Pemanipulasian data laporan keuangan
tersebut dapat berupa praktek manajemen laba dengan cara pemilihan metode akuntansi atau
kebijakan akrual, yaitu dengan mengendalikan transaksi akrual sehingga laba terlihat tinggi/
rendah, akan tetapi transaksi tersebut tidak mempengaruhi aliran kas. Menurut Hidayati &
Zulaikha
(2003)
dalam
Wahyuningsih
(2007),
konsep
akrual
ini
memungkinkan
dilakukannya rekayasa laba atau earning management oleh manajer untuk menaikkan atau
menurunkan angka akrual dalam laporan laba rugi. Menurut Fischer & Rozenzweig (1995)
masih dalam Wahyuningsih (2007), perekayasaan laba juga dapat dilakukan dengan
mendistorsi laba dengan cara menggeser periode pengakuan biaya dan pendapatan. Dengan
demikian, informasi terkait laporan keuangan yang akan diterima investor tidaklah laporan
keuangan yang memberikan informasi keadaan perusahaan yang sebenarnya. Pengetahuan terkait akrual dapat membantu investor dalam mendeteksi adanya manajemen
laba pada suatu perusahaan (Frankle, 2018). Investor dapat menggunakan akrual untuk melihat
aspek oportunistik manajmen. Selain itu, akrual dapat membantu investor mengetahui arus
kas dan pendapatan dimasa depan. REVIU LITERATUR DAN HIPOTESIS
A
Th Hal tersebut terlihat bagaimana akrual berperan dalam
menyeimbangi arus kas periode saat ini dan faktor ekonomi periode sebelumnya, seperti
piutang awal periode. Akrual periode berjalan yang menyelaraskan arus kas periode
berikutnya dan faktor ekonomi periode berjalan, seperti piutang akhir periode, memberikan
informasi tentang bagian sementara dari satu komponen arus kas periode berikutnya. Dengan demikian, investor akan mengetahui jika terdapat pemanipulasian data laporan
keuangan melalui kebijakan akrual dengan memprediksi akrual. Siklus Kas Operasi Siklus kas operasi adalah jumlah atau putaran waktu yang dibutuhkan oleh kas dari pengeluaran
kas tunai untuk pembelian persediaan hingga penerimaan kas tunai dari penjualan
persediaan tersebut. Lamanya waktu yang terjadi pada siklus kas operasi ini terkait dengan
asumsi tentang proses menghasilkan penjualan, karena kontrak yang dibuat pada saat
penjualan menentukan waktu dan jumlah arus kas masuk atau arus kas keluar dan
pengakuan pendapatan. Kontrak penjualan menentukan kapan dan dalam kondisi apa
pelanggan harus membayar. Kondisi-kondisi tersebut menentukan pola penerimaan kas,
sehingga sangat erat kaitannya dengan siklus kas operasi perusahaan (Dechow, 1998). Siklus kas operasi dalam perubahan pendapatan dapat meningkatkan atau menguatkan
hubungan positif yang ada antara akrual dengan pendapatan. Penelitian yang dilakukan oleh
Frankle (2018) menemukan bahwa hubungan positif antara akrual modal kerja dan pendapatan
menguat seiring siklus kas operasi diperpanjang. Dengan siklus operasi yang lebih lama, arus kas
perusahaan memiliki masalah waktu dan pencocokan yang lebih serius. Akibatnya, untuk
perubahan pendapatan yang diberikan, diperlukan tingkat akrual yang lebih besar untuk
mengimbangi arus kas yang direalisasikan dalam periode yang berbeda dari inovasi dalam laba
kotor. Akrual Dalam akuntansi dikenal accrual basis dan cash basis. Menurut PSAK No. 1 paragraf 27 dan 28
(IAI, 2018), entitas dalam menyusun laporan keuangan menggunakan dasar akrual, kecuali
laporan arus kas. Besaran akrual adalah besaran pendapatan diakui pada saat hak kesatuan usaha
timbul lantaran penyerahan barang ke pihak luar dan biaya diakui pada saat kewajiban timbul
lantaran penggunaan sumber ekonomik yang melekat pada barang yang diserahkan tersebut
(Dechow dan Dichev, 2002). Subramanyam dan Wild (2010) menyatakan bahwa akuntansi akrual bertujuan untuk
memberikan informasi kepada pemakai laporan keuangan mengenai konsekuensi aktivitas
usaha terhadap arus kas perusahaan di masa depan secepat mungkin dengan tingkat kepastian
yang layak. Akuntansi akrual dapat mengurangi masalah ketepatan waktu dan pengaitan yang
terdapat pada akuntansi kas. Masalah timing mengacu pada arus kas yang tidak selalu terjadi
bersamaan dengan aktivitas yang menghasilkan kas tersebut. Masalah matching mengacu pada
arus kas masuk dan keluar yang disebabkan oleh aktivitas usaha tetapi tidak dapat dikaitkan
dengan waktu terjadinya. 3273 Penelitian Terdahulu Penelitian terdahulu yang meneliti tentang memprediksi akrual meggunakan property arus kas
adalah penelitian yang dilakukan oleh Frankel (2018), Richard M. Frankel dan Yan Sun (2016),
Dechow, P., S. P. Kothari, and R. Watts. (1998), Dechow, P. (1994), Jones (1991), dengan
ringkasan hasil penelitian sebagai berikut: g
p
g
a. Perubahan pendapatan memiliki hubungan positif dengan akrual (Frankel, 2018; Richard M. Frankel dan Yan Sun, 2016; Dechow, P., S. P. Kothari, and R. Watts, 1998; Dechow, P.,
1994; Jones, 1991) a. Perubahan pendapatan memiliki hubungan positif dengan akrual (Frankel, 2018; Richard M. Frankel dan Yan Sun, 2016; Dechow, P., S. P. Kothari, and R. Watts, 1998; Dechow, P.,
1994; Jones, 1991) b. Perubahan pendapatan memiliki hubungan yang positif dengan akrual, hubungan positif ini
akan menguat dengan adanya siklus kas operasi yang semakin lama (Frankel, 2018; Richard
M. Frankel dan Yan Sun, 2016; Dechow, P., S. P. Kothari, and R. Watts, 1998). b. Perubahan pendapatan memiliki hubungan yang positif dengan akrual, hubungan positif ini
akan menguat dengan adanya siklus kas operasi yang semakin lama (Frankel, 2018; Richard
M. Frankel dan Yan Sun, 2016; Dechow, P., S. P. Kothari, and R. Watts, 1998). Perubahan Pendapatan p
Pendapatan merupakan salah satu unsur yang paling utama dalam pembentukan laporan laba rugi
perusahaan. Definisi pendapatan dalam PSAK No.23 pargf 07 (2018) adalah arus masuk bruto
dari manfaat ekonomi yang timbul dari aktivitas normal entitas selama suatu periode jika arus
masuki tersebut mengakibatkan kenaikan ekuitas yang tidak berasal dari kontribusi penanaman
modal. Perubahan pendapatan adalah fluktuasi arus masuk atau peningkatan lainnya atas aktiva
sebuah entitas atau penyelesaian kewajiban selama satu periode dari pengiriman atau produksi
barang, penyediaan jasa, atau aktivitas lain yang merupakan operasi utama atau sentral entitas
yang sedang berlangsung. Hubungan perubahan pendapatan yang dimoderasi oleh siklus kas operasi dalam
memprediksi akrual j
H1: Perubahan pendapatan berkorelasi positif dengan tingkat akrual. H1: Perubahan pendapatan berkorelasi positif dengan tingkat akrual. H2: Perubahan pendapatan berkorelasi positif dengan tingkat akrual, hubungan ini semakin
positif seiring dengan perubahan siklus kas operasi. Hubungan perubahan pendapatan yang dimoderasi oleh siklus kas operasi dalam
memprediksi akrual Asumsi dasar akrual menggambarkan efek transaksi dan peristiwa dan keadaan lain pada
sumber daya ekonomi perusahaan, yaitu, aset, dan klaim terhadap sumber daya berupa
kewajiban dan ekuitas, dalam periode di mana efek tersebut terjadi, bahkan jika penerimaan 3274 dan pembayaran kas yang dihasilkan terjadi dalam periode yang berbeda. Menurut Frankle
(2018), fungsi utama akrual adalah untuk mengimbangi komponen arus kas yang tidak terkait
dengan kinerja periode saat ini. Komponen akrual yang mengimbangi arus kas adalah akrual
yang berasal dari perubahan modal kerja karena perubahan penjualan periode berjalan yang
diperkirakan akan kembali. Ini menunjukkan bahwa akrual adalah fungsi dari perubahan
penjualan saat ini dan sebelumnya. Koefisiennya adalah fungsi dari kebijakan perusahaan atas
persediaan dan persyaratan kredit dari piutang dan hutang, yang merupakan siklus kas operasi
perusahaan (Dechow, 1998). Dengan demikian, prediksi akrual dapat didasarkan pada perubahan
pendapatan yang menggabungkan siklus kas operasi karena panjang siklus kas operasi dapat
menangkap sifat timing dan matching arus kas yang diimbangi oleh akrual (Frankle, 2018). Penelitian yang dilakukan oleh Frankle (2018) menemukan bahwa terdapat hubungan
positif antara perubahan pendapatan dalam memprediksi akrual. Dengan menggabungkan
panjang
siklus
kas
operasi
pada
hubungan
antara
akrual
dan
perubahan
pendapatan
meningkatkan kekuatan penjelas dari semua model akrual (yaitu, model Jones, Ball dan
Shivakumar, McNichols, dan Jeter dan Shivakumar). Perusahaan yang memiliki siklus kas
operasi lebih lama, perubahan yang diberikan dalam penjualan atau operasi menyebabkan
perubahan yang lebih besar dalam modal kerja. Dengan siklus kas operasi yang lebih lama, arus
kas perusahaan memiliki masalah waktu dan pencocokan yang lebih serius. Akibatnya, untuk
perubahan pendapatan yang diberikan, diperlukan tingkat akrual yang lebih besar untuk
mengimbangi arus kas yang direalisasikan dalam periode yang berbeda dari inovasi dalam laba
kotor. Hasil penelitian Jones (1991); Dechow, P. (1994); Dechow et al. (1998); dan Frankle (2018)
menunjukkan bahwa perubahan pendapatan mempengaruhi perubahan akrual dari modal kerja
(working capital) seperti piutang, persediaan, dan utang. Menurut Dechow et al. (1998) dan
Frankle (2018), akrual modal kerja adalah fungsi dari perubahan penjualan pada periode saat ini
dan memberikan dukungan analitis untuk dimasukkannya perubahan pendapatan periode
berjalan dalam model yang dibentuk Jones (1991). Frankle (2018) dalam penelitiannya juga
menunjukkan bahwa hubungan antara perubahan pendapatan dan akrual meningkat seiring
dengan perubahan siklus kas operasi. Ini konsisten dengan kesimpulan oleh Dechow (1994)
bahwa ketika suatu perusahaan memiliki siklus kas operasi yang lebih lama, perubahan yang
diberikan dalam penjualan atau operasi menyebabkan perubahan yang lebih besar dalam modal
kerja. METODE PENELITIAN
Jenis Penelitian Jenis Penelitian Populasi dan Sampel p
p
Populasi dalam penelitian ini adalah seluruh perusahaan manufaktur yang terdaftar di Bursa Efek
Indonesia dari tahun 2016-2018. Jumlah sampel dalam penelitian ini adalah 167 perusahaan
manufaktur. Pengambilan sampel dalam penelitian ini dilakukan secara purposive sampling
dengan kriteria tertentu, dengan rincian sebagai berikut: Tabel 1
Kriteria Pengambilan Sampel
Keterangan
Jumlah
Perusahaan manufaktur yang tercatat di Bursa Efek
Indonesia tahun 2014-2018
167
Perusahaan manufaktur yang tidak memiliki data
lengkap terkait dengan variabel penelitian
(112)
Perusahaan manufaktur yang menyajikan laporan
keuangan menggunakan mata uang rupiah
(12)
Total perusahaan sampel (per tahun)
43
Total perusahaan sampel (2016-2018)
129
(Sumber: www.idx.co.id, data diolah) Tabel 1
Kriteria Pengambilan Sampel Berdasarkan kriteria tersebut, sampel yang menjadi fokus penelitian adalah sebanyak 43
perusahaan pertahun atau 129 sampel untuk 3 tahun pengamatan. Teknik Pengumpulan Data g
p
Penelitian ini menggunakan teknik dokumentasi dengan melihat laporan tahunan perusahaan
sampel. Teknik dokumentasi ditujukan untuk memperoleh data langsung yang diperlukan dalam
penelitian ini. Adapun dokumentasi dalam penelitian ini berupa laporan keuangan perusahaan
manufaktur yang terdaftar di Bursa Efek Indonesia (BEI) tahun 2016-2018. Data tersebut
diperoleh melalui situs resmi Bursa Efek Indonesia (BEI) dan situs lain yang diperlukan. Jenis Penelitian Jenis penelitian yang dilakukan adalah asosiatif kausal dengan pendekatan kuantitatif. Penelitian
asosiatif kausal adalah penelitian yang bertujuan untuk menganalisis hubungan antara satu
variabel dengan variabel lainnya atau bagaimana suatu variabel memengaruhi variabel lain. Penelitian ini bertujuan untuk menjelaskan apakah perubahan pendapatan dapat memprediksi
akrual. 3275 ACCt =
οܣffncti ܴ݁f݁༠푎ܾ݈݁+ οܫt݁tin⿏ݕ㷟οܣffnctiݏܲ푎ݕ푎ܾ݈݁㷟
οܶ푎h݁ݏܲ푎ݕ푎ܾ݈݁+ οܱi݄݁⿏ܣݏݏ݁iݏ
Assets Totals Akrual Variabel dependen dalam penelitian adalah akrual modal kerja. Mengikuti Frankle (2018),
penelitian ini mendefinisikan akrual modal kerja sebagai perubahan Account Receivable,
perubahan Inventory, perubahan Account Payable, perubahan Taxes Payable dan perubahan
Other Assets (net) dibagi dengan total aset awal. ACCt =
οܣffncti ܴ݁f݁༠푎ܾ݈݁+ οܫt݁tin⿏ݕ㷟οܣffnctiݏܲ푎ݕ푎ܾ݈݁㷟
οܶ푎h݁ݏܲ푎ݕ푎ܾ݈݁+ οܱi݄݁⿏ܣݏݏ݁iݏ
Assets Totals Dimana ACCt adalah akrual modal kerja, οܣffncti ܴ݁f݁༠푎ܾ݈݁
adalah perubahan
piutang dari tahun sebelumnya ke tahun berjalan. οܫt݁tin⿏ݕadalah perubahan persediaan dari
tahun sebelumnya ke tahun berjalan. οܣffnctiݏܲ푎ݕ푎ܾ݈݁adalah perubahan utang usaha dari
tahun sebelumnya ke tahun berjalan. οܶ푎h݁ݏܲ푎ݕ푎ܾ݈݁adalah perubahan utang pajak dari tahun
sebelumnya ke tahun berjalan. οܱi݄݁⿏ܣݏݏ݁iݏ
adalah perubahan aset lainnya dari tahun
sebelumnya ke tahun berjalan. Assets Totals merupakan jumlah aset tahun berjalan. 3276 Metode Analisis Data Adapun tahap-tahap dalam melakukan analisis data pada penelitian ini adalah sebagai berikut: Adapun tahap-tahap dalam melakukan analisis data pada penelitian ini adalah sebagai berikut: Perubahan Arus Kas Operasi Variabel kontrol dalam penelitian ini adalah perubahan arus kas operasi. Perubahan arus kas
operasi adalah fluktuasi aliran kas masuk dan aliran kas keluar serta sumber dan pemakaian kas
dalam suatu perusahaan pada periode tertentu. Perubahan arus kas operasi diukur dengan
menggunakan perubahan nilai arus kas operasi perusahaan i pada akhir periode t dengan t-1
dibagi dengan total aset (Frankle, 2018). ∆CFOt = CFO t – CFO t₋₁
Total Assets ∆CFOt = CFO t – CFO t₋₁
Total Assets Dimana ∆CFOt merupakan perubahan arus kas tahun berjalan. CFOt
merupakan arus kas
operasi tahun berjalan, CFOt-1 merupakan arus kas operasi tahun sebelumnya dan Total assets
merupakan total asset tahun berjalan. Perubahan Pendapatan p
Variabel dependen dalam penelitian adalah perubahan pendapatan. Mengikuti Frankle (2018),
penelitian ini mendefinisikan perubahan pendapatan sebagai perubahan pendapatan bersih dari
periode t-1 ke periode t yang berasal dari penjualan dibagi dengan total aset awal. οREVₜ =
Revₜ - Revt-₁
Assets Total Dimana ∆REVt merupakan perubahan pendapatan tahun berjalan. REVt merupakan pendapatan
tahun berjalan, REVt-1 merupakan pendapatan tahun sebelumnya dan Total assets merupakan
jumlah aset tahun berjalan. Siklus kas Operasi Variabel moderasi dalam penelitian ini adalah siklus kas operasi. Untuk menghitung siklus kas
operasi dalam penelitian ini, peneliti mengikuti penelitian yang dilakukan oleh Frankel (2018)
yang mengukur siklus kas operasi dengan cara mencari rank persentil dari siklus kas operasi dari
rata-rata 3 tahun terakhir. Frankel (2018) mendefinisikan siklus kas operasi sebagai piutang rata-
rata dibagi dengan pendapatan bersih ditambah persediaan rata-rata dibagi dengan harga pokok
penjualan dikurangi dengan rata-rata hutang dibagi dengan pembelian. rCYCLEt = Rata- rata Piutang + Rata- rata Persediaan – Rata-rata Hutang
Pendapatan Bersih
HPP
Pembelian
Dimana ∆rCYCLEt merupakan siklus kas operasi tahun berjalan. rCYCLEt = Rata- rata Piutang + Rata- rata Persediaan – Rata-rata Hutang
Pendapatan Bersih
HPP
Pembelian
Dimana ∆rCYCLEt merupakan siklus kas operasi tahun berjalan. Dimana ∆rCYCLEt merupakan siklus kas operasi tahun berjalan. Analisis Deskriptif p
Analisis deskriptif yang digunakan dalam penelitian ini adalah untuk menginterpretasikan data
rata-rata (mean), median, standar deviasi, nilai minimum, dan nilai maksimum. p
Analisis deskriptif yang digunakan dalam penelitian ini adalah untuk menginterpretasikan data
rata-rata (mean), median, standar deviasi, nilai minimum, dan nilai maksimum. 3277 C t = α0 + β1∆REVt + β2∆rCYCLEt + β3∆REVt * ∆rCYCLEt + β4∆OCFt+ εt ….(2) Model 2 digunakan untuk menguji pengaruh siklus kas operasi terhadap hubungan perubahan
pendapatan dalam memprediksi akrual. Dimana β3∆REVt * ∆rCYCLEt memberikan gambaran
besaran pengaruh siklus kas operasi yang memoderasi hubungan perubahan pendapatan dalam
memprediksi akrual. Model 2:
ACC t = α0 + β1∆REVt + β2∆rCYCLEt + β3∆REVt * ∆rCYCLEt + β4∆OCFt+ εt ….(2) Model 2: Analisis Induktif Metode yang digunakan dalam penelitian ini untuk menguji hipotesis penelitian adalah model
regresi data panel. Model regresi data panel merupakan model yang melibatkan lebih dari satu
variabel bebas (independen) dan terdiri dari beberapa tahun pengamatan. Model yang digunakan
pada penelitian ini adalah sebagai berikut: Model 1:
ACC t = α0 + β1∆REVt + β2∆CFOt…………………..………………..(1) Model 1: ACC t = α0 + β1∆REVt + β2∆CFOt… Model 1 digunakan untuk menguji pengaruh perubahan pendapatan dalam memprediksi akrual. Dimana β1∆REVt memberikan gambaran besaran pengaruh perubahan pendapatan dalam
memprediksi akrual. HASIL DAN PEMBAHASAN Adapun hasil Hausman test adalah sebagai berikut: Berdasarkan hasil Chow Test atau Likelyhood Test pada Tabel 2, maka dibutuhkan uji
Hausman pada persamaan regresi untuk hipotesi pertama (Model 1) dan hipotesis kedua (Model p
p
g
p
p
(
)
p
(
2). Adapun hasil Hausman test adalah sebagai berikut: Tabel 4
Hasil Hausman Test
Model
Penelitian
Cross-section random
Chi-Sq. Chi-Sq. Statistic
d.f
Prob
Model 1
0.01551
2
0.9923
Model 2
0.29752
4
0.9900
(Sumber: Data Olahan Eviews 10 tahun 2020) Berdasarkan hasil Hausman Test dengan menggunakan eviews10, Nilai probabilitas Model 1
adalah
sebesar
0,9923,
dan
nilai
probabilitas
Model
2
adalah
sebesar
0,9900. Nilai
probabilitasnya lebih besar dari level signifikansinya (α = 0,05), maka H0 untuk kedua model ini
diterima dan Ha ditolak. Model estimasi yang tepat digunakan dalam model ini adalah Random
Effect Model (REM) untuk Model 1 dan Model 2, sehingga tidak perlu dilakukan uji asumsi
klasik terhadap kedua model tersebut. HASIL DAN PEMBAHASAN Statistik Deskriptif Statistik Deskriptif
Hasil analisis deskriptif variabel yang digunakan pada penelitian ini adalah sebagai berikut:
Tabel 2
Statistik Deskriptif
Deskriptive statistics
Variabel
Mean
Median
Maximum
Minimum
Std. Deviasi
Accrual (t)
0.0233
0.0100
1.0300
-0.9200
0.1610
∆REV (t)
0.0591
0.0600
1.1800
-0.8200
0.2104
rCYCLE (t)
2.3395
-0.1600
38.2000
-17.710
7.6065
∆OCF (t)
0.0020
0.0000
0.3100
-0.3300
0.0807
n = 129
(Sumber: Data Olahan E-views 10, 2020) p
alisis deskriptif variabel yang digunakan pada penelitian ini adalah sebagai berikut:
Tabel 2
Statistik Deskriptif
Deskriptive statistics (Sumber: Data Olahan E-views 10, 2020) Berdasarkan tabel 1 diatas, variabel Accrual(t) sebagai variabel dependen memiliki rata-
rata 0,0233 dengan standar deviasi 0,1610. Nilai Accrual(t) maksimum sebesar 1,0300 dan nilai
minimum sebesar -0,9200. Variabel perubahan pendapatan (∆REV) sebagai variabel independen
memiliki rata-rata 0,0591 dengan standar deviasi 0,2104. Nilai ∆REV maksimum sebesar 1,1800
dan nilai minimum sebesar -0,8200. Variabel siklus kas operasi (rCYCLE) sebagai variabel
moderasi memiliki rata-rata 2,3395 dengan standar deviasi 7,6065. Nilai rCYCLE maksimum
sebesar 38,2000 dan nilai minimum sebesar -17,7100. Variabel perubahan arus kas operasi
(∆OCF) sebagai variabel kontrol memiliki rata-rata 0,0020 dengan standar deviasi 0,0807. Nilai
∆OCF maksimum sebesar 0,3100 dan nilai minimum sebesar -0,3300. 3278 Analisis Induktif
Estimasi Model Regresi
Untuk mengetahui model yang tepat untuk dalam pengolahan data penelitian, maka dilakukan uji
Chow test dan hausman test. Berikut hasil Chow test dan hausman test untuk variabel penelitian: Analisis Induktif
Estimasi Model Regresi
Untuk mengetahui model yang tepat untuk dalam pengolahan data penelitian, maka dilakukan uji
Chow test dan hausman test. Berikut hasil Chow test dan hausman test untuk variabel penelitian: Analisis Induktif
Estimasi Model Regresi Tabel 3
Hasil Chow Test atau Likelyhood Test
Cross-section Chi-square
Model Penelitian
Statistic
d.f
Prob
Model 1
11.743898
42
1.0000
Model 2
12.114828
42
1.0000
(Sumber: Data Olahan Eviews 10 tahun 2020) Tabel 3
Hasil Chow Test atau Likelyhood Test Berdasarkan hasil Chow-Test dengan menggunakan eviews10, Nilai probabilitas Model 1
dan 2 adalah lebih besar dibanding level signifikansinya (α = 0,05). Maka H0 untuk model ini
diterima dan Ha ditolak sehingga estimasi yang lebih baik digunakan dalam Model 1 dan 2 ini
adalah Common Effect Model (CEM). Untuk itu perlu dilanjutkan ke Hausman Test. Berdasarkan hasil Chow Test atau Likelyhood Test pada Tabel 2, maka dibutuhkan uji
Hausman pada persamaan regresi untuk hipotesi pertama (Model 1) dan hipotesis kedua (Model
2). Uji Model j
Berdasarkan hasil regresi pada Tabel 4 nilai adjusted R2 yang diperoleh untuk model pertama
sebesar 6,07%. Hal ini mengindikasikan bahwa kontribusi variabel independen terhadap variabel
dependen pada model persamaan pertama untuk hipotesis pertama menjadi 6,07% dan sebesar
93,93% ditentukan oleh variabel lain yang tidak dianalisis dalam model penelitian ini. Pada
model kedua nilai adjusted R2 yang diperoleh sebesar 4,74%. Hal ini mengindikasikan bahwa
kontribusi variabel independen terhadap variabel dependen pada model persamaan kedua untuk
hipotesis kedua menjadi 4,75% dan sebesar 95,25% ditentukan oleh variabel lain yang tidak
dianalisis dalam model penelitian ini. Uji F digunakan untuk menguji secara bersama-sama pengaruh variabel independen
terhadap variabel dependen, apakah model yang digunakan dapat memberikan pengaruh
signifikan atau tidak yang dapat dilihat dari probabilitas (F-statistic). Jika F-stat lebih besar dari
nilai kritis artinya variabel independen dalam model persamaan tersebut secara bersama-sama
tidak berpengaruh signifikan terhadap variabel dependen dan sebaliknya. Berdasarkan Tabel 4,
model 1 memiliki nilai probabilitas (F-statistic) < 0,05 = 0,0072, sehingga dapat disimpulkan
bahwa variabel independen dalam model persamaan tersebut secara bersama-sama berpengaruh
signifikan terhadap variabel dependen. Model 2 memiliki nilai probabilitas (F-statistic) < 0,05 =
0,0395, sehingga dapat disimpulkan bahwa variabel independen dalam model persamaan ini
secara bersama-sama berpengaruh signifikan terhadap variabel dependen Uji t digunakan
untuk
mengetahui
pengaruh
masing-masing
variabel
independen
berpengaruh terhadap variabel dependen, apakah model yang digunakan dapat memberikan
pengaruh signifikan atau tidak. Pada model regresi pertama, dilakukan estimasi pada pengaruh
perubahan
pendapatan
dalam
memprediksi
akrual
yang
disimbolkan
dengan
α1∆REVt. Berdasarkan Tabel 4 diketahui bahwa koefisien α1∆REVt bernilai positif dan nilai prob sebesar
0,1151. Hal ini berarti perubahan pendapatan tahun berjalan positif namun tidak berpengaruh
signifikan dalam memprediksi akrual, karena nilai prob > 0,05, yaitu 0,1151 > 0,05. Berdasarkan hasil regresi dari model pertama dapat disimpulkan bahwa hipotesis pertama
ditolak. Dimana hipotesis pertama berbunyi “perubahan pendapatan berkorelasi positif dengan
tingkat akrual”. Model persamaan regresi kedua merupakan model persamaan regresi yang
digunakan untuk mengestimasi pengaruh siklus kas operasi yang memoderasi hubungan
perubahan pendapatan dalam memprediksi akrual. Pada model regresi kedua, dilakukan estimasi
pada pengaruh siklus kas operasi yang memoderasi hubungan perubahan pendapatan dalam
memprediksi akrual yang disimbolkan dengan α3∆REVt x rCYCLEt. Berdasarkan Tabel 4
diketahui bahwa koefisien α3∆REVt x rCYCLEt bernilai negatif dengan nilai prob sebesar
0,7109. Hal ini berarti siklus kas operasi tidak mampu memperkuat atau memperlemah
hubungan perubahan pendapatan dalam memprediksi akrual, karena nilai signifikansi > 0,05
yaitu 0,7109 > 0,05. Hasil Regresi Panel Berdasarkan hasil estimasi model regresi pada Chow Test (Tabel 2) dan Hausman Test (Tabel 3),
maka digunakan pendekatan random effect model untuk mengestimasi pengaruh perubahan
pendapatan dalam memprediksi akrual (Model 1) dan pengaruh siklus kas operasi dalam
memoderasi hubungan perubahan pendapatan dalam memprediksi akrual (Model 2). Adapun
hasil regresi panel dengan menggunakan pendekatan random effect model untuk regresi
pengaruh perubahan pendapatan dalam memprediksi akrual (Model 1) dapat dilihat pada Tabel 5. 3279 Tabel 5
Hasil Regresi Panel
Persamaan Regresi Model 1
ACCₜ = α₀+ ߚΌοܴܧܸₜ + ߚtοܥܨܱₜ
Persamaan Regresi Model 2
ACCₜ = α₀+ ߚΌοܴܧܸₜ + ߚt⿏ܥdܥlܧₜ + ߚtοܴܧܸₜ כ ⿏ܥdܥlܧₜ +
ߚtܥܨܱt+Ɛₜ
Variabel
Prediksi
Model 1
Model 2
Konstanta
Coef. 0.0169
0.0178
t-stat
1.0133
1.0004
∆REV (t)
Coef. +
0.1231*
0.1334*
t-stat
1.5997
1.5869
rCYCLE (t)
Coef. -0.0006
t-stat
-0.2768
∆REV x
Coef. +
₋
-0.0045
rCYCLE (t)
t-stat
₋
-0.3714
∆CFO (t)
Coef. +
-0.4703**
-0.4693**
t-stat
-2.3429
-2.3107
Adj R-Square
0.0607
0.0475
F-Statistic
5.1336***
2.5971**
Keterangan
∆REV (t)
: Perubahan pendapatan
rCYCLE (t) : Siklus Kas Operasi
∆CFO (t)
: Arus Kas Operasi
n
: 129
***,**,*
: Signifikan pada α 1%, 5%, dan 10%
(Sumber: Data Olahan Eviews 10 tahun 2020) Tabel 5 Hasil Regresi Panel Hasil estimasi model regresi dengan pendekatan Random Effect Model (REM) pada tabel 4
diatas, diketahui bahwa besaran pengaruh perubahan pendapatan dalam memprediksi akrual
(model 1) diwakili oleh koefisien regresi ∆REVt. Dimana berdasarkan Tabel 4 diketahui
koefisien regresi ∆REVt adalah sebesar 0,1231, namun pengaruhnya tidak signifikan. Hasil Adj
R-square sebesar 0,0607, yang berarti seluruh variabel bebas berpengaruh dalam memprediksi
akrual sebesar 0,0607 atau 6,07%. Untuk hasil estimasi model regresi dengan pendekatan Random Effect Model (REM) pada
model 2, berdasarkan tabel 4 diketahui bahwa besaran pengaruh siklus kas operasi dalam
memoderasi hubungan perubahan pendapatam dalam memprediksi akrual diwakili oleh koefisien
∆REVt x rCYCLEt. Dimana berdasarkan Tabel 4 diketahui koefisien regresi ∆REVt x rCYCLEt
adalah sebesar -0,0045, namun pengaruhnya tidak signifikan. Hasil Adj R-square sebesar 0,0475,
yang berarti seluruh variabel bebas berpengaruh dalam memprediksi akrual sebesar 0,0475 atau
4,75%. 3280 Uji Model Berdasarkan hasil regresi dari model kedua dapat disimpulkan bahwa
hipotesis kedua ditolak. Dimana hipotesis kedua berbunyi “Perubahan pendapatan berkorelasi
positif dengan tingkat akrual, hubungan ini semakin positif dengan adanya siklus kas operasi”. Pembahasan Hasil Penelitian Pengaruh Perubahan Pendapatan dalam Memprediksi Akrual Hipotesis pertama pada penelitian ini adalah perubahan pendapatan berkorelasi positif dengan
tingkat akrual, dimana semakin besar perubahan pendapatan maka tingkat akrual yang ada dalam
laporan keuangan semakin besar. Variabel akrual pada penelitian ini dihitung dengan akrual 3281 modal kerja, kemudian variabel perubahan pendapatan pada penelitian ini diukur dengan
menggunakan ∆REVt, yang dihitung dengan mengurangi pendapatan pada tahun berjalan
dengan pendapatan tahun sebelumnya (Frankel, 2018). g
p
p
y (
)
Berdasarkan hasil penelitian pada perusahaan sektor manufaktur yang terdaftar di BEI
pada tahun 2016-2018, ditemukan bahwa hipotesis (H1) ditolak. Hasil penelitian ini tidak dapat
membuktikan bahwa perubahan pendapatan berkorelasi dengan tingkat akrual. Hal ini dapat
dilihat dari hasil regresi panel pada tabel 10, yang menunjukkan bahwa nilai signifikan dari
variabel ∆REVt lebih besar dari α 0,05%, yaitu 0,1151 > 0,05%. Hasil ini menunjukkan bahwa
perubahan pendapatan positif namun tidak signifikan dalam memprediksi akrual. p
p
p
p
g
p
Hasil penelitian ini berbeda dengan hasil penelitian yang dilakukan oleh Frankel (2018)
tentang Predicting Accruals Based on Cash-Flow Propertie, yang mana menunjukkan bahwa
perubahan pendapatan berkorelasi positif signifikan dengan tingkat akrual. Hasil penelitian
Frankel (2018) menyimpulkan bahwa perubahan pendapatan cukup untuk menjelaskan
perubahan akrual modal kerja. Sehingga, dengan mengetahui perubahan pada pendapatan dalam
suatu periode dapat memprediksi akrual modal kerja dimasa yang akan datang. Hasil penelitian ini juga berbeda dengan penellitian Jones (1991) yang mengatakan bahwa
perubahan pendapatan mempengaruhi perubahan akrual dari modal kerja (working capital)
seperti piutang, persediaan, dan utang. Perubahan pendapatan yang terjadi dalam suatu
perusahaan saat ini mengarah ke komponen arus kas yang terkait dengan perubahan akrual
modal kerja yang diimbangi untuk lebih mengukur kinerja perusahaan. Sehingga, peningkatan
yang terjadi dalam perubahan pendapatan akan meningkatkan akrual modal kerja seperti piutang,
persediaan, dan utang. Peneliti sepakat dengan penelitian Frankle (2018) dan Jones (1991), namun hasil penelitian
ini tidak dapat membuktikan bahwa perubahan pendapatan mampu memprediksi akrual. Hasil
penelitian ini juga tidak dapat mengindikasikan bahwa tinggi rendahnya perubahan yang terjadi
dalam pendapatan saat ini dapat memprediksi akrual. Beberapa faktor yang menyebabkan
hipotesis pertama ini ditolak yang pertama, karena variabel perubahan pendapatan pada
perusahaan manufaktur tahun 2016-2018 menunjukkan angka yang rendah, hal ini tercermin dari
jauhnya besaran perubahan anatara pendapatan dan total aset yang dimiliki perusahaan, sehingga
membuat perubahan pendapatan menjadi rendah. p
p
p
j
Selain itu, besaran perubahan pendapatan yang dihasilkan tidak sebanding dengan akrual
modal kerja yang dihasilkan seperti piutang dan persediaan. Pembahasan Hasil Penelitian Ketika perubahan pendapatan yang
dihasilkan meningkat, akrual modal kerja yang dihasilkan tidak terlalu mengalami perubahan. Hal ini bisa terjadi diakibatkan oleh adanya kebijakan piutang (kredit), kebijakan persediaan, dan
kebijakan hutang yang diterapkan perusahaan sebagai akibat terjadinya penjualan dalam
perusahaan. Kedua, data tahun pengamatan yang digunakan dalam pengambilan sampel
penelitian cukup pendek hanya mencakup tiga tahun yaitu dari tahun 2016-2018, sehingga
hipotesis pertama ditolak. Pengaruh siklus kas operasi yang memoderasi hubungan perubahan pendapatan dalam
memprediksi akrual. Hipotesis kedua dalam penelitian ini adalah perubahan pendapatan berkorelasi positif dengan
tingkat akrual, hubungan ini semakin positif dengan adanya siklus kas operasi yang positif. Variabel siklus kas operasi dihitung menggunakan rank persentil dari rata-rata tiga tahun terakhir
siklus kas operasi sesuai dengan penelitian yang telah dilakukan oleh Frankel (2018). 3282 Berdasarkan hasil uji hipotesis kedua, ditemukan dalam penelitian ini bahwa hipotesis
kedua ditolak. Hasil penelitian ini tidak membuktikan bahwa siklus kas operasi memperkuat atau
memperlemah hubungan antara perubahan pendapatan dengan akrual. Hal ini dapat dilihat dari
hasil regresi panel pada tabel 10, yang menunjukkan bahwa nilai signifikasi variabel t yang tidak
signifikan pada α 5%, yaitu koefisien α3∆REVt x rCYCLEt bernilai negatif dengan nilai prob
sebesar 0,7109 atau 0,7109 > 0,05. Hasil ini menunjukkan bahwa siklus kas operasi tidak mampu
memperkuat atau memperlemah hubungan perubahan perubahan pendapatan dalam memprediksi
akrual. Hasil penelitian ini berbeda dengan hasil penelitian yang dilakukan oleh Frankel (2018). Hasil penelitian Frankel (2018) menemukan bahwa koefisien positif yang signifikan untuk siklus
kas operasi. Temuan Frankel (2018) ini menunjukkan bahwa hubungan positif antara perubahan
arus kas dengan akrual menjadi lebih kuat karena siklus kas operasi yang meningkat. Menurut
Frankel (2018) hubungan positif antara perubahan pendapatan dengan akrual yang diperkuat
oleh siklus kas operasi yang meningkat menunjukkan bahwa perubahan yang diberikan dalam
penjualan atau operasi menyebabkan perubahan yang lebih besar dalam akrual modal kerja. Dengan siklus kas operasi yang lebih lama, arus kas perusahaan memiliki masalah waktu dan
pencocokan yang lebih serius. Akibatnya, untuk perubahan pendapatan yang diberikan, diperlukan tingkat akrual yang
lebih besar untuk mengimbangi arus kas yang direalisasikan dalam periode yang berbeda dari
inovasi dalam laba kotor. Tapi dalam penelitian ini hubungan antara perubahan pendapatan
dengan akrual yang dimoderasi oleh siklus kas operasi tidak menunjukkan hasil yang signifikan. Hasil ini menandakan bahwa pada arus kas yang ada dalam perusahaan manufaktur yang
menjadi sampel penelitian ini tidak terdapat masalah timing dan matching yang serius. Sehingga
perubahan pendapatan yang diberikan, tidak diperlukan tingkat akrual yang besar untuk
mengimbangi arus kas yang direalisasikan dalam periode yang berbeda dari inovasi dalam laba
kotor. Oleh karena itu, hipotesis kedua penelitian ini ditolak. Beberapa faktor yang menyebabkan hipotesis kedua ditolak diantaranya adalah karena
perusahaan yang menjadi sampel penelitian tidak mengungkapkan data pembelian bahan baku
dan barang jadi secara keseluruhan, sehingga hasil utuk siklus kas operasi menjadi tidak
signifikan. Pengaruh siklus kas operasi yang memoderasi hubungan perubahan pendapatan dalam
memprediksi akrual. Kemudian perusahaan manufaktur yang dijadikan sampel penelitian diambil secara
keseluruhan dan tidak membedakan karakteristik masing-masing perusahaan, sehingga data yang
digunakan menjadi sangat beragam terutama untuk data pembelian perusahaan. Data tahun
pengamatan yang digunakan dalam pengambilan sampel penelitian cukup pendek hanya
mencakup tiga tahun yaitu dari tahun 2016-2018, sehingga hipotesis kedua ditolak. Berdasarkan
penelitian ini siklus kas operasi merupakan variabel yang tidak berpengaruh terhadap perubahan
pendapatan dalam memprediksi akrual. Saran Bagi peneliti selanjutnya diharapkan dapat 1) menggunakan jenis perusahaan yang berbeda-beda
dan memakai ruang lingkup sampel yang lebih luas. 2) menambah tahun pengamatan dengan
meneliti lebih dari 3 tahun agar hasil yang diperoleh lebih berkualitas. 3) sebaiknya
mempertimbangkan dan mencari variabel independen lainnya yang berhubungan dengan
memprediksi akrual. Dechow, P., S. P. Kothari, dan R. Watts. 1998. The relation between earnings and cash flow.
Journal of Accounting and Economics 25 (1998). pp. 133 —168. KESIMPULAN, KETERBATASAN, SARAN ,
Kesimpulan dan Implikasi Kesimpulan dan Implikasi Berdasarkan hasil temuan penelitian dan pengujian hipotesis yang diajukan sebelumnya dapat
disimpulkan bahwa perubahan pendapatan tidak berpengaruh signifikan terhadap akrual, yang
artinya semakin tinggi perubahan pendapatan maka tidak akan meningkatkan akrual. Siklus kas
operasi yang memoderasi hubungan antara perubahan pendapatan dan akrual dalam penelitian
juga tidak dapat memperkuat atau memperlemah hubungan perubahan pendapatan dalam
memprediksi akrual, yang artinya hubungan antara akrual dan perubahan pendapatan periode 3283 berjalan tidak bervariasi dengan siklus kas operasi perusahaan sehingga kurang tepat dijadikan
sebagai variabel moderasi dalam penelitian ini. Selain itu, implikasi dari penelitian ini bahwa siklus kas operasi perusahaan tidak terbukti
dapat memperkuat atau memperlemah hubungan antara perubahan pendapatan dan akrual. Interaksi antara perubahan pendapatan dan rank persentil dari siklus kas operasi rata-rata tiga
tahun
terakhir
ditemukan
memiliki
koefisein
negatif
dan
tidak
signifikan. Hal
ini
mengindikasikan siklus kas operasi yang menangkap masalah timing dan matching pada arus kas,
tidak membantu menjelaskan akrual melalui pengaruhnya terhadap hubungan antara perubahan
pendapatan dan akrual. Hasil penelitian ini berlawan dengan penelitian yang dilakukan oleh Frankle (2018) dan
Dechow, P. (1994) yang menyatakan bahwa hubungan antara perubahan pendapatan dan akrual
meningkat seiring dengan perubahan siklus kas operasi. Hal ini dapat terjadi karena perusahaan
yang menjadi sampel penelitian tidak mengungkapkan data pembelian bahan baku dan barang
jadi secara keseluruhan pada laporan keuangannya, perhitungan data untuk siklus kas operasi
menjadi terganggu dan perusahaan tersebut dikeluarkan dari sampel penelitian yang membuat
sampel penelitian menjadi lebih sedikit sehingga hasil untuk siklus kas operasi menjadi tidak
signifikan. Keterbatasan Keterbatasan dari penelitian ini pertama adalah tidak semua perusahaan yang dijadikan sebagai
sampel penelitian menyajikan data pembelian bahan baku dan pembelian barang jadi pada
laporan keuangannya, sehingga perhitungan data untuk siklus kas operasi (rCYCLE) menjadi
terganggu dan perusahaan tersebut dikeluarkan dari sampel penelitian. Kedua, penelitian ini hanya penggunakan sampel pada sektor manufaktur, sehingga data
penelitian tidak dapat mewakili keseluruhan perusahaan pada BEI. Ketiga, tahun pengamatan
yang digunakan dalam pengambilan sampel hanya mencakup 3 tahun yaitu dari tahun 2016-2018. Hal ini dikarenakan keterbatasan informasi dan keterbatasan data yang tidak lengkap. Dechow, P., dan I. Dichev. 2002. The Quality of Accruals and Earnings: The Role of Accrual
Estimation Errors. The Accounting Review. Vol. 77, Supplement: Quality of Earnings
Conference (2002). pp. 35-59. Ball, Ray. 2013. Accounting Informs Investors and EarningsManagement is Rife: Two
Questionable Beliefs. Accounting Horizons. Vol.27. No 4. pp. 847-853. Bodroastuti, Tri. 2009. Pengaruh Struktur Corporate Governance terhadap Financial Distress.
Jurnal Riset Akuntansi, Manajemen, Ekonomi, Vol. 1, No.1, h. 87-105. DAFTAR PUSTAKA Ball, Ray. 2013. Accounting Informs Investors and EarningsManagement is Rife: Two
Questionable Beliefs. Accounting Horizons. Vol.27. No 4. pp. 847-853. Bodroastuti, Tri. 2009. Pengaruh Struktur Corporate Governance terhadap Financial Distress. Jurnal Riset Akuntansi, Manajemen, Ekonomi, Vol. 1, No.1, h. 87-105. Dechow, P., dan I. Dichev. 2002. The Quality of Accruals and Earnings: The Role of Accrual
Estimation Errors. The Accounting Review. Vol. 77, Supplement: Quality of Earnings
Conference (2002). pp. 35-59. Dechow, P., S. P. Kothari, dan R. Watts. 1998. The relation between earnings and cash flow. Journal of Accounting and Economics 25 (1998). pp. 133 —168. 3284 Dechow, P. 1994. Accounting earnings and cash flows as measures of firm performance The role
of accounting accruals. Journal of Accounting and Economics 18 (1994). pp. 3-42. Ellen dan Juniarti, 2013, “Penerapan Good Corporate Governance, Dampaknya Terhadap
Prediksi Financial Distress pada Sektor Aneka Industri dan Barang Konsumsi periode
2008-2010”. Journal Business Accounting Review. Vol. 1, No. 2. 2013. g
Frankel, Richard .M dan Yan Sun. 2018. Predicting Accruals Based on Cash-Flow Properties. The Accounting Review. Vol.93. No.5. pp. 165-186. Hribar, Paul dan Daniel W Collins. 2002. Errors in Estimating Accruals: Implications for
Empirical Research. Jurnal of Accounting Research. Vol.40. No.1. Ikatan Akuntansi Indonesia. PSAK No. 1 Tentang Laporan Keuangan– edisi revisi 2018. Penerbit Dewan Standar Akuntansi Keuangan: PT. Raja Grafin. Ikatan Akuntan Indonesia. 2010. Standar Akuntansi Keuangan. Jakarta: Salem Akuntan Indonesia. 2010. Standar Akuntansi Keuangan. Jakarta: Salemba Empat
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l f
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Jones, J., 1991. Earnings management during import relief investigations. Journal of Accounting
Research 29, 193–228. Kothari, S., Leone, A., Wasley, C. 2005. Performance matched discretionary accrual measures. Journal of Accounting and Economics 39, 163–197. Lam, Nelson dan Peter Lau. 2015. Akuntansi keuangan intermediate financial report . Ed 2.1
Jakarta: salemba empat. McNichols, M., 2000. Research design issues in earnings management studies, Journal of
Accounting and Public Policy 19, 313-345 McNichols, M. 2002. Discussion of The Quality of Accruals and Earnings: The Role of Accrual
Estimation Errors. The Accounting Review, Vol. 77 (Supplement), pp. 61–9. Owens, E., J. Wu, dan J. Zimmerman, 2013. Business model shocks and abnormal accrual
models, Working paper, University of Rochester. Scott, W.R. 1997. Financial Accounting Theory. Prentice Hall Inc. New Jersey. g
y
y
Subramanyam, K.R dan J.J. Wild. 2010. Analisis Laporan Keuangan. Edisi 10 Buku 1.Jakarta :
Salemba Empat Suganda, William dan Firman Syarif. 2015. DAFTAR PUSTAKA Analisis Pengaruh Kualitas Akrual (Accruals
Quality) terhadap Sinkronitas Harga Haham (Stock Price Synchronicity). Simposiun
Nasional Akuntansi (SNA) XVIII. (
)
Suranggane, Zulaikha. 2007. ”Analisis Aktiva Pajak Tangguhan dan Akrual Sebagai Prediktor
Manajemen Laba: Kajian Empiris Pada Perusahaan Manufaktur Yang Terdaftar di BEJ”. Jurnal Akuntansi dan Keuangan Indonesia Vol.4, No.1, Hal 77-94. Wahyuningsih, Dwi R, 2007. Hubungan Praktk Manajemen Laba dengan Reaksi Pasar Atas
Pengumuman Informasi Laba Perusahaan Manufaktur di Bursa Efek Indonesia Jakarta. Thesis. Universitas Diponegoro Semarang 3285
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Caring for palliative care patients at home: medicines management principles and considerations
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Caring for palliative care patients at home: medicines management principles and
considerations Standfirst: This article outlines how and when medicine support may be required by
palliative care patients and family caregivers, alongside practical steps pharmacy
teams can take to deliver supportive care. Palliative care is defined by the National Institute for Health and Care Excellence (NICE) as the active holistic
care of patients with advanced, progressive illness, where the management of pain and other symptoms and
provision of psychological, social and spiritual support is paramount. With many aspects of palliative care
applicable earlier in the course of illness, the main goal is achievement of the best quality of life for patients
and their families [1]. Access to palliative care is highly uneven and the quality variable, even in high-income countries such as the
UK [2]. Only 14% of patients deemed able to benefit from palliative care currently receive it [3,4] while the
demand for palliative care is growing. For example, in England and Wales, the need for palliative care is
estimated to increase by 25-47% by 2040 [3]. Policy and commissioning drives are required to support
excellent care for all patients, including those being care for and dying at home [5]. This is assumed to be the
preference for the majority [6], but may not be appropriate for all [7]. Patients and family caregivers require appropriate, tailored and rapid access to end of life care. An important
aspect of this is support is to better manage medicines, including potent analgesics prescribed to help control
pain. The needs of terminally ill patients can change quickly and be unpredictable. Responding to symptoms
and advising on care involves a multi-disciplinary whole system approach. Community pharmacists have
occupied a marginal role in patient care, being mainly involved in medicine supply. However, current policy
promotes a greater involvement of pharmacists in direct care as a means of reducing pressure on GP services
and increasing patient access to care. There is great scope for pharmacists to contribute, for example,
counselling on medicine use for seriously ill patients who may be homebound, identifying key family members
and carers responsible for care, and liaising with other health professionals about prescribing changes,
including deprescribing and use of anticipatory medicines, close to the end of life. This article aims to consider how pharmacists can better support terminally ill patients, and the family
caregivers who support them, to manage medicines at home. Caring for palliative care patients at home: medicines management principles and
considerations Specifically, it will highlight the day-to-day
challenges patients and family caregivers face and offer practical information and ways that medicines
optimisation can be applied to support palliative care patients being cared in the community setting. Caring for palliative care patients at home: medicines management principles and
considerations Caring for palliative care patients at home: medicines management principles and
considerations The end-of-life care pathway The NHS Long Term Plan recommends improved proactive, personalised and well-coordinated care for all
people in their final year of life [8]. Care should be patient-centered and sensitive to the physical, The NHS Long Term Plan recommends improved proactive, personalised and well-coordinated care for all
people in their final year of life [8]. Care should be patient-centered and sensitive to the physical,
psychological, social and spiritual needs of the patient and family. Healthcare professionals should have the ychological, social and spiritual needs of the patient and family. Healthcare professionals should have th competence and confidence to hold ‘difficult conversations’ with patients and family caregivers about end of
life care. However, foreseeing when and how to initiate discussions, considering uncertainty around
prognostication, is challenging and was covered in a previous learning article that detailed the communication
and cultural competence skills required by pharmacists [9]. The government’s end-of-life care strategy sets out
recommendations governing the six key elements that characterise the end-of-life pathway (see Figure and
Box 1) [10]. Figure 1: Department of Health: End of Life (EoL) care pathway The six key elements of an EoL pathway. It is important that support and advice, including access to spiritual
care services are provided alongside these steps to patients and family caregivers. Source: [10] ///Box 1: How pharmacists should communicate and have open discussions about end-of-life with the
patient, carers and their families//// ///Box 1: How pharmacists should communicate and have open discussions about end-of-life with the
patient, carers and their families//// The extent to which discussions around end-of-life care are undertaken varies, but open and honest
communication that is sensitive to the situation, commences early and continues through the patient’s
journey. The aims of these discussions include: The extent to which discussions around end-of-life care are undertaken varies, but open and honest
communication that is sensitive to the situation, commences early and continues through the patient’s
journey. The aims of these discussions include:
Eliciting the patient’s level of understanding, main problem(s) or concern(s) about their medicines
(especially those that are anticipatory) and any impact (physical, emotional or social) that these are
having on the patient;
Determining how much information the patient wishes to receive and providing this to ensure
medicine optimisation;
Ascertaining whether the patient wishes more support to engage in medicines management or end-
of-life conversations with other family members or carers, and responding to these needs as
necessary [9]. ////End Box/// How community pharmacists can support patients and families at the end-of-life
With workplace pressures and lack of remuneration, community pharmacy has had limited involvement in
supporting palliative care, but there is a growing need and so great scope for greater input. Patients and family
caregivers often find it hard to cope with rapidly changing symptoms and associated prescription changes. This
may be particularly important in the last days of life – where pharmaceutical care needs relating to timely
supply, advice on anticipatory medicines [11] and managing symptoms may become more pressing. Effective communication with patients alongside effective collaboration with a range of care providers is
therefore central to achieving medicines optimisation. Given their ease of access, community pharmacists and
their teams could support palliative care in the community. At present most pharmacists have limited
interaction, are not formally integrated into the multi-disciplinary team or are not appropriately trained to
fully contribute to palliative patient-centered care [12]. A previous article provides pharmacists with simple
strategies and techniques to help manage difficult conversations and situations related to palliative care and
end-of-life [9]. The following sections outline how and when medicine support may be required alongside
practical steps pharmacy teams can take to deliver supportive care. Opportunities for pharmaceutical care Opportunities for pharmaceutical care
In accordance with National Institute for Clinical Excellence (NICE) guidelines [14], pharmacists should
recognise when a person may be in the last days of life and provide advice and support both to patients /
family caregivers and health care professionals. This advice should extend to the use of anticipatory medicines
(i.e. ‘just in case’ medicines, see Box 2) [13] and that sufficient stocks of these are available. Shared-decision
making is important, as will being mindful of cultural, religious or spiritual preferences. Care will need to be
carefully tailored and individualized; examples of the type of advice / interventions that could be given are
included in Table 1. Table 1: Examples of pharmacological interventions at the end of life [14]
Symptom management at the
end of life
Example of pharmacological intervention / considerations
Managing pain
Following discussion with the patient / family caregiver, if pain is identified, initiate
discussion with the prescriber about optimal management. Ensure a match between the medicine and severity of pain. Advise patient / family caregiver to monitor breakthrough pain episodes; ensure
appropriate extended-release formulations are prescribed and counsel on correct
use. Consider possible non-pharmacological pain management. Managing nausea and
vomiting. Ideally, the cause should be determined through discussion with the patient / family
caregiver. Table 1: Examples of pharmacological interventions at the end of life [14] Ensure prescribing (where necessary) and counsel on the use of a suitable
antiemetic. Invite the patient / family caregiver to contact the pharmacist after 24 hours to
measure progress. Contact the prescriber if substitution or additional anti-emetic is
needed. Consider possible non-pharmacological management. Managing anxiety, delirium
and agitation
Identify and explore possible causes of anxiety or delirium. The dose and frequency should be reviewed according to levels of patient distress
and response. Managing troublesome
respiratory secretions
‘The death rattle’ is a distinctive sound that a person may make as they are coming
to the end of their life and may no longer be able to swallow or cough effectively
enough to clear their saliva. Reassure family members / friends that, although the noise can be distressing, it is
unlikely to cause discomfort. If a medicine is indicated, periodic review for efficacy and adverse effects is needed,
whereas some non-pharmacological advice may be needed (e.g. repositioning). Other symptoms
Invite the patient to discuss other symptoms that could arise, such as
anorexia/cachexia (i.e. Further details (for non-specialists) on how to manage end of life symptoms can be found in the NICE guideline
‘Care of dying adults in the last days of life’ [14]. Further details (for non-specialists) on how to manage end of life symptoms can be found in the NICE guideline
‘Care of dying adults in the last days of life’ [14]. Opportunities for pharmaceutical care wasting of the body), fatigue, constipation, diarrhea, oral
health issues, depression and difficulty sleeping. Discuss solutions with the patient and offer advice to the prescriber / appropriate
member of the wider care team. Maintain hydration: Advise the family caregiver to support the dying person to drink
if they wish to and are able to. Rational use of medicines and
deprescribing
Where appropriate, and with discussion with the patient / family caregiver and
prescriber, suggest to the prescriber to stop any previously prescribed medicines
that are not providing symptomatic benefit (e.g. if the patient is on a statin) or that
may cause harm. ///Box 2: Just in case boxes/// Many areas of the UK have implemented ‘Just in case box’ initiatives to support anticipatory prescribing,
where dispensed medicines are stored in a readily identifiable container along with the appropriate equipment
(see ‘Typical contents’) and the necessary documentation to facilitate prompt administration if symptoms
develop [15]. Procedures should be established to minimise the risk of medicines being unlawfully diverted
[16]. [16].
Anticipatory medicines for subcutaneous use (including diluent);
Needles and syringes;
Prescribing guidance;
Authorisation to administer medication document;
Patient and carer information leaflet;
Contact details for advice [11]. Best practice principles The prescriber must accept responsibility for prescribing in anticipation of need and be mindful that the
availability of medication does not replace the need for clinical assessment when the patient’s clinical
condition changes. They must: Agree the list of anticipatory medicines locally with key stakeholders; Reduce the risk of prescription errors by agreeing the recommended starting doses and making them
readily available to prescribers on pre-printed sheets;
Balance the quantity supplied between adequate supply and potential waste; Include equipment and documentation to facilitate the administration of medicines in the just in case
box;
Ensure that all healthcare professionals involved in the care of the patient are aware of the clinical
situation and the availability of anticipatory medicines, including those providing the out-of-hours
services [15]. ////End Box//// With counselling on dispensed medicines or through offering an advanced service (Medicines Use Review
(MUR) / New Medicines Service (NMS)), patients along with family / carers, can be enabled to make more
informed decisions about their pharmaceutical care; for guidance on how to structure these conversations, see
[9]. However, most seriously ill patients will either have their medicines delivered or collected by family
caregivers and this could undermine the extent to which medication review and support services are offered
or for opportunities for patient-pharmacist personal contact. In these cases, the pharmacist will need to
ensure family / carers feel fully supported to manage medicines. Where clinical interventions have been
proactively discussed with patients / carers and are then considered to be outside the pharmacists’ remit,
referrals should be made to the appropriate health professionals. Medicine optimisation Medicine optimisation p
It is essential that pharmacists actively support patients to take their medicines as intended. Pharmacists
should routinely initiate discussions with patients about how they are using their medicines, and any concerns
and questions they may have about these. This should include offering appropriate advice on the management
of multi-morbidities and polypharmacy [17, 18]. In managing the care of patients at the end-of-life clinical
decision-making may become more complex as new symptoms develop and become more prevalent and even emotionally distressing for families and carers. Guidance on exercising professional judgement can be found in
the ‘Medicine Ethics and Practice’ (MEP) [19] and through guidance issued by the RPS on ethical and
professional decision making in the COVID-19 Pandemic [20]. This includes making informed decisions about
the care of the patient by taking into account the law, ethical considerations relevant factors related to the
surrounding circumstances (e.g. facts, relevant laws, standards and good practice guidance). The patient
should remain the first concern of the pharmacist and you must ensure that you can justify your decisions. Where appropriate, make a written record of the decision-making process and your reasons leading to your
particular course of action. The principles of medicine optimisation should be employed to ensure patients get the best possible outcomes
from their medicines (see Figure) [21]. The principles of medicine optimisation should be employed to ensure patients get the best possible outcomes
from their medicines (see Figure) [21]. Principle 1 “Aim to understand the patient’s experience” o It is paramount to understand patients’ perspectives for any given
intervention or treatment. Pharmacists can encourage palliative care patients
and their carers to express their views in an open dialogue about their
experience and any concerns. This needs to be an ongoing two-way process
to elicit patients’ choices as they progress through different phases of their
illness. An important part of this process will be active listening to
understand the patient/family experience of specific medicines i.e. concerns
about side effects, dislike of certain medicines/formulations and respond
constructively. These discussions will help ensure the patients’ preferences
are understood and promote an environment of shared decision; Principle 2 “Evidence based choice of medicines” Principle 2 “Evidence based choice of medicines” o Medicines regimens should be regularly reviewed to ensure they remain
clinically appropriate and cost effective while meeting the needs of the
patient. Medicine optimisation Such reviews can help identify medicines which are no longer
necessary and can be stopped as symptomatic treatment progressively takes
precedence. Principle 3 “Ensure medicines use is as safe as possible” o All aspects of safety should be considered, for example side effects,
interactions and safe processes for handling. Medicines in palliative care
carry significant potential for harm if mishandled or not used as advised. Pharmacists can engage patients and carers in improving their health literacy. carry significant potential for harm if mishandled or not used as advised. Pharmacists can engage patients and carers in improving their health literacy. Pharmacists can engage patients and carers in improving their health literacy. This can ensure that unnecessary incidents of harm do not occur and the
patient feels confident using their prescribed medicine; Principle 4 “Make medicines optimisation part of routine practice” o Pharmacists should routinely initiate conversations with patients or their
carers about their medicines. If they are homebound, a considerate
telephone call to enquire how the patient is would be welcome. Additionally,
effective medicines optimisation strategies require routine discussion and
collaboration across professional boundaries. Patient permission for
information sharing between services should be obtained. Insights and lessons from the ‘Managing medicines for patients with serious illness being
cared for at home’ study Insights and lessons from the ‘Managing medicines for patients with serious illness being
cared for at home’ study The ‘Managing medicines at home’ study was a three-year qualitative investigation examining how patients
and family givers cope with managing medicines at home, how they felt about doing this and the manner in
which they were supported by different health care professionals and services involved in providing their care. [22]. Three main themes emerged and their application to medicines optimisation is described below. These
themes can act as further areas where recommendations for community pharmacists can be considered. 1. Help required to enable patients to manage their medicines at home
Patients were being asked to manage complex medicines regimes at home with little support or guidance on
their use [22]. A range of simple recommendations were identified to improve the support and management
of medicines in the home, including:
Encouraging greater awareness that people can approach the pharmacist for help and advice about
medicines; the pharmacist was an untapped and under-used resource. Medicine optimisation
Provide patients with accessible written and verbal information about what medicines are for and
how to take them – especially in relation to pain relief;
Suggest alternative routes of administration where appropriate (e.g. move to liquid formulations);
When applicable, provide dose administration aids, such as using multi-compartment compliance
aids, to help reduce confusion about what tablets a patient should take and when; review their use on
refill to ensure medicine adherence;
Whenever possible, inform patients and carers when a tablet changes in size, shape or colour; these
small changes were seen to be the cause of much confusion and anxiety;
Keep adequate stocks of palliative care medicines in the pharmacy (as appropriate), for example
medicines to relieve pain (e.g. various formulations of fentanyl and morphine) and manage confusion
or restlessness (e.g. midazolam);
Rationalise the use of or deprescribing of medicines where appropriate [23-25]. Prescribing changes often become more frequent during end-of-life, family caregivers may become anxious or
confused if they are not properly informed and supported thereby resulting in potential non-adherence and
medicine waste. This may be partly mitigated using dosette boxes, however advice on how to manage liquids,
creams, patches and ‘when required’ doses will need to be discussed with the patient and carefully monitored
by the pharmacist to ensure medicines are used appropriately [26]. Prescribing changes often become more frequent during end-of-life, family caregivers may become anxious or
confused if they are not properly informed and supported thereby resulting in potential non-adherence and
medicine waste. This may be partly mitigated using dosette boxes, however advice on how to manage liquids,
creams, patches and ‘when required’ doses will need to be discussed with the patient and carefully monitored
by the pharmacist to ensure medicines are used appropriately [26]. Improving awareness of pharmacy support Patients and families are often unaware of the help and support the pharmacy team is able to provide. A
simple and effective way of alerting patients /family caregivers about what the pharmacy can offer could be
made possible through a poster displayed in the pharmacy or local GP surgery. This could encourage patients
and family caregivers to take the first step to seek help (Figure). For example, when a patient asks the following questions, you could respond as follows:
“Why have I been given this medicine?” o You should ask them what conversations they had with the prescriber and check
their understanding of why this has been prescribed. If applicable, you can reinforce
what the medicine is used for, explaining to the patient how the medicine works and
why it is needed can help ensure patients feel comfortable with the prescribing
decision.
“Why have my medicines been changed?” o You can discuss their condition and how they feel it has been managed. If a medicine
has been changed, explore the reasons why this change has occurred and
communicate this to the patient. Has there been a GP practice formulary change? Did the patient report a side-effect, certain medicines are more or less appropriate
for any given patient.
“What if I miss a dose?”
“What if I miss a dose?” o Explain what the patient should do in this circumstance. This may be taking one as
soon as they remember, or skipping a single dose. Advising the patient or their carer
to check the patient information leaflet that typically has information relating to
missed doses. Remind them that you or the pharmacist on duty for questions, and
they may see you or ring you during opening times. o Questions like this will depend on the medicine being taken. Explaining the rationale
behind dosage regime can alleviate concerns. Where available, consider offering a
Medicine Use Review (MUR) where more detailed discussions are warranted.
“I’m finding my medicines difficult to take?” o Try to understand the reason it is difficult to take, consider whether it is mechanical
(e.g. are they unable to open the packaging), a scheduling issue (i.e. are they
struggling to access their medicines at night) or physical (e.g. is the medicine simply
too large to swallow). Explore intentional non-adherence issues too i.e. aversion to
taking medicines. 2. Diversity and Disadvantage Emerging evidence suggests that patients from marginalised groups may have greater un-met palliative and
end-of-life care needs [27]. The study outlined how community pharmacy can improve the care delivered to
people from diverse backgrounds (e.g. people who are unable to speak English may require support through a
translator or a bilingual member of pharmacy staff) [28, 29]. It is crucial that patients and family caregivers
feel supported to ensure optimum use of their medicines and avoidance of adverse effects. A range of
disadvantaged patient groups who may require additional support include:
Those with disability, including people with physical disability; visual impairment; hearing
impairments or learning impairment;
Those from Black, Asian and Minority Ethnic (BAME) communities and whose first language is not
English;
Those from Gypsy, Roma and Traveller communities;
The homeless or those with no fixed address;
Refugees or those seeking asylum;
Refugees or those seeking asylum;
Those with mental health and stigmatised medical conditions (e.g. HIV);
Those from rural communities;
Those from rural communities;
Those with alcohol/drug dependency;
Those with poor literacy [30]. 3. System complexity for patients Complexity, especially when patient care involves a large number of professionals from different services is
challenging to co-ordinate and manage. Patients sometimes felt frustration with a complex system that was
experienced as fragmented and bureaucratic [22]. Pharmacists should facilitate and offer navigational support
to sign-post to appropriate healthcare professionals or care agencies, particularly in a crisis. As patients’ health deteriorates, they gradually relinquish responsibility for medicines management, even to
the point of requiring physical assistance with administration. Pharmacists should work closely with key family
caregivers to maintain quality of care and safety of medicines and be mindful that sometimes family caregivers
can themselves be in poor health. Better communication, such as regular phone calls, between healthcare professionals and with families,
especially GPs and pharmacists, is important to enable effective continuity of care. Patients may welcome
pharmacists as the 'go to' professional whenever difficulties arise to proactively fix issues related to symptom
management and medicines use. For example, pharmacists can carry out early palliative care discussions with
patients and their family caregivers. It is important for pharmacists to develop a relationship so they have a
good rapport with them that is useful in facilitating potential future difficult discussions. 2. Diversity and Disadvantage However, currently
there is a lack of awareness of the pharmacist as a potential resource. In this case, the pharmacist should be
proactive and can periodically check the wellbeing of palliative patients and their family members by
organising routine review appointments, either in person or remotely. Conducting medication reviews could be particularly valuable during end-of-life care, where achieving
medicine optimisation may be more imperative. Pharmacists could consider conducting audits / research to
build this evidence base. Improving awareness of pharmacy support
“Where should I keep my medicines?” o Advise the patient on the appropriate location to store their medicine and add that
the patient can read the PIL for directions of a particular medicine. Display the poster in your pharmacy – this can be freely downloaded. Consider printing an A5 flyer
to be inserted in dispensed medicine bags. Be especially mindful of people from marginalised groups
who find navigating health services challenging and may experience additional barriers to access the
pharmacy for support. Improving awareness of pharmacy support Once this is understood the pharmacist can make
recommendations on how to best manage the individual issues.
“Where should I keep my medicines?” o Advise the patient on the appropriate location to store their medicine and add that
the patient can read the PIL for directions of a particular medicine. ///INSERT POSTER HERE (SEE ATTACHED FILE)/// Figure: Example of a poster that could be displayed in pharmacies and GP surgeries to facilitate access to
care. This poster was co-produced by patient-professionals from the ‘managing medicines at home’ study. It
can be downloaded and displayed in your pharmacy. Multidisciplinary collaboration Multidisciplinary collaboration The current involvement of community pharmacists in palliative and end-of-life care is predominantly with
their role in medicine supply. As this learning article demonstrates, the pharmacist could significantly
contribute and support dying patients and their families. Common barriers that contribute towards effective
inter-professional collaboration include:
limited access to medical care records;
lack of remunerations for extended services;
limited integration within the multidisciplinary team [31]. However, newer roles have promoted the potential for greater integration, for example, the ‘Macmillan
Pharmacist Project’, ‘Macmillan Pharmacy Service’, the ‘Community Macmillan Pharmacist Project’ and
‘Macmillan Rural Palliative Care Pharmacist Practitioner Project’ [32-36]. These projects demonstrate that
clinic-based, hospice and home medicine support is feasible and can help patients with complex palliative care
needs. The following approaches can help towards improved pharmacy inclusion in palliative care services:
Engage with Quality Improvement audits and review and action outcomes. For care pathways, the
aim will be to enable more efficient information exchanges between different health care
professionals engaged in patient care;
Delegate more routine work to other pharmacy team members to allow for greater clinical input for
patients with palliative care needs
Promote your pharmacy as a ‘medicines information hub’ for other healthcare professionals, patients,
and family carergivers;
Invest in wider staff training to enable them to engage more effectively with palliative care patients
and their families
Explore the role of independent prescriber pharmacists in palliative care. This could be tailored for
the appropriate and timely supply of anticipatory medicines. For a summary of best practice principles for community pharmacists in relation to palliative care, see Box 3. ////Box 3: Best practice/// For a summary of best practice principles for community pharmacists in relation to palliative care, see Box 3. ////Box 3: Best practice/// ////Box 3: Best practice/// Community pharmacists should undertake certain responsibilities in order to ensure the best possible care for
palliative patients: Community pharmacists should undertake certain responsibilities in order to ensure the best possible care for
palliative patients: •
Act as a central point of contact and information about medicines for patients and their carers; •
Provide adherence aids (i.e. reminder charts) and patient information sheets about medicines that are
used in palliative care; •
Provide advice to patients receiving palliative care and their carers post-discharge from hospital. This
could be a useful trigger to ensure patients are aware of the pharmacist and are able to ask for advice
to manage any changes that may have been made; •
Recommend the discontinuation of long-term medicines that are not needed during palliative car •
Support the MDT about changes to medicines, providing prescribing advice and medicines
information on complex medication issues (e.g. medicines compatibility with syringe drivers). ////End box/// ////End box/// ////End box/// Response to COVID-19 This article illustrates the significant opportunities (including new avenues for remuneration) for pharmacist
involvement in palliative and end-of-life care. It highlights that pharmacists can play a key role in reducing the
burden of medicines management for patients and their families. This learning article is written at a time when
the world is struggling to overcome the effects of the COVID-19 pandemic. Rapid guidance from NICE has
already been issued, prompting changes in how anticipatory medicines are prescribed and used. To cope with
the pressures of rising demand for end-of-life care medicines and on professional time, it is suggested that
prescribers should consider the use of long acting formulations which can be administered by subcutaneous
injection, buccally, sublingually or rectally [37] and that these may need to be administered by family
members. This proposal will likely result in extending the role and responsibility of FCGs in administering end
of life care medicines in relation to the type of drug and method of delivery. It is the pharmacist’s
responsibility to advise on the correct use of these medicines and to adopt strategies that ensure effective
counselling can be undertaken where people are in isolation (i.e. via remote consultations) and to those who
are from marginalized groups who may find accessing pharmacy service more challenging [38]. References 1. National Institute for Clinical Excellence. Guidance on cancer services: improving supportive and
palliative care for adults with cancer. The manual. London: NICE, 2004. 2. Connor SR, Bermedo MCS. Global atlas of palliative care at the end of life, Worldwide hospice
palliative care alliance. 2014. http://www.thewhpca.org/resources/global-atlas-on-end-of-life-care. [Accessed 04th Nov 2019]. 3. Age UK. Briefing: Health and care of older people in England 2017. Age UK: London, UK. 2017 Aug. Found at: https://www.ageuk.org.uk/Documents/EN-GB/For-
professionals/Research/The_Health_and_Care_of_Older_People_in_England_2016.pdf?dtrk=true
[Accessed 10th Oct 2019]. 4. Etkind SN, Bone AE, Gomes B, Lovell N, Evans CJ, Higginson IJ, Murtagh FE. How many people will
need palliative care in 2040? Past trends, future projections and implications for services. BMC
medicine. 2017 Dec;15(1):102. 5. Department of Health. End of life care strategy—Promoting high quality care for all adults at the end
of life Department of Health, London. (2008) 6. Gomes B, Calanzani N, Higginson IJ. Local preferences and place of death in regions within Englan
2010. London: Cicely Saunders International. 2011. Available at: 6. Gomes B, Calanzani N, Higginson IJ. Local preferences and place of death in regions within England
2010. London: Cicely Saunders International. 2011. Available at:
https://www.palliativecarescotland.org.uk/content/publications/Local-preferences-and-place-of-
death-in-regions-within-England-2010.pdf [Accessed 10th Oct 2019]. 7. Pollock K. Is home always the best and preferred place of death? BMJ 2015;351:h4855. doi:
10.1136/bmj.h4855 8. Alderwick H, Dixon J. The NHS long term plan, 2019. 9. https://www.pharmaceutical-journal.com/learning/learning-article/communicating-with-palliative-
care-patients-nearing-the-end-of-life-their-families-and-carers/20202154.article 10. Department of Health. End of life care strategy: promoting high quality care for all adults at the end of
life. London: Department of Health; 2008. Available at:
https://www.gov.uk/government/uploads/system/uploads/attachment_data/file/136431/End_of_lif
e_strategy.pdf 11. https://www.pharmaceutical-journal.com/cpd-and-learning/learning-article/facilitating-anticipatory-
prescribing-in-end-of-life-care/20202703.article 12. O'Connor M, Pugh J, Jiwa M, Hughes J, Fisher C. The palliative care interdisciplinary team: where is the
community pharmacist? Journal of Palliative Medicine 2011;14(1)7-11. 13. Wilcock A. Pre-emptive prescribing in the community. Palliative Care Formulary (PCF4). 2011. 14. National Institute for Clinical Excellence. Care of dying adults in the last days of life. NICE guideline
[NG31]. 2015 Dec 16. 14. National Institute for Clinical Excellence. Care of dying adults in the last days of life. NICE guideline
[NG31]. 2015 Dec 16. 15. British Medical Association. Focus on anticipatory prescribing for end-of-life care. General
Practitioners Committee guidance. April 2012. Available
at: https://www.bma.org.uk/advice/employment/gp-practices/service-provision/prescribing/focus-
on-anticipatory-prescribing-for-end-of-life-care 15. British Medical Association. Focus on anticipatory prescribing for end-of-life care. General
Practitioners Committee guidance. April 2012. Available
at: https://www.bma.org.uk/advice/employment/gp-practices/service-provision/prescribing/focus-
on-anticipatory-prescribing-for-end-of-life-care 16. The Gold Standards Framework. Examples of good practice resource guide—Just in case boxes. August 2006. References Available at: http://www.goldstandardsframework.org.uk/cd-
content/uploads/files/Library%2C%20Tools%20%26%20resources/ExamplesOfGoodPracticeResource
GuideJustInCaseBoxes.pdf 17. Latif A, 2019. Medicines Management: The core of pharmacy practice. In: Zaheer-Ud-Din Babar, ed.,
Encyclopedia of Pharmacy Practice and Clinical Pharmacy Academic Press. Ch. 701. 18. National Institute for Health and Care Excellence. Medicines optimisation: the safe and effective u
of medicines to enable the best possible outcomes. NG5. 2015. 18. National Institute for Health and Care Excellence. Medicines optimisation: the safe and effective use
of medicines to enable the best possible outcomes. NG5. 2015. 9. Boyd M. RPS guidance on ethical, professional decision making in the Covid-19 Pandemic. Available
at: 19. Boyd M. RPS guidance on ethical, professional decision making in the Covid-19 Pandemic. Available
at:
https://www.rpharms.com/Portals/0/RPS%20document%20library/Open%20access/Coronavirus/002
39%20001a%202004%20COVID19%20Ethical%20guide%20document%20WEB.pdf?ver=2020-04-09-
111739-353 Accessed [17th Apr 2020] 0. Council GP. Medicine Ethics and Practice. The professional guide for pharmacists. Edition. 2019 21. Picton C, Wright H. Medicines optimisation: helping patients to make the most of medicines. Good
practice guidance for healthcare professionals in England. Royal Pharmaceutical Society. 2013 May. 21. Picton C, Wright H. Medicines optimisation: helping patients to make the most of medicines. Good
practice guidance for healthcare professionals in England. Royal Pharmaceutical Society. 2013 May. 22. Wilson E, Caswell G, Latif A, Anderson C, Faull C, Pollock K. An exploration of the experiences of
professionals supporting patients approaching the end of life in medicines management at home. A
qualitative study. BMC Palliative Care [in press] 22. Wilson E, Caswell G, Latif A, Anderson C, Faull C, Pollock K. An exploration of the experiences of
professionals supporting patients approaching the end of life in medicines management at home. A
qualitative study. BMC Palliative Care [in press] 23. John C. The changing role of the pharmacist in the 21st century. The Pharmaceutical Journal 2018. Available at: https://www.pharmaceutical-journal.com/your-rps/the-changing-role-of-the-
pharmacist-in-the-21st-century/20204131.article [Accessed 10th Oct 2019]. 23. John C. The changing role of the pharmacist in the 21st century. The Pharmaceutical Journal 2018. Available at: https://www.pharmaceutical-journal.com/your-rps/the-changing-role-of-the-
pharmacist-in-the-21st-century/20204131.article [Accessed 10th Oct 2019]. 4. https://www.pharmaceutical-journal.com/cpd-and-learning/cpd-article/polypharmacy-a-framework
for-theory-and-practice/20207246.cpdarticle 24. https://www.pharmaceutical-journal.com/cpd-and-learning/cpd-article/polypharmacy-a-framework-
for-theory-and-practice/20207246.cpdarticle 5. https://www.pharmaceutical-journal.com/cpd-and-learning/cpd-article/polypharmacy-putting-the-
framework-into-practice/20207368.cpdarticle 6. Elliott RA. Appropriate use of dose administration aids. Aust Prescr. 2014 Apr 1;37(2):46-50. 27. Muslim Council of Britain, Elderly & End of Life Care for Muslims in the UK. 2019 Available at
https://mcb.org.uk/wp-content/uploads/2019/08/MCB_ELC_Web.pdf [Accessed 10th Oct 2019]. 27. Muslim Council of Britain, Elderly & End of Life Care for Muslims in the UK. 2019 Available at
https://mcb.org.uk/wp-content/uploads/2019/08/MCB_ELC_Web.pdf [Accessed 10th Oct 2019]. 28. References Latif A, Waring J, Chen LC, Pollock K, Solomon J, Gulzar N, Gulzar S, Anderson E, Choudhary S, Abbasi
N, Wharrad HJ. Supporting the provision of pharmacy medication reviews to marginalised (medically
underserved) groups: a before/after questionnaire study investigating the impact of a patient–
professional co-produced digital educational intervention. BMJ open. 2019 Sep 1;9(9):e031548. 28. Latif A, Waring J, Chen LC, Pollock K, Solomon J, Gulzar N, Gulzar S, Anderson E, Choudhary S, Abbasi
N, Wharrad HJ. Supporting the provision of pharmacy medication reviews to marginalised (medically
underserved) groups: a before/after questionnaire study investigating the impact of a patient–
professional co-produced digital educational intervention. BMJ open. 2019 Sep 1;9(9):e031548. 29. Latif A, Waring J, Pollock K, Solomon J, Gulzar N, Choudhary S, Anderson C. Towards equity: a
qualitative exploration of the implementation and impact of a digital educational intervention for
pharmacy professionals in England. International journal for equity in health. 2019 Dec 1;18(1):151. 29. Latif A, Waring J, Pollock K, Solomon J, Gulzar N, Choudhary S, Anderson C. Towards equity: a
qualitative exploration of the implementation and impact of a digital educational intervention for
pharmacy professionals in England. International journal for equity in health. 2019 Dec 1;18(1):151. 30. Latif A, Tariq S, Abbasi N, Mandane B. Giving voice to the medically under-served: a qualitative co-
production approach to explore patient medicine experiences and improve services to marginalized
communities. Pharmacy. 2018 Mar;6(1):13. 30. Latif A, Tariq S, Abbasi N, Mandane B. Giving voice to the medically under-served: a qualitative co-
production approach to explore patient medicine experiences and improve services to marginalized
communities. Pharmacy. 2018 Mar;6(1):13. 31. O'Connor M, Pugh J, Jiwa M, Hughes J, Fisher C. The palliative care interdisciplinary team: where is the
community pharmacist? Journal of Palliative Medicine. 2011;14(1):7-11. 32. Akram G, Bennie M, McKellar S, Michels S, Hudson S, Trundle J. Effective delivery of pharmaceutical
palliative care: challenges in the community pharmacy setting. Journal of palliative medicine. 2012
Mar 1;15(3):317-21. 33. Bennie M, Akram G, Corcoran ED, Maxwell D, Trundle J, Afzal N. Macmillan Pharmacist Facilitator
Project-Final Evaluation Report. Strathclyde Institute of Pharmacy and Biomedical Sciences: NHS
GG&C. 2012. 34. Bennie M, McCusker C, Harris E, Akram G, Corcoran E. Macmillan Pharmacy Service 2015: Evaluation
of Impact of Community Pharmacy Palliative Care Training Programme. 5. Mellor K. The community Macmillan pharmacist project. Prescriber. 2014 Oct 5;25(19):32-5. 36. Akram G, Corcoran E, MacRobbie A, Harrington G, Bennie M. References Developing a model for pharmaceutical
palliative care in rural areas—experience from Scotland. Pharmacy. 2017 Mar;5(1):6. 37. NICE. COVID-19 rapid guideline: managing symptoms (including at the end of life) in the community
[NG163]. 3 Apr 2020. Available at https://www.nice.org.uk/guidance/NG163 [Accessed 22nd April
2020]. 38. Latif A, Mandane B, Ali A, Ghumra S, Gulzar N. A Qualitative Exploration to Understand Access to
Pharmacy Medication Reviews: Views from Marginalized Patient Groups. Pharmacy 2020, 8, 73.
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rsc.li/professional-development Registered charity number: 207890 Showcasing research at the Department of Chemical
Metals Science at the Max Plank Institute for Chemical
Physics of Solids in Dresden. Ordering by cation replacement in the system Na 2− x Li x Ga 7
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COMMUNICATION
Yuto Suga and Yusuke Sunada
Reduction-induced hapticity increase in a silacycle-bridged
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Elevated Phospholipase A2 Activities in Plasma Samples from Multiple Cancers
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Abstract Only in recent years have phospholipase A2 enzymes (PLA2s) emerged as cancer targets. In this work, we report the first
detection of elevated PLA2 activities in plasma from patients with colorectal, lung, pancreatic, and bladder cancers as
compared to healthy controls. Independent sets of clinical plasma samples were obtained from two different sites. The first
set was from patients with colorectal cancer (CRC; n = 38) and healthy controls (n = 77). The second set was from patients
with lung (n = 95), bladder (n = 31), or pancreatic cancers (n = 38), and healthy controls (n = 79). PLA2 activities were analyzed
by a validated quantitative fluorescent assay method and subtype PLA2 activities were defined in the presence of selective
inhibitors. The natural PLA2 activity, as well as each subtype of PLA2 activity was elevated in each cancer group as compared
to healthy controls. PLA2 activities were increased in late stage vs. early stage cases in CRC. PLA2 activities were not
influenced by sex, smoking, alcohol consumption, or body-mass index (BMI). Samples from the two independent sites
confirmed the results. Plasma PLA2 activities had approximately 70% specificity and sensitivity to detect cancer. The marker
and targeting values of PLA2 activity have been suggested. Citation: Cai H, Chiorean EG, Chiorean MV, Rex DK, Robb BW, et al. (2013) Elevated Phospholipase A2 Activities in Plasma Samples from Multiple Cancers. PLoS
ONE 8(2): e57081. doi:10.1371/journal.pone.0057081 Editor: Mohammad O. Hoque, Johns Hopkins University, United States of America Editor: Mohammad O. Hoque, Johns Hopkins University, United States of America Received September 27, 2012; Accepted January 17, 2013; Published February 22, 2013 Received September 27, 2012; Accepted January 17, 2013; Published February 22, 2013 Copyright: 2013 Cai et al. This is an open-access article distributed under the terms of the Creative Commons Attribution
use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: 2013 Cai 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 is supported in part by the Cancer Care Engineering Project, Department of Defense, USAMRMC, W81XWH-08-1-0065 and W81XWH-10-1-
0540 (http://www.grants.gov/search/search.do?mode = AGENCYSEARCH&agency = DOD), as well as National Institutes of Health R21 CA133744 (http://www. cancer.gov/researchandfunding) and the Mary Fendrich Hulman Charitable Trust to YX. Abstract Funding for the Hoosier Oncology Group Study was provided by the
Walther Cancer Institute Foundation, Indianapolis, IN. 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. The authors have filled a PCT Patent Application ‘‘PLA2 Activity as a Marker
for Ovarian and Other Gynecologic Cancers’’ in 2011. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials. * E-mail: xu2@iupui.edu * E-mail: xu2@iupui.edu Elevated Phospholipase A2 Activities in Plasma Samples
from Multiple Cancers Hui Cai1,2, Elena G. Chiorean3, Michael V. Chiorean3, Douglas K. Rex3, Bruce W. Robb3, Noah M. Hahn3,4,
Ziyue Liu5, Patrick J. Loehrer3, Marietta L. Harrison6, Yan Xu1* 1 Department of Obstetrics and Gynecology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America, 2 Department of Thoracic
Oncosurgery, First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, People’s Republic of China, 3 Department of Medicine, Indiana University Melvin and Bren Simon
Cancer Center, Indiana University School of Medicine, Indianapolis, Indiana, United States of America, 4 Hoosier Oncology Group, Indianapolis, Indiana, United States of
America, 5 Department of Biostatistics, Indiana University School of Medicine, Indianapolis, Indiana, United States of America, 6 Medicinal Chemistry and Molecular
Pharmacology, Oncological Sciences Center, Purdue University Center for Cancer Research, West Lafayette, Indiana, United States of America Introduction Demographic data for participants from IUSM and HOG study. Age (years)
Sex (%)
Race (%)
No. Mean
SD
P value
Male
Female
P value
White
African
American
Others
P value
Participants from IUSM
Healthy
77
51.6
13.5
40.3
59.7
89.6
9.1
1.3
CRC
38
56.0
13.4
0.1075
44.7
55.3
0.6470
86.8
5.3
7.9
0.4501
Participants from HOG
Healthy
79
42.7
12.0
77.2
22.8
89.3
2.7
8.0
LC
95
63.2
9.5
,0.0001
64.2
35.8
0.0621
89.5
8.4
2.1
0.0654
BC
31
70.5
10.0
,0.0001
87.1
12.9
0.2438
93.6
6.5
0.0
0.1868
PC
38
68.7
8.7
,0.0001
63.2
36.8
0.1102
92.1
5.3
2.6
0.4327
CRC: colorectal cancer; LC: Lung cancer; BC: Bladder cancer; PC: Pancreatic cancer; SD, standard deviation; HOG: Hoosier Oncology Group. In the HOG set: N
For gender, the overall P value is 0.0339. N
For Age, the overall P value is ,0.0001. N
For race, the overall P value is 0.2352. Since it was not
significant, it was not recommended to perform pair-wise comparisons. Nonetheless, P values for pair-wise comparisons are given to be consistent with the other two
variables. All pair-wise comparisons are against healthy controls. doi:10.1371/journal.pone.0057081.t001 CRC: colorectal cancer; LC: Lung cancer; BC: Bladder cancer; PC: Pancreatic cancer; SD, standard deviation; HOG: Hoosier Oncology Group. In the HOG set: N
For gender, the overall P value is 0.0339. N
For Age, the overall P value is ,0.0001. N
For race, the overall P value is 0.2352. Since it was not
significant, it was not recommended to perform pair-wise comparisons. Nonetheless, P values for pair-wise comparisons are given to be consistent with the other two
variables. All pair-wise comparisons are against healthy controls. doi:10.1371/journal.pone.0057081.t001 138’’. Blood samples were centrifuged at 3,500 rpm for 30 min. The aliquoted samples were stored at 270uC. HOG is a non-
profit medical research organization. Although it is a separate
entity, IUSM is a supporting organization for board appointments
and IRB approvals for HOG. Three separate IRB protocols for
collecting and/or use the blood samples related to this work have
been approved by the same IUSM Institutional Review Board
(Protocol numbers: 0670-81, 0808-24, 0905-20). Written informed
consent forms were obtained from all subjects and all clinical
investigation had been conducted according to the principles
expressed in the Declaration of Helsinki. PLA2 (Lp-PLA2). In cancer, most of the attention has been focused
on sPLA2 and cPLA2 [8]. Reagents and inhibitors In the current work we have focused on PLA2 activities rather
than expression of individual PLA2 enzymes and examined PLA2
activities in blood plasma samples from patients with different
cancers, in comparison with those from healthy controls. We used
our recently validated quantitative, convenient, highly reproduc-
ible PLA2 assay method [16] and showed for the first time that
plasma PLA2 activities from patients with CRC, LC, PC, and
bladder cancer (BC) were significantly higher than those of healthy
controls. In addition, PLA2 activities were correlated with tumor
stages in CRC. Other potential influential factors, including sex,
age, smoking and alcohol consumption were also examined in this
study. The PLA2 substrate 1-O-(6-Dabcyl-Aminohexanoyl)-2-O-(6-
(12-BODIPY-Dodecanoyl) Aminohexanoyl)-sn-3-Glyceryl Phos-
phatidylcholine (DBPC) was from Echelon Bioscience (Salt Lake
City, UT, USA). Bromoenol lactone (BEL) and methyl arachido-
nylfluorophosphonate (MAFP) were from Santa Cruz Biotechnol-
ogy (Santa Cruz, CA, USA). PLA2 enzymatic activity analyses PLA2 activities were analyzed using the fluorescent substrate
DBPC, a fluorogenic phosphatidylcholine substrate [17]. Plasma
samples (0.1 mL) were mixed with DBPC (0.2 mg in PBS) to final
volume 200 mL. The fluorescence was read at intervals over
several hours on a Victor3V plate reader (Perkin Elmer, Waltham,
MA,
USA). PLA2
activities
were
expressed
as
change
in
fluorescence intensity/min/mL of plasma. As previously described
[16], we selected conditions to distinguish PLA2 activity derived
from different subtypes: a) the ‘‘natural’’ PLA2 activity without any
exogenous additives, b) the iPLA2 activity in the presence of 5 mM
EDTA (a divalent cation chelator to block all PLA2s requiring
calcium, including sPLA2 and cPLA2), c) the sPLA2 activity in the
presence of 1.2 mM calcium chloride (the natural ionized calcium
concentration in blood [18]) and MAFP (10 mM, a dual inhibitor
of cPLA2 and iPLA2), and d) the cPLA2 activity in the presence of
100 mM calcium chloride and bromoenol lactone (BEL, 10 mM, a
selective inhibitor for iPLA2). Introduction We and others have shown that iPLA2 is
functionally involved in promoting the development of ovarian
cancer (OC) and other cancers, in vitro and in vivo [9–13]. sPLA2
and Lp-PLA2 are secreted enzymes. In contrast, both cPLA2 and
iPLA2 are cytosolic enzymes and their extracellular existence has
only been shown to be related to exosomes from RBL-2H3 cells (a
mast and basophil cell line) [14]. Exosomes are 40–100 nm
diameter membrane vesicles released from multivesicular bodies
by intact cells and are known to participate in intercellular
signaling [15]. We have recently detected extracellular- and
exosome-free iPLA2 and cPLA2 activities in ascites and tissues
from OC patients [16]. Introduction discovery of lung nodules, many of which are falsely positive, but
still calling for additional and sometimes painful examinations [5]. Molecular approaches to cancer diagnosis through biomarkers
measured by non-invasive means could significantly improve the
specificity and sensitivity of cancer detection [5]. In particular,
serologic biomarkers may be the most useful in early detection due
to the minimal invasiveness, cost-effectiveness, and convenience. Moreover, serological biomarkers identifying novel cancer-related
genes and/or molecules may provide new insight into the biology
of cancers and may also become attractive targets for treatment
[3]. Cancer is one of the major health burdens worldwide. More
than 1.6 million new cancer cases and over 577,000 deaths from
cancer are projected to occur in the United States in 2012 [1]. Lung cancer (LC) and colorectal cancer (CRC) are among the
most frequent types of cancers. Pancreatic and bladder cancers
count for (3–7% of all cancers), but pancreatic cancer (PC) is one
of the deadest cancers, with a dismal 5-year survival rate of 6%
[1]. The success of current cancer treatments depends strongly on
the time of diagnosis, with early detection and identification of
high risk patient populations resulting in the most favorable overall
survivals in these diseases [1,2]. It is expected that early detection
and screening of high risk patient populations may have the most
significant impact on altering overall survival in these diseases. Thus, there is an urgent need for efficient and sensitive early
detection methods [3]. While imaging-based detection methods,
including
spiral
computerized
axial
tomography
(CT)
and
colonoscopy are effective early detection methods for LC and
CRC, respectively [4], they are rather expensive and inconvenient. In addition, modern imaging techniques have a high incidence of Phospholipase A2 enzymes (PLA2s) are the major enzymes
producing the cyclooxygenase-2 (COX-2) substrate, arachidonic
acid (AA), as well as lysophospholipids. Both of these classes of
products are signaling molecules involved in cancers. However,
only in recent years have PLA2s emerged as cancer targets [6]. More than 30 enzymes that possess PLA2 or related activity have
been identified in mammals [7]. They are divided into four groups
based on their cellular localization, substrate specificity, and
calcium-dependence [8], including cytosolic (cPLA2), calcium-
independent (iPLA2), secreted (sPLA2), and lipoprotein-associated February 2013 | Volume 8 | Issue 2 | e57081 PLOS ONE | www.plosone.org 1 February 2013 | Volume 8 | Issue 2 | e57081 Elevation of Plasma PLA2 Activity in Cancers Table 1. Human sample collection and processing The mean values of PLA2 activities were presented by the ‘‘+’’ in the figure. *P,0.05; **P,0.01;
***P,0.001. doi:10.1371/journal.pone.0057081.g001 Figure 1. Plasma PLA2 activities were elevated in cancer groups vs. healthy controls. Comparison of PLA2 activities in the healthy control
(normal colonoscopy; n = 77) and CRC (n = 38) groups. A. Comparison of PLA2 activities in the healthy (n = 79) and other cancer groups. Lung cancer
(LC, n = 95), bladder cancer (BC, n = 31) and pancreatic cancer (PC, n = 38). The distribution of the natural and individual groups of PLA2 activities were
analyzed as described in Materials and Methods. The mean values of PLA2 activities were presented by the ‘‘+’’ in the figure. *P,0.05; **P,0.01;
***P,0.001. doi:10.1371/journal.pone.0057081.g001 doi:10.1371/journal.pone.0057081.g001 between disease statuses and categorical covariates such as sex,
race, smoking, and alcohol drinking. For continuous covariates
such as age and BMI, one-way ANOVA was used to test the
overall difference and Student t-test was used to test the pair-wise
difference across disease statuses. Linear regression was used to
assess the univariate association between PLA2 measurements and Human sample collection and processing Human sample collection and processing p
p
g
Two sets of independently collected clinical plasma samples
were obtained for this study. The first set from the Indiana
University School of Medicine (IUSM) focused on CRC patients
and healthy controls screened by colonoscopy and found negative
for adenomatous polyps and CRC. Blood samples collected in the
presence of EDTA were centrifuged at 1,750 g for 15 min at 15–
24uC, aliquoted in siliconized Eppendorf tubes and stored at
280uC. The second set of samples was from patients with lung,
bladder, or pancreatic cancers, as well as healthy controls. These
samples were collected by the Hoosier Oncology Group (HOG) in
Indianapolis, IN as part of the study entitled, ‘‘A Biological Sample
Collection Protocol of Patients with and without Metastatic Solid
Organ Malignancies: Hoosier Oncology Group Study BANK09- February 2013 | Volume 8 | Issue 2 | e57081 PLOS ONE | www.plosone.org 2 Elevation of Plasma PLA2 Activity in Cancers Statistical Analysis
Categorical variables were summarized as counts with percent-
ages and continuous variables were summarized as means with
between disease statuses and categorical covariates such as se
race, smoking, and alcohol drinking. For continuous covariat
such as age and BMI one-way ANOVA was used to test t
Figure 1. Plasma PLA2 activities were elevated in cancer groups vs. healthy controls. Comparison of PLA2 activities in the healthy cont
(normal colonoscopy; n = 77) and CRC (n = 38) groups. A. Comparison of PLA2 activities in the healthy (n = 79) and other cancer groups. Lung canc
(LC, n = 95), bladder cancer (BC, n = 31) and pancreatic cancer (PC, n = 38). The distribution of the natural and individual groups of PLA2 activities we
analyzed as described in Materials and Methods. The mean values of PLA2 activities were presented by the ‘‘+’’ in the figure. *P,0.05; **P,0.0
***P,0.001. doi:10.1371/journal.pone.0057081.g001 Figure 1. Plasma PLA2 activities were elevated in cancer groups vs. healthy controls. Comparison of PLA2 activities in the healthy control
(normal colonoscopy; n = 77) and CRC (n = 38) groups. A. Comparison of PLA2 activities in the healthy (n = 79) and other cancer groups. Lung cancer
(LC, n = 95), bladder cancer (BC, n = 31) and pancreatic cancer (PC, n = 38). The distribution of the natural and individual groups of PLA2 activities were
analyzed as described in Materials and Methods. The natural PLA2, cPLA2, iPLA2, and sPLA2 activities were
elevated in cancer samples vs. healthy controls Blood iPLA2 and cPLA2 activities have not been previously
reported in human samples. We analyzed the natural PLA2
(defined as the PLA2 activities measured under the conditions
without any modifiers, which may be lower than the sum of the
subgroup PLA2 activities measured under modified conditions), as
well as cPLA2, iPLA2, and sPLA2 activities in plasma samples. Except for the healthy vs. CRC cPLA2 activity, all other
comparisons showed that PLA2 activities were significantly
elevated in the cancer groups (Fig. 1A and 1B). PLA2 activity
values across plasma samples from LC, BC, and PC patients were
not significantly different (P values .0.05). Statistical Analysis Categorical variables were summarized as counts with percent-
ages and continuous variables were summarized as means with
standard deviations (SD) across the healthy control, and cancer
groups. The Chi-square test was used to test the associations PLOS ONE | www.plosone.org February 2013 | Volume 8 | Issue 2 | e57081 3 Elevation of Plasma PLA2 Activity in Cancers Figure 2. PLA2 activities were higher in late stage CRC samples. The natural and individual groups of PLA2 activities were progressively
increased by tumor stage. Stage I and II (n = 7), Stage III and IV (n = 31). *Student t-test, P,0.05. doi:10.1371/journal.pone.0057081.g002 cancer groups (Table 1). Although males were dominant in each
group, they were not significantly different from the second set of
healthy controls. 71.2% of the overall participants were male,
90.4% were white, and the mean age was 58.3 years (Table 1). cancer groups (Table 1). Although males were dominant in each
group, they were not significantly different from the second set of
healthy controls. 71.2% of the overall participants were male,
90.4% were white, and the mean age was 58.3 years (Table 1). Reproducibility and stability of the PLA2 assays p
y
y
2
y
We have recently validated the quantitative nature of the
DBPC-based PLA2 assays and optimized the conditions for
biological fluids and tissues samples [16]. When each of 8
representative plasma samples were analyzed three times, the
PLA2 activity values reproducible, with an average standard
deviation (SD) 13.5 (1.3%). We also tested the PLA2 activity
stability by comparing them in samples before storage to those in
the same samples stored at 280uC for one month, as well as the
effect of freeze-and-thaw on the PLA2 activities in 8 representative
samples. As previously reported [16], PLA2 activities were stable
with SDs ,35.8 (5.8%). In addition, we showed that in the
presence of all other assay components including inhibitor(s) used
in this study, but in the absence of blood or other biological
samples, the non-specific hydrolysis of the DBPC substrate was
negative and negligible (data not shown). Figure 2. PLA2 activities were higher in late stage CRC samples. The natural and individual groups of PLA2 activities were progressively
increased by tumor stage. Stage I and II (n = 7), Stage III and IV (n = 31). *Student t-test, P,0.05. doi:10.1371/journal.pone.0057081.g002 covariates. Logistic regression was used to evaluate the classifica-
tion performance of PLA2 measurements. Bootstrap was used to
internally validate the classification performance using 1000
samples [19]. Each sample had the same size as the original data
set and was used to develop the model. The results were then
tested in the original data. Areas under the curve (AUCs) were
calculated for each repeat. All tests were two-sided. Given the
exploratory nature of this study, no adjustments for multiple
comparisons were adopted. P values ,0.05 were considered to be
statistically significant. All analyses were performed using SAS
software Version 9.3 (SAS Institute Inc., Cary, NC, USA). The natural PLA2, cPLA2, iPLA2, and sPLA2 activities were
elevated in cancer samples vs. healthy controls February 2013 | Volume 8 | Issue 2 | e57081 CT: chemotherapy; RT: radiotherapy. Subject demographic data The demographic data for the study participants are summa-
rized in Table 1. For the IUSM CRC set samples, 41.7% of the
overall participants were male, 88.7% were white, and the mean
age was 53.1 years. The age, sex, and race composition were not
significantly different in control vs. CRC groups (P values .0.05;
Table 1). For the second set of samples, the age in the healthy
control group was significantly younger than each of the three Since sPLA2 is secreted, it was expected that sPLA2 activity
would account for the major portion of the PLA2 activity detected
in the blood. However, in our results, high levels of iPLA2 and
cPLA2 and low levels of sPLA2 activities were observed in all
plasma samples. Blood exosomes may be the carriers of these
activities [15]. We used differential centrifugation steps as we
described recently [16] to determine whether these activities could Table 2. Clinical characteristics and PLA2 activities in the CRC cases. Table 2. Clinical characteristics and PLA2 activities in the CRC cases. PLA2
iPLA2
cPLA2
sPLA2
No. Mean
SD
P value
Mean
SD
P value
Mean
SD
P value
Mean
SD
P value
Primary site
0.6856*
0.7407*
0.6419*
0.3410*
Colon
24
1412.3
473.75
1302.8
428.78
1424.0
480.16
476.3
199.33
Rectum
14
1348.0
460.33
1254.7
428.33
1347.7
489.95
408.3
195.50
Previous treatment
0.6543**
0.3081**
0.3348**
0.6212**
Surgery only
7
1340.3
382.29
1193.1
286.55
1303.4
424.30
435.5
92.75
CT and RT
3
1614.8
542.80
1599.5
626.64
1755.4
707.50
495.9
219.34
Surgery and CT
10
1496.8
596.54
1399.5
543.97
1521.1
633.55
519.8
264.44
Surgery, CT and RT
5
1459.0
437.46
1402.9
322.07
1511.8
322.27
472.8
200.95
Untreated
13
1252.2
404.71
1128.6
341.99
1221.8
327.50
388.5
183.68
*Student t-test. **One-way ANOVA. CT: chemotherapy; RT: radiotherapy. doi:10.1371/journal.pone.0057081.t002
PLOS ONE | www.plosone.org
4
February 2013 | Volume 8 | Issue 2 | e57081 February 2013 | Volume 8 | Issue 2 | e57081 PLOS ONE | www.plosone.org Elevation of Plasma PLA2 Activity in Cancers Table 3. Clinical characteristics and PLA2 activities in the LC, BC, and PC cases. PLA2
iPLA2
cPLA2
sPLA2
No. Subject demographic data Mean
SD
P value
Mean
SD
P value
Mean
SD
P value
Mean
SD
P value
Lung
cancer
T stage
0.4760**
0.5001**
0.4960**
0.6129**
T1
19
1714.2
548.5
1626.6
514.2
2190.2
752.6
457.1
280.7
T2
21
1569.3
671.8
1490.6
640.6
1924.1
754.0
478.7
261.4
T3
9
1437.9
479.0
1347.4
460.7
1721.3
480.5
356.2
166.6
T4
35
1803.5
878.2
1691.8
823.9
2141.7
1105.9
471.5
228.4
N stage
0.9973**
0.9685**
0.9758**
0.8009**
N0
14
1626.7
566.2
1559.5
522.6
2061.3
584.4
386.3
143.2
N1
11
1574.8
508.1
1442.7
479.8
1960.3
671.2
404.1
200.4
N2
21
1613.9
643.7
1524.1
631.4
1961.6
883.6
434.1
222.9
N3
29
1604.2
671.7
1510.2
620.6
1944.3
888.0
454.8
270.0
Bladder
cancer
T stage
0.7482**
0.7320**
0.5790**
0.1793**
Tis
2
1736.6
429.9
1568.4
406.3
2046.9
355.0
395.2
119.1
T1
4
1326.0
525.4
1306.3
517.3
1717.4
525.6
299.2
222.9
T2
10
1833.5
832.3
1710.5
759.0
2207.7
1074.9
451.0
160.6
T3
7
1812.3
522.3
1833.4
506.3
2541.3
630.6
444.4
141.7
T4
8
1800.7
582.9
1630.9
544.2
2098.4
638.9
596.2
263.3
N stage
0.8224**
0.7579**
0.7786**
0.1010**
N0
10
1665.1
479.6
1535.2
446.2
2158.4
619.1
436.1
168.2
N1
5
1602.0
482.3
1549.8
507.6
1872.4
726.4
411.2
173.2
N2
8
1813.4
964.3
1806.0
881.1
2333.5
1107.6
400.4
180.1
N3
5
1937.5
428.0
1761.7
394.4
2135.4
234.1
671.9
287.7
Pancreatic
cancer
T stage
0.7748**
0.8479**
0.8488**
0.8453**
T1
1
1398.3
1294.2
1400.7
419.7
T2
12
1823.2
708.6
1730.9
647.8
2413.7
897.1
476.3
139.5
T3
8
2031.6
1477.7
1891.1
1421.5
2693.9
2009.0
556.0
286.2
T4
15
2279.0
1509.8
2077.8
1423.9
2741.7
1919.3
595.7
506.3
N stage
0.1928*
0.1448*
0.1243*
0.0671*
N0
9
2663.9
1855.8
2564.3
1749.6
3475.3
2290.2
699.2
377.1
N1
19
1756.4
837.8
1598.2
738.6
2133.6
964.7
428.6
152.1
*Student t-test. **One-way ANOVA. d
l Clinical parameters and PLA2 activities in cancer plasma
samples be associated with platelets, exosomes, or other microvesicles. Two
blood samples were first centrifuged at 1,750 g for 15 min to
remove most blood cells and the supernatant was termed S1. S1
was centrifuged at 20,000 g for 20 min, which resulted in S2 and
the pellet 2 (P2; cell fragments and large vesicles). S2 was
ultracentrifuged at 110,000 g for 2 hr, which resulted in S3 and P3
(exosomes). A final centrifugation of S3 at 200,000 g for 2 hr
resulted in S4 and P4 (other microvesicles). Subject demographic data We found that all
PLA2 activities were retained in the S1 through S4 fractions,
suggesting they were ‘‘free’’ and not associated with microvesicles
(detailed data not shown). This is similar to what we have recently
reported in human ovarian cancer ascites and novel secretion
mechanisms for iPLA2s and cPLA2s may be involved [16]. be associated with platelets, exosomes, or other microvesicles. Two
blood samples were first centrifuged at 1,750 g for 15 min to
remove most blood cells and the supernatant was termed S1. S1
was centrifuged at 20,000 g for 20 min, which resulted in S2 and
the pellet 2 (P2; cell fragments and large vesicles). S2 was
ultracentrifuged at 110,000 g for 2 hr, which resulted in S3 and P3
(exosomes). A final centrifugation of S3 at 200,000 g for 2 hr
resulted in S4 and P4 (other microvesicles). We found that all
PLA2 activities were retained in the S1 through S4 fractions,
suggesting they were ‘‘free’’ and not associated with microvesicles
(detailed data not shown). This is similar to what we have recently
reported in human ovarian cancer ascites and novel secretion
mechanisms for iPLA2s and cPLA2s may be involved [16]. Interestingly, the natural, iPLA2, and sPLA2 activities in plasma
of CRC patients were increased in subjects with late stages (III and
IV) as compared to earlier stage (I and II) disease (P = 0.0335,
0.0367, 0.0778, and 0.0345 for PLA2, iPLA2, cPLA2, and sPLA2,
respectively; Fig. 2), suggesting that these enzymatic activities may
be involved in CRC progression. We compared the PLA2 activities
in colon (n = 24) vs. rectal (n = 14) samples. Even though the
nature, as well the sub-group PLA2 activities in colon cancer
patients were higher than those from rectal cancer patients, the
differences were not statistically significant (P.0.05 in all cases;
Table 2). In addition, no difference was found in PLA2 activities February 2013 | Volume 8 | Issue 2 | e57081 5 PLOS ONE | www.plosone.org February 2013 | Volume 8 | Issue 2 | e57081 Elevation of Plasma PLA2 Activity in Cancers Figure 3. The influence of smoking on plasma PLA2 activities. PLA2 activities in healthy and CRC samples with different smoking
statuses(a)and second hand smoking statuses(b). A. Comparison of PLA2 activities between different smoking statuses(a) and second hand smoking
statuses(b) in participants with LC, BC, and PC. doi:10.1371/journal.pone.0057081.g003 Figure 3. The influence of smoking on plasma PLA2 activities. Subject demographic data PLA2 activities in healthy and CRC samples with different smoking
statuses(a)and second hand smoking statuses(b). A. Comparison of PLA2 activities between different smoking statuses(a) and second hand smoking
statuses(b) in participants with LC, BC, and PC. doi:10.1371/journal.pone.0057081.g003 Table 4. Smoking, alcohol consumption, and the BMI status
of participants from IUSM*. Healthy
CRC
No. %
No. %
P value
Smoking status
Current smoker 2
3.8
1
3.7
0.4609
Past smoker
18
34.0
13
48.2
Never smoked
33
62.3
13
48.2
Second hand
smoke
0.2185
No
48
90.6
21
80.8
Yes
5
9.4
5
19.2
Alcohol
0.2542
No
6
11.3
1
3.7
Yes
47
88.4
26
96.3
BMI (Mean 6 SD) 28.266.96
25.464.01
0.0272
*These data are available from a subset of the participants. doi:10.1371/journal.pone.0057081.t004 among different subgroups of CRC patients with previous
treatments (surgery, chemotherapy, radiotherapy, or untreated;
Table 2). In contrast, we found that PLA2 activities were not
correlated to either T or N stages of LC, BC, or PC (Table 3). Table 4. Smoking, alcohol consumption, and the BMI status
of participants from IUSM*. Table 4. Smoking, alcohol consumption, and the BMI status
of participants from IUSM*. The classification performance of plasma PLA2 activities The classification performance of plasma PLA2 activities supporting the concept obtained from the first set of study that
plasma PLA2 activities are not affected by smoking (Fig. 3B). Logistic regression was used to evaluate the classification
performance of PLA2 activities in cancers. All four PLA2
measurements
were
kept
as
predictors
regardless
of
their
significance. The classification performances were summarized
by receiver operating characteristic (ROC) curves (Fig. 8). Prediction formulas were generated using the parameter estimates. Alcohol consumption in the control and CRC groups were not
significantly different (Table 4). The alcohol consumers in the
CRC groups tend to have higher levels of plasma PLA2 activities,
but the differences were not statistically significant (Fig. 4). However, due to the low numbers in non-alcohol consumption
group (n = 7), we should interpret these data with caution. For
other cancers, similar results were obtained. Although there were
significant higher percentages of alcohol consumption subjects in
other cancer groups (Table 5), the PLA2 activities were not
significantly related to alcohol (Fig. 5). The prediction formula to separate CRC cases from healthy
subjects is: ln
PCRC
1{PCRC
~{2:7372z0:00329|PLA2{0:00286|
cPLA2z0 000362|iPLA2z0 00344|sPLA ln
PCRC
1{PCRC
~{2:7372z0:00329|PLA2{0:00286| The differences in PLA2 activities between sexes did not reach a
statistical significance in the control or any cancer groups in either
set of studies (Figs. 5A and 5B). Similarly, there was no difference
or correlation in PLA2 activities in subjects with BMI ,25 vs. BMI
$25 groups in the CRC study (Figs. 6A and 6B), although the
mean BMI in the CRC and PC groups were significantly less than
those in control groups (Tables 4 and 5). cPLA2z0:000362|iPLA2z0:00344|sPLA2 cPLA2z0:000362|iPLA2z0:00344|sPLA2 where ‘‘PCRC’’ stands for the probability of having CRC. The
ROC curve shown in Fig. 8A has an area under the curve
(AUC) = 0.7502. where ‘‘PCRC’’ stands for the probability of having CRC. The
ROC curve shown in Fig. 8A has an area under the curve
(AUC) = 0.7502. Since external validations would require an independent study,
we instead adopted internal methods to validate the classification
performances. Among different internal validation methods, it has
been shown that bootstrap outperforms jackknife, cross-validation
and data-splitting methods [19]. Therefore, bootstrap was adopted
for validation. In particular, the prediction formula was obtained
by fitting the same model for each bootstrapped sample. The
formula was applied to the original data to calculate the prediction
probabilities. Impact of other factors on PLA2 activity All P values are comparisons between the cancer group and the healthy group. doi:10.1371/journal.pone.0057081.t005 Impact of other factors on PLA2 activity We have collected other factors, which might influence PLA2
activities, including smoking, alcohol consumption, and body-mass
index (BMI). For the CRC study, from the 80 subjects with
available smoking information (27 CRC and 53 healthy cases), the
majority (96%) of people did not smoke when the samples were
collected, with an average 50.5% people never smoked and 45.7%
of past smoker, and only 9.4% and 19.2% people had second hand
smoking exposure in each group. The smoking statuses were
similarly distributed in the two groups (P values .0.05; detailed
data in Table 4) and there were no statistical differences in PLA2
activities among different smoking statuses (Fig. 3A). In the second
set of study, the smoking statuses were significantly different
between healthy controls and each of the cancer groups with
higher percentages of people who were current, past, or second-
hand smokers in cancer groups (P values ,0.05, except in one
comparison, Table 5). Nevertheless, no statistical differences were
observed in the PLA2 activities among different smoking statuses, *These data are available from a subset of the participants. doi:10.1371/journal.pone.0057081.t004 *These data are available from a subset of the participants. doi:10.1371/journal.pone.0057081.t004 February 2013 | Volume 8 | Issue 2 | e57081 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 6 Elevation of Plasma PLA2 Activity in Cancers Table 5. Smoking and alcohol consumption status of participants from HOG study. Healthy
LC
BC
PC
No. %
No. %
P value
No. %
P value
No. %
P value
Smoking status
,0.0001
0.0017
0.016
Current smoker
4
5.1
29
30.5
4
12.9
7
18.4
Past smoker
25
31.6
58
61.1
19
61.3
16
42.1
Never smoked
50
63.3
8
8.4
8
25.8
15
39.5
Second hand smoke
,0.0001
0.0091
0.0741
No
32
40.5
10
10.5
4
13.8
9
23.7
Yes
47
59.5
85
89.5
25
86.2
29
76.3
Alcohol
,0.0001
0.0018
,0.0001
No
23
29.6
67
70.5
19
61.3
31
81.6
Yes
56
70.4
28
29.5
12
38.7
7
18.4
BMI (Mean 6 SD)
28.365.55
26.566.76
0.0707
28.365.01
0.9625
25.966.17
0.0374
LC: Lung cancer; BC: Bladder cancer; PC: Pancreatic cancer. All P values are comparisons between the cancer group and the healthy group. doi:10.1371/journal.pone.0057081.t005 LC: Lung cancer; BC: Bladder cancer; PC: Pancreatic cancer. All P values are comparisons between the cancer group and the healthy group. doi:10.1371/journal.pone.0057081.t005 LC: Lung cancer; BC: Bladder cancer; PC: Pancreatic cancer. The classification performance of plasma PLA2 activities The performance for this particular bootstrap
sample was summarized by AUC of the ROC curve. The overall
performance was evaluated by summarizing AUCs across 1,000
bootstrapped samples. In 95% of the 1000 bootstrapped samples,
AUCs are higher than 0.7143 for CRC vs. healthy. Therefore,
these results internally confirm the classification performances of
the models developed above. Age is one of the most clearly identified risk factors for
developing CRC and other cancers. The American Cancer
Society and the U.S. Preventive Services Task Force recommend
that people receive colonoscopy screenings every 10 years
beginning at age 50. We thus analyzed the age effects on PLA2
activities by dividing the subjects into two groups (age ,50 years,
n = 37 and $50 years, n = 78). Although there was a trend of
increased PLA2 activities in the $50 years-old group in the CRC
group, the differences were not statistically significant (Fig. 7A). Similarly, no difference was detected between the two age groups
in the LC, BC, and PC set, although when all the subjects
(including healthy controls) were combined, there were significant
increases in PLA2 activates in the older subjects (Fig. 7B) increases in PLA2 activates in the older subjects (Fig. 7B). A direct comparison of the two sets of healthy controls showed
that although their sex, age, second-hand smoking, and alcohol
consumption were different, there were no differences in any PLA2
activity measured (Table 6). This further supports that plasma
PLA2 activities are not significantly affected by age, sex, alcohol
consumption, and/or smoking. 2
j
(
g
)
A direct comparison of the two sets of healthy controls showed
that although their sex, age, second-hand smoking, and alcohol
consumption were different, there were no differences in any PLA2
activity measured (Table 6). This further supports that plasma
PLA2 activities are not significantly affected by age, sex, alcohol
consumption, and/or smoking. The prediction formula and the ROC curve to separate all
cancer cases from healthy subjects in the second set of study are as
follows and shown in Fig. 8B, with an AUC = 0.8337. February 2013 | Volume 8 | Issue 2 | e57081 7 PLOS ONE | www.plosone.org 7 Elevation of Plasma PLA2 Activity in Cancers ln
P
1{P
~{2:6398{0:00035|PLA2{0:00169|iPLA2z
0:00311|cPLA2z0:00276|sPLA2
0.8205 for LC, PC, BC combined vs. the second set of healthy
controls. The classification performance of plasma PLA2 activities The prediction formulas and the ROC curves for the separate
LC, BC, and PC cases from healthy subjects in the second set of
study are listed below and Figs. 8C to 8E, with AUCs .0.811,
Figure 4. The influence alcohol consumption on plasma PLA2 activities. Comparison of PLA2 activities between ‘‘non-alcohol’’ and ‘‘alcohol’’
groups with the healthy and CRC participants. A. Comparison of PLA2 activities between ‘‘non-alcohol’’ and ‘‘alcohol’’ groupsin the second set of
studies with healthy, LC, BC, and PC participants. doi:10.1371/journal.pone.0057081.g004 umption on plasma PLA2 activities. Comparison of PLA2 activities between ‘‘non-alcohol’’ and ‘‘alcohol’’
pants. A. Comparison of PLA2 activities between ‘‘non-alcohol’’ and ‘‘alcohol’’ groupsin the second set of
ticipants. Figure 4. The influence alcohol consumption on plasma PLA2 activities. Comparison of PLA2 activities between ‘‘non-alcohol’’ and ‘‘alcohol’’
groups with the healthy and CRC participants. A. Comparison of PLA2 activities between ‘‘non-alcohol’’ and ‘‘alcohol’’ groupsin the second set of
studies with healthy, LC, BC, and PC participants. doi:10.1371/journal.pone.0057081.g004 0.8205 for LC, PC, BC combined vs. the second set of healthy
controls. ln
P
1{P
~{2:6398{0:00035|PLA2{0:00169|iPLA2z
0:00311|cPLA2z0:00276|sPLA2 The prediction formulas and the ROC curves for the separate
LC, BC, and PC cases from healthy subjects in the second set of
study are listed below and Figs. 8C to 8E, with AUCs .0.811,
indicating good classification performances of the tests. For LC
versus healthy control, the AUC = 0.8119 (Fig. 8C). In 95% of the
1000 bootstrapped sample, AUCs are higher than 0.7923. 0:00311|cPLA2z0:00276|sPLA2 This performance was validated in 1000 bootstrapped samples. In
95% of the 1000 bootstrapped samples, AUCs are higher than February 2013 | Volume 8 | Issue 2 | e57081 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 8 Elevation of Plasma PLA2 Activity in Cancers Figure 5. PLA2activities were not significantly different between male and female. PLA2 activity comparison in males and females in the
healthy and the CRC groups. A. PLA2 activity comparison in males and females in the healthy and other cancer groups. Data from all subjects in each
set of studies are also presented. doi:10.1371/journal.pone.0057081.g005 Figure 5. PLA2activities were not significantly different between male and female. PLA2 activity comparison in males and females in the
healthy and the CRC groups. A. PLA2 activity comparison in males and females in the healthy and other cancer groups. Data from all subjects in each
set of studies are also presented. The classification performance of plasma PLA2 activities doi:10.1371/journal.pone.0057081.g005 February 2013 | Volume 8 | Issue 2 | e57081 PLOS ONE | www.plosone.org 9 Elevation of Plasma PLA2 Activity in Cancers ln
P
1{P
~{2:9998{0:00024|PLA2{0:00136|iPLA2z
0:00278|cPLA2z0:00230|sPLA2
F
BC
h
lth
t
l th AUC =0 8624 (Fi
8D) I 95%
ln
P
1{P
~{5:3597{0:002|PLA2z0:00086|iPLA2z
0:00313|cPLA2z0:00236|sPLA2
F
PC
h
lth
t
l th
AUC= 0 8671 I
95%
f th
Figure 6. Comparison of PLA2 activities among different BMI groups. The subjected in healthy and CRC groups were divided into two
groups: BMI 18.5 to ,25 (For all subjects, n = 43) and BMI.25 (For all subjects, n = 80). A. The subjected in healthy and other cancer groups were
divided into two groups: BMI#25 (For all subjects, n = 80) and BMI.25 (For all subjects, n = 148). doi:10.1371/journal.pone.0057081.g006 Figure 6. Comparison of PLA2 activities among different BMI groups. The subjected in healthy and CRC groups were divided into two
groups: BMI 18.5 to ,25 (For all subjects, n = 43) and BMI.25 (For all subjects, n = 80). A. The subjected in healthy and other cancer groups were
divided into two groups: BMI#25 (For all subjects, n = 80) and BMI.25 (For all subjects, n = 148). doi:10.1371/journal.pone.0057081.g006 ln
P
1{P
~{5:3597{0:002|PLA2z0:00086|iPLA2z
0:00313|cPLA2z0:00236|sPLA2 ln
P
1{P
~{2:9998{0:00024|PLA2{0:00136|iPLA2z
0:00278|cPLA2z0:00230|sPLA2 0:00313|cPLA2z0:00236|sPLA2 0:00278|cPLA2z0:00230|sPLA2 For PC versus healthy control, the AUC= 0.8671. In 95% of the
1000 bootstrapped sample, AUCs are higher than 0.8426. For BC versus healthy control, the AUC =0.8624 (Fig. 8D). In 95%
of the 1000 bootstrapped sample, AUCs are higher than 0.8161. February 2013 | Volume 8 | Issue 2 | e57081 February 2013 | Volume 8 | Issue 2 | e57081 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 10 Elevation of Plasma PLA2 Activity in Cancers Finally, since we have f
Figure 7. PLA2activities were not significantly correlated to ages. The participants in healthy and CR
age,50 (For all subjects, n = 42) and age $50 (For all subjects, n = 153). Student t-test was performed to an
groups. A. The participants in healthy and other cancer groups were divided into two groups: age,50 (For
subjects, n = 144). Student t-test was performed to analyze the differences between these two groups. The classification performance of plasma PLA2 activities ***
doi:10.1371/journal.pone.0057081.g007 ln
P
1{P
~{4:6897z0:00263|PLA2{0:00411|iPLA2z
0:00263|cPLA2z0:00342|sPLA2
Finally, since we have found that the PLA2 activities were
essentially no difference in the two sets of healthy controls, we
combined all healthy controls and all cancer cases in both sets of
studies and generated a combined formula, with an AUC = 0.8011
(Fig. 8F). In 95% of the 1000 bootstrapped samples, AUCs are
Figure 7. PLA2activities were not significantly correlated to ages. The participants in healthy and CRC groups were divided into two groups:
age,50 (For all subjects, n = 42) and age $50 (For all subjects, n = 153). Student t-test was performed to analyze the differences between these two
groups. A. The participants in healthy and other cancer groups were divided into two groups: age,50 (For all subjects, n = 61) and age $50 (For all
subjects, n = 144). Student t-test was performed to analyze the differences between these two groups. ***P,0.001. doi:10.1371/journal.pone.0057081.g007 Figure 7. PLA2activities were not significantly correlated to ages. The participants in healthy and CRC groups were divided into two groups:
age,50 (For all subjects, n = 42) and age $50 (For all subjects, n = 153). Student t-test was performed to analyze the differences between these two
groups. A. The participants in healthy and other cancer groups were divided into two groups: age,50 (For all subjects, n = 61) and age $50 (For all
subjects, n = 144). Student t-test was performed to analyze the differences between these two groups. ***P,0.001. doi:10.1371/journal.pone.0057081.g007 Finally, since we have found that the PLA2 activities were
essentially no difference in the two sets of healthy controls, we
combined all healthy controls and all cancer cases in both sets of
studies and generated a combined formula, with an AUC = 0.8011
(Fig. 8F). In 95% of the 1000 bootstrapped samples, AUCs are
higher than 0.7907. ln
P
1{P
~{4:6897z0:00263|PLA2{0:00411|iPLA2z
0:00263|cPLA2z0:00342|sPLA2 PLOS ONE | www.plosone.org February 2013 | Volume 8 | Issue 2 | e57081 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 11 Elevation of Plasma PLA2 Activity in Cancers Table 6. Comparison of two independent sets of healthy
controls. 1st set
2nd set
No. %
No. 0:00222|cPLA2z0:00254|sPLA2 ln
PB
1{PB
~{7:6200{0:00424|PLA2{0:00010|iPLA2z
0:00538|cPLA2z0:00292|sPLA2z5:9791|
(Age§50){2:9782|Alcohol The sensitivities and specificities can be obtained from the ROC
curves shown in Fig. 8. For the CRC set of study, 60.5%
sensitivity and 77.9% of specificity were obtained (Table 7). For
the 2nd set of study and the data combined for both sets,
sensitivities .80% and the specificities .66% were obtained for
all cases (Table 7). For PC versus healthy control, the AUC = 0.9907. In 95% of the
1000 bootstrapped sample, AUCs are higher than 0.9740. The classification performance of plasma PLA2 activities %
P value
Gender
,0.0001
Male
31
40.3
61
77.2
Female
46
59.7
18
22.8
Smoking status
Current smoker
2
3.8
4
5.1
0.889
Past smoker
19
35.8
25
31.6
Never smoked
32
60.4
50
63.3
Second hand smoke
,0.0001
No
48
90.6
32
40.5
Yes
5
9.4
47
59.5
Alcohol
,0.0001
No
6
11.3
23
29.6
Yes
47
88.4
56
70.4
Age (Mean 6 SD)
51.6613.5
42.7612.03
,0.0001
PLA2 activities(Mean 6 SD)
PLA2
1135.96266.2
1156.36311.8
0.660
iPLA2
1082.06286.1
1105.46301.6
0.620
cPLA2
1246.16318.7
1287.86425.9
0.488
sPLA2
299.16164.7
293.96137.3
0.828
doi:10.1371/journal.pone.0057081.t006 Table 6. Comparison of two independent sets of healthy
controls. The prediction formula and the ROC curve to separate all cancer
cases from healthy subjects in the second set of study are as follows
and shown in Fig. 9B, with an AUC = 0.9728. ln
P2nd,all
1{P2nd,all
~{6:7670{0:00034zPLA2{0:00193|
iPLA2z0:00415|cPLA2z0:00173|
sPLA2z5:3434|(Age§50){3:4392|
Alcoholz2:8792|(Currently Smoke)z
1 4151|(Currently not but used to smoke) ln
P2nd,all
1{P2nd,all
~{6:7670{0:00034zPLA2{0:00193| iPLA2z0:00415|cPLA2z0:00173|
sPLA2z5:3434|(Age§50){3:4392|
Alcoholz2:8792|(Currently Smoke)z 1:4151|(Currently not but used to smoke) 1:4151|(Currently not but used to smoke) This performance was validated in 1000 bootstrapped samples. In
95% of the 1000 bootstrapped samples, AUCs are higher than
0.9625 for LC, PC, and BC combined vs. the second set of healthy
controls. The prediction formulas and the ROC curves for the separate
LC, BC, and PC cases from healthy subjects in the second set of
study are listed below and Figs. 9C to 9E, with AUCs .0.968,
indicating high sensitivities and specifies of the tests. For LC versus
healthy
control, the
AUC = 0.9742. In
95% of
the 1000
bootstrapped sample, AUCs are higher than 0.9598. ln
PL
1{PL
~{7:5757z0:00136|PLA2{0:00373|iPLA2z
0:00423|cPLA2z0:00207|sPLA2z4:3789|
Age§50
ð
Þ{3:4043|Alcoholz4:0312|
Currently Smoke
ð
Þz2:3753|
Currently not but used to smoke
ð
Þ Currently not but used to smoke
ð
Þ Currently not but used to smoke
ð
Þ ln
P
1{P
~{3:0732z0:000461|PLA2{0:00131|iPLA2z ln
P
1{P
~{3:0732z0:000461|PLA2{0:00131|iPLA2z For BC versus healthy control, the AUC = 0.9682. In 95% of the
1000 bootstrapped sample, AUCs are higher than 0.9398. 0:00222|cPLA2z0:00254|sPLA2 Discussion In this work, we have presented the first measurements of
plasma natural and subtype PLA2 activities and their performanc-
es as potential markers for four different cancers, CRC, LC, BC,
and PC. We have measured the PLA2 activities in more than 20
blood samples with or without adding 1.2 mM calcium (the
natural ionized calcium concentration in blood) to the final assay
mixtures and did not detect any significant differences. Hence, we
define the PLA2 activities obtained without any additives as the
‘‘natural’’ PLA2 activity. We have selected optimal conditions to
measure each sub-family of PLA2 activities. These modified
conditions do not co-exist in the blood. Therefore, it is not
surprising that the sum of the subfamily PLA2 activities is greater
than the ‘‘natural’’ PLA2 activity. However, it is important to note
that although modified conditions were used to obtain the
activities for subfamilies of PLA2s, the final results are from the
natural PLA2s present in the blood samples. Is a marker which may detect multiple types of cancers useful? The answer is likely to be yes. Even after decades of efforts, the
success rate in finding highly specific markers for specific cancers
has been low. Prostate-specific antigen is an exception, but even
the value of this marker as a screening tool in prostate cancer has
been recently questioned [22]. Many tumor markers are known to
affect multiple cancers. Sensitive, minimally invasive, reproduc-
ible, and cost-effective blood biomarkers to detect multiple
malignancies are likely to be clinically significant and highly
valuable as routine first line detection. In addition, the PLA2 While the advantages of using convenient serologic markers are
obvious, no reliable blood markers for any of these cancers are
currently available. For CRC, developing/validating non-invasive
or minimally invasive detection methods is a major focus in the
field. Current methods include stool and blood tests, such as fecal
immunochemical tests (FITs) and guaiac-based fecal occult blood
testing (got). Although the specificity is high (.85%), these tests Table 7. Sensitivities and specificities of PLA2 to distinguish different cancers from healthy cases. Table 7. Sensitivities and specificities of PLA2 to distinguish different cancers from healthy cases. Without other parameters
With other parameters
Sensitivity
Specificity
Sensitivity
Specificity
First set
CRC vs. Healthy
60.5%
77.9%
63.0%
75.5%
Second set
All cancer vs. healthy
81.1%
72.2%
87.2%
88.6%
LC vs. healthy
82.1%
67.1%
85.3%
91.1%
BC vs. healthy
80.6%
75.9%
87.1%
87.3%
PC vs. The potential contributions of other factors The majority
solid cancers are highly heterogeneous, which is one of the major
reasons that any single or small set of markers can hardly detect
any specific cancer with very high sensitivity and specificity. Testing the potential complementary values of other identified
markers in CRC and other cancers will be of high significance. Interestingly, our results suggest that PLA2 activities are indepen-
dent of several common demographic and environmental factors. In addition, we have compared 11 CRC blood samples collected
before and after surgeries and found that 7 of 11 had reduced
PLA2 activities (26.1610.9% reduction) and 4 of 11 had increased
PLA2 activities (37.7%611.7%). Among them, disease progression
data was available for only 6 subjects (3 from each group). Interestingly, all 3 subjects from the reduced PLA2 activity group
had chemotherapy and complete response and all 3 subjects from
the increased PLA2 activity group had chemotherapy and disease
progression. These data were from a very limited number of
subjects, but suggest that the prognostic value of PLA2 activities
warrants further testing. We are fully aware that our studies are
limited to the cohort size and external validation in independent
and larger scale studies will be critical and hope that our report
will promote such studies. ln
Pboth,all
1{Pboth,all
~{3:5290z0:000371|PLA2{0:00066|
iPLA2z0:0015|cPLA2z0:00219|sPLA2
z1:9213|(Age§50){1:7107|Alcohol
z1:4619|(Currently Smoke)z1:0802|
(Currently not but used to smoke) The sensitivities and specificities obtained with these ROC curves
with additional parameters are increased to .85% (except CRC
vs. healthy cases; Table 7). The sensitivities and specificities obtained with these ROC curves
with additional parameters are increased to .85% (except CRC
vs. healthy cases; Table 7). The potential contributions of other factors Although most other demographic and/or environmental
factors tested were not significantly different in cancer and control
groups (Figs 3–7), we tested their potential contributions to the
performance when they are evaluated at individual levels. We kept
all four PLA2 measurements in the models, but let other predictors
subject to model selections at the significant level 0.05. ln
PP
1{PP
~{3:1927z0:00229|PLA2{0:00384|iPLA2z
0:00179|cPLA2z0:00105|sPLA2z2:9716|
(Age§50){2:1084|Alcohol The prediction formula and the ROC curve to separate CRC
from healthy subjects in the first set of study are as follows and
shown in Fig. 9A, with an AUC = 0.8162. In 95% of the 1000
bootstrapped sample, AUCs are higher than 0.7589. Finally, since we found that the PLA2 activities were essentially
no difference in the two sets of healthy controls, we combined all
healthy controls and all cancer cases in both sets of studies and
generated a combined formula, with an AUC = 0.9140 (Fig. 9F). In 95% of the 1000 bootstrapped samples, AUCs are higher than
0.9018. ln
PL
1{PL
~{7:1140{0:0006|PLA2z0:00909|iPLA2{
0:00612|cPLA2z0:00327|sPLA2z3:5428|
Alcohol ln
PL
1{PL
~{7:1140{0:0006|PLA2z0:00909|iPLA2{ Alcohol February 2013 | Volume 8 | Issue 2 | e57081 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 12 Elevation of Plasma PLA2 Activity in Cancers PLOS ONE | www.plosone.org
13
February 2013 | Volume 8 | Issue 2 | e57081 February 2013 | Volume 8 | Issue 2 | e57081 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 13 Elevation of Plasma PLA2 Activity in Cancers Figure 8. ROC curves of plasma PLA2 activities in different groups. CRC cases vs. healthy subjects. A. All cancers vs. healthy subjects in the
2nd set. B. Lung cancer vs. healthy subjects. C. Bladder cancer vs. healthy subjects. D. Pancreatic cancer vs. healthy subjects. E. All cancers vs. all
healthy subjects with combined two sets. doi:10.1371/journal.pone.0057081.g008 have a low sensitivity (,23%) for colorectal adenomas and are
thus unlikely to be able to increase the early detection of CRC
[20,21]. Genetic stool tests detect mutations in stool that can be
found in CRC. A 21 genetic change test was found to be superior
to guaiac fecal occult blood test (gFOBT) for the detection of CRC
[20]. However, the sensitivity only reached 51.6% [20]. The
plasma PLA2 activities tests presented show promising initial
results in separating healthy controls from cancer patients with
sensitivities and specificities approximately 70%. Discussion healthy
81.6%
77.2%
86.8%
96.2%
Combined two sets
Cancer vs. healthy
80.2%
66.7%
83.2%
81.1%
doi:10.1371/journal.pone.0057081.t007 14 Elevation of Plasma PLA2 Activity in Cancers PLOS ONE | www.plosone.org
15
February 2013 | Volume 8 | Issue 2 | e57081 February 2013 | Volume 8 | Issue 2 | e57081 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 15 15 Elevation of Plasma PLA2 Activity in Cancers Figure 9. ROC curves of plasma PLA2 activities with additional parameters. CRC cases vs. healthy subjects. A. All cancer cases vs. healthy
subjects in the second set. B. Lung cancer vs. healthy subjects. C. Bladder cancer vs. healthy subjects. D. Pancreatic cancer vs. healthy subjects. E. All
cancers vs. all healthy subjects with combined two sets. doi:10.1371/journal.pone.0057081.g009 Figure 9. ROC curves of plasma PLA2 activities with additional parameters. CRC cases vs. healthy subjects. A. All cancer cases vs. healthy
subjects in the second set. B. Lung cancer vs. healthy subjects. C. Bladder cancer vs. healthy subjects. D. Pancreatic cancer vs. healthy subjects. E. All
cancers vs. all healthy subjects with combined two sets. doi:10.1371/journal.pone.0057081.g009 shown not coincidental but may indeed be causal [23]. In
addition, non-steroidal anti-inflammatory drugs has been associ-
ated with reduced risk to developing many types of cancers [25]. Thus, future studies on the significance of elevated PLA2 activity in
disease detection and progression prediction will be highly
interesting. activity test is very easy to perform in any laboratory with a
fluorescent plate reader, and would be feasible to develop into an
automated test. Moreover, very small amounts samples (1–10 mL
of plasma) are needed to perform the test and the results can be
obtained
in
1–2 hrs. Additional
markers
or
more
specific
modalities, such as colonoscopy or imaging are likely to be needed
to detect specific cancers. Our results also strongly imply that the PLA2 activities (which
may or may not correlate to their RNA or protein expression
levels) are potential targets for cancer treatment tested here. Most,
if not all previous PLA2 assays conducted in tissues or cell lines
mainly focus on their expression levels using PCR and IHC, which
are gene/protein specific, time-consuming, costly, and require
relatively large amount of samples to cover different isoforms of
PLA2s. Since there are more than 30 PLA2s, none of the previous
studies provide an overall picture of PLA2s in any cancer. Acknowledgments A potential caveat of the clinical usage of PLA2 activity is that
these enzymes are known to be involved in inflammation and may
be elevated in patients with benign inflammatory diseases. This
needs to be experimentally tested in clinical samples. However,
there are mounting lines of evidence supporting the strong causal
connections between inflammation and cancers [23]. In particular,
chronic inflammation plays a pivotal role in the development of
CRC in patients with inflammatory bowel disease [24]. The
connection between inflammation and lung cancer has been We highly appreciate the proof-reading and editing of Dr. Caryl Antalis. References 13. Li H, Zhao Z, Wei G, Yan L, Wang D, et al. (2010) Group VIA phospholipase
A2 in both host and tumor cells is involved in ovarian cancer development. FASEB J 24: 4103–4116. 1. Siegel R, Naishadham D, Jemal A (2012) Cancer statistics, 2012. CA
Cancer J Clin 62: 10–29. 2. Khochikar MV (2011) Rationale for an early detection program for bladder
cancer. Indian J Urol 27: 218–225. 14. Subra C, Grand D, Laulagnier K, Stella A, Lambeau G, et al. (2010) Exosomes
account for vesicle-mediated transcellular transport of activatable phospholipases
and prostaglandins. J Lipid Res 51: 2105–2120. 3. Risch A, Plass C (2008) Lung cancer epigenetics and genetics. Int J Cancer 123:
1–7. 4. Pastorino U (2010) Lung cancer screening. Br J Cancer 102: 1681–1686. 15. Iguchi H, Kosaka N, Ochiya T (2010) Secretory microRNAs as a versatile
communication tool. Commun Integr Biol 3: 478–481. 5. Mutti A (2008) Molecular diagnosis of lung cancer: an overview of recent
developments. Acta Biomed 79 Suppl 1: 11–23. 16. Cai Q, Zhao Z, Antalis C, Yan L, Del Priore G, et al. (2012) Elevated and
secreted phospholipase A2 activities as new potential therapeutic targets in
human epithelial ovarian cancer. FASEB J. 6. Cummings BS (2007) Phospholipase A2 as targets for anti-cancer drugs. Biochem Pharmacol 74: 949–959. 7. Murakami M, Taketomi Y, Sato H, Yamamoto K (2011) Secreted phospho-
lipase A2 revisited. J Biochem 150: 233–255. 17. Zhao X, Wang D, Zhao Z, Xiao Y, Sengupta S, et al. (2006) Caspase-3-
dependent activation of calcium-independent phospholipase A2 enhances cell
migration in non-apoptotic ovarian cancer cells. J Biol Chem 281: 29357–29368. p
8. Kudo I, Murakami M (2002) Phospholipase A2 enzymes. Prostaglandins Other
Lipid Mediat 68–69: 3–58. 18. Murakami M, Kudo I (2001) Diversity and regulatory functions of mammalia
secretory phospholipase A2s. Adv Immunol 77: 163–194. 9. Xu Y, Xiao YJ, Zhu K, Baudhuin LM, Lu J, et al. (2003) Unfolding the
pathophysiological role of bioactive lysophospholipids. Curr Drug Targets
Immune Endocr Metabol Disord 3: 23–32. 19. Steyerberg EW, Harrell FE, Jr., Borsboom GJ, Eijkemans MJ, Vergouwe Y, et
al. (2001) Internal validation of predictive models: efficiency of some procedures
for logistic regression analysis. J Clin Epidemiol 54: 774–781. 10. Sengupta S, Kim KS, Berk MP, Oates R, Escobar P, et al. (2007) Lysopho-
sphatidic acid downregulates tissue inhibitor of metalloproteinases, which are
negatively involved in lysophosphatidic acid-induced cell invasion. Oncogene 26:
2894–2901. Discussion More
importantly, their enzymatic activities, but not necessarily their
RNA and/or protein expression levels, are directly related to the
biological effects, since PLA2 activities are well-known to be
regulated post-transcriptionally [26,27]. This concept has been
supported in our recent pre-clinical (mouse models) and human
sample studies in ovarian cancer [16] and remain to be tested
further in other cancers. Another major advantage of the test is that it is reproducible,
robotic, and stable. It is well-recognized that many blood markers
are sensitive to how blood samples are handled, processed, and
stored. It is almost impossible to unify these procedures in the USA
or anywhere in the world. Freeze-and-thaw is another well-known
factor affecting marker stability. These reasons account, at least in
part, for the fact that although thousands of markers have been
reported in the past decades, seldom have any of them been cross-
validated in different centers and moved to the clinic. We have
used two completely independent sets of human plasma samples,
which were processed somewhat differently (see Method for
details) and show that the PLA2 activities are highly compatible,
consistent in healthy controls, and are independent of several
demographic and environmental factors, making them more likely
to be useful markers. More independent studies with large samples
sizes need to be conducted to further evaluate the clinical
significance of our finding reported here. Author Contributions Conceived and designed the experiments: YX. Performed the experiments:
HC. Analyzed the data: HC ZL. Contributed reagents/materials/analysis
tools: EGC MVC DKR BWR NMH PJL MLH. Wrote the paper: YX
HC. Conceived and designed the experiments: YX. Performed the experiments:
HC. Analyzed the data: HC ZL. Contributed reagents/materials/analysis
tools: EGC MVC DKR BWR NMH PJL MLH. Wrote the paper: YX
HC. 25. Vendramini-Costa DB, Carvalho JE (2012) Molecular Link Mechanisms
between Inflammation and Cancer. Curr Pharm Des 18: 3831–3852. 24. Moossavi S, Bishehsari F (2012) Inflammation in sporadic colorectal cancer.
Arch Iran Med 15: 166–170.
25. Vendramini-Costa DB, Carvalho JE (2012) Molecular Link Mechanisms
between Inflammation and Cancer. Curr Pharm Des 18: 3831–3852. 26. Burke JE, Dennis EA (2009) Phospholipase A2 biochemistry. Cardiovasc Drugs
Ther 23: 49–59.
27. Burke JE, Dennis EA (2009) Phospholipase A2 structure/function, mechanism,
and signaling. J Lipid Res 50 Suppl: S237–242. Elevation of Plasma PLA2 Activity in Cancers 24. Moossavi S, Bishehsari F (2012) Inflammation in sporadic colorectal cancer.
Arch Iran Med 15: 166–170. 26. Burke JE, Dennis EA (2009) Phospholipase A2 biochemistry. Cardiovasc Drugs
Ther 23: 49–59. 27. Burke JE, Dennis EA (2009) Phospholipase A2 structure/function, mechanism,
and signaling. J Lipid Res 50 Suppl: S237–242. Elevation of Plasma PLA2 Activity in Cancers 26. Burke JE, Dennis EA (2009) Phospholipase A2 biochemistry. Cardiovasc Drugs
Ther 23: 49–59.
27. Burke JE, Dennis EA (2009) Phospholipase A2 structure/function, mechanism,
and signaling. J Lipid Res 50 Suppl: S237–242. References g
g
y
J
p
20. Pox C (2011) Colon Cancer Screening: Which Non-Invasive Filter Tests? Digestive Diseases 29: 56–59. 21. Wong MC, John GK, Hirai HW, Lam TY, Luk AK, et al. (2012) Changes in the
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Ovarian cancer. 2 ed. New York, Dordrecht, Heidelberg, London: Springer. pp. 241–268. 22. Chou R, Croswell JM, Dana T, Bougatsos C, Blazina I, et al. (2011) Screening
for prostate cancer: a review of the evidence for the U.S. Preventive Services
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inflammation in the pathogenesis of lung cancer. Expert Opin Ther Targets 15:
1127–1137. February 2013 | Volume 8 | Issue 2 | e57081 February 2013 | Volume 8 | Issue 2 | e57081 PLOS ONE | www.plosone.org 16 February 2013 | Volume 8 | Issue 2 | e57081 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 17
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Impact of an evidence-based intervention on urinary catheter utilization, associated process indicators, and infectious and non-infectious outcomes
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The Journal of hospital infection/Journal of hospital infection
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Impact of an evidence-based intervention on urinary
catheter utilization, associated process indicators, and
infectious and non-infectious outcomes a Swissnoso, National Centre for Infection Control, Bern, Switzerland
b Department of Infectious Diseases and Hospital Epidemiology, Basel University Hospital, Basel, Switzerland
c Zug Cantonal Hospital, Zug, Switzerland
d Division of Infectious Diseases and Hospital Epidemiology, University and University Hospital Zurich, Zurich, Switzerland
e Swiss Patient Safety Foundation, Zurich, Switzerland
f Lucerne Cantonal Hospital, Lucerne, Switzerland
g Ente Ospedaliero Cantonale, Bellinzona, Switzerland
h Department of Internal Medicine, Hoˆpital Neuchaˆtelois, Neuchaˆtel, Switzerland
i University of Geneva, Geneva, Switzerland
j CTU Bern, University of Bern, Bern, Switzerland
k Department of Infectious Diseases, Inselspital, Bern University Hospital, Bern, Switzerland
l Institute for Social and Preventive Medicine, University of Bern, Bern, Switzerland https://doi.org/10.1016/j.jhin.2020.07.002
0195-6701/ª 2020 The Author(s). Published by Elsevier Ltd on behalf of The Healthcare Infection Society. This is an open access article
under the CC BY license (http://creativecommons.org/licenses/by/4.0/). * Corresponding author. Address: Department of Infectious Diseases,
Bern University Hospital, Friedbu¨hlstrasse 53, 3010 Bern, Switzerland.
Tel.: þ41 31 632 9992; fax: þ41 31 632 2120. journal homepage: www.elsevier.com/locate/jhin journal homepage: www.elsevier.com/locate/jhin E-mail address: jonas.marschall@insel.ch (J. Marschall). Zurich Open Repository and
Archive
University of Zurich
University Library
Strickhofstrasse 39
CH-8057 Zurich
www.zora.uzh.ch Year: 2020 Impact of an evidence-based intervention on urinary catheter utilization, associated
process indicators, and infectious and non-infectious outcomes Schweiger, A ; Kuster, S P ; Maag, J ; Züllig, S ; Bertschy, S ; Bortolin, E ; John, G ; Sax, H ; Limacher, A
Atkinson, A ; Schwappach, D ; Marschall, J Schweiger, A ; Kuster, S P ; Maag, J ; Züllig, S ; Bertschy, S ; Bortolin, E ; John, G ; Sax, H ; Limacher, A ;
Atkinson, A ; Schwappach, D ; Marschall, J DOI: https://doi.org/10.1016/j.jhin.2020.07.002 DOI: https://doi.org/10.1016/j.jhin.2020.07.002 Posted at the Zurich Open Repository and Archive, University of Zurich
ZORA URL: https://doi.org/10.5167/uzh-196204
Journal Article
Published Version Posted at the Zurich Open Repository and Archive, University of Zurich
ZORA URL: https://doi.org/10.5167/uzh-196204
Journal Article
Published Version Originally published at:
Schweiger, A; Kuster, S P; Maag, J; Züllig, S; Bertschy, S; Bortolin, E; John, G; Sax, H; Limacher, A; Atkinson,
A; Schwappach, D; Marschall, J (2020). Impact of an evidence-based intervention on urinary catheter utiliza-
tion, associated process indicators, and infectious and non-infectious outcomes. Journal of Hospital Infection,
106(2):364-371.t DOI: https://doi.org/10.1016/j.jhin.2020.07.002 Journal of Hospital Infection 106 (2020) 364e371 Available online at www.sciencedirect.com A R T I C L E
I N F O Measuring infections per 1000 catheter-days, the rate was 1.02 (baseline) and
1.33 (post intervention) (P¼0.60). Non-infectious complications decreased significantly,
from 0.79 to 0.56 events per 100 patient-days (P<0.001), and from 39.4 to 35.4 events per
1000 catheter-days (P¼0.23). Indicated catheters increased from 74.5% to 90.0%
(P<0.001). Re-evaluations increased from 168 to 624 per 1000 catheter-days (P<0.001). Conclusion: A straightforward bundle of three evidence-based measures reduced catheter
utilization and non-infectious complications, whereas the proportion of indicated urinary
catheters and daily evaluations increased. The CAUTI rate remained unchanged, albeit at
a very low level. ª 2020 The Author(s). Published by Elsevier Ltd ( )
y
on behalf of The Healthcare Infection Society. This is an open access article
under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Methods A before/after non-randomized multi-centre intervention
study, corresponding to a quasi-experimental study type 1, was
conducted [23]. The primary outcome was urinary catheter
utilization. Secondary outcomes were symptomatic CAUTIs,
non-infectious outcomes and process indicators, such as the
proportion of indicated catheters and the frequency of cath-
eter evaluation. It was hypothesized that the intervention
would significantly reduce catheter utilization, and infectious
and non-infectious complications, and significantly improve
process indicator endpoints. The study design and results
presented here were an integral part of the ‘Progress! Safe
urinary catheterization’ pilot programme that ran from 2015 to
2018 in Switzerland. In addition to the awareness campaign
that accompanied the programme and started in 2016 after
completion of the baseline surveillance, two large surveys
were launched in parallel to the baseline and intervention
periods, respectively; their goal was to better understand
knowledge, perceptions, attitudes and behaviours of health-
care workers (HCWs) surrounding the topic of urinary cathe-
terization [24,25]. The use of transurethral urinary catheters is common in
acute care hospitals. With catheterization proportions of
12e25% [1e5] and nearly 1.5 million hospitalizations in Swit-
zerland in 2016 (Source: Federal Statistics Office, accessed 27th
February 2018), it is estimated that more than 200,000 urinary
catheters are placed in Switzerland each year, eventually
causing infectious and non-infectious complications, increased
morbidity and mortality, and additional healthcare costs. Catheter-associated urinary tract infections (CAUTIs) are a
well-known complication of urinary tract catheterization, with
rates ranging from 0.2 to 4.8 per 1000 catheter-days. Non-
infectious
complications
from
catheterization
have
not
received widespread attention to date [6,7], although they
may be as common as CAUTIs [8,9]; they include mechanical
trauma to the lower urinary tract, false passage, and acci-
dental inflation of a catheter balloon in the urethra or prostate. Up to 5% of all catheterized patients develop acute gross
haematuria, and approximately 3% suffer urethral strictures in
the long term [8]. The presence of a urinary catheter and the duration of
catheterization
are
the
main
risk
factors
for
catheter-
associated complications; however, 21e65% of all catheter
insertions are not necessary [10e12], and prolonged cathe-
terization without clear indication is common [5,13]. Sample size calculation A baseline prevalence of urinary catheterization of 15% and
a potential relative reduction of 10% following the intervention
were assumed. Given a two-sided statistical significance of 5%
and statistical power of 80%, at least 8524 patients were
required in each group (17,048 overall) to detect a significant
difference. Therefore, prior intervention studies have focused on the
following main pillars: (1) strict indications for catheter
insertion; (2) rapid removal of unnecessary urinary catheters;
and (3) proper insertion of and care for urinary catheters. For
example, the Keystone Bladder Bundle Initiative implemented
a protocol for catheter utilization, reminders and stop orders,
use of alternative strategies for urinary management, portable
ultrasound to measure bladder volume, and adherence to
protocols for insertion of and care for urinary catheters. Con-
sequently, the CAUTI rate decreased by 25% and 6% in the state
of Michigan and the entire USA, respectively [14,15]. Based on
this success, several programmes started worldwide and pro-
duced similar results [16e19]. In Switzerland, a few single-
centre
studies
have
addressed
urinary
catheterization
[20e22]; however, a uniform nationwide approach has been
lacking to date. As such, the aim of this study was to reduce
urinary catheterization by implementing a three-fold bundle:
(1) a nationally endorsed list of indications for urinary cathe-
terization; (2) daily evaluation of the need for ongoing cathe-
terization;
and
(3)
education
on
proper
insertion
and
maintenance of a urinary catheter. A R T I C L E
I N F O Article history:
Received 19 April 2020
Accepted 2 July 2020
Available online 10 July 2020 Background: Multi-centre intervention studies tackling urinary catheterization and its
infectious and non-infectious complications are lacking. Aim: To decrease urinary catheterization and, consequently, catheter-associated urinary
tract infections (CAUTIs) and non-infectious complications. Methods: Before/after non-randomized multi-centre intervention study in seven hospitals
in Switzerland. Intervention bundle consisting of: (1) a concise list of indications for uri-
nary catheterization; (2) daily evaluation of the need for ongoing catheterization; and (3)
education on proper insertion and maintenance of urinary catheters. The primary outcome
was urinary catheter utilization. Secondary outcomes were CAUTIs, non-infectious com-
plications and process indicators (proportion of indicated catheters and frequency of
catheter evaluation). Keywords:
Healthcare-associated infection
Infection control
CAUTI
Urinary catheter
Haematuria )
Findings: In total, 25,880 patients were included in this study [13,171 at baseline (August
eOctober 2016) and 12,709 post intervention (AugusteOctober 2017)]. Catheter uti-
lization decreased from 23.7% to 21.0% (P¼0.001), and catheter-days per 100 patient-days
decreased from 17.4 to 13.5 (P¼0.167). CAUTIs remained stable at a low level with 0.02
infections per 100 patient-days (baseline) and 0.02 infections (post intervention) A. Schweiger et al. / Journal of Hospital Infection 106 (2020) 364e371 365 (P¼0.98). Measuring infections per 1000 catheter-days, the rate was 1.02 (baseline) and
1.33 (post intervention) (P¼0.60). Non-infectious complications decreased significantly,
from 0.79 to 0.56 events per 100 patient-days (P<0.001), and from 39.4 to 35.4 events per
1000 catheter-days (P¼0.23). Indicated catheters increased from 74.5% to 90.0%
(P<0.001). Re-evaluations increased from 168 to 624 per 1000 catheter-days (P<0.001). Conclusion: A straightforward bundle of three evidence-based measures reduced catheter
utilization and non-infectious complications, whereas the proportion of indicated urinary
catheters and daily evaluations increased. The CAUTI rate remained unchanged, albeit at
a very low level. (P¼0.98). Measuring infections per 1000 catheter-days, the rate was 1.02 (baseline) and
1.33 (post intervention) (P¼0.60). Non-infectious complications decreased significantly,
from 0.79 to 0.56 events per 100 patient-days (P<0.001), and from 39.4 to 35.4 events per
1000 catheter-days (P¼0.23). Indicated catheters increased from 74.5% to 90.0%
(P<0.001). Re-evaluations increased from 168 to 624 per 1000 catheter-days (P<0.001). Conclusion: A straightforward bundle of three evidence-based measures reduced catheter
utilization and non-infectious complications, whereas the proportion of indicated urinary
catheters and daily evaluations increased. The CAUTI rate remained unchanged, albeit at
a very low level. (P¼0.98). (P¼0.98). Measuring infections per 1000 catheter-days, the rate was 1.02 (baseline) and
1.33 (post intervention) (P¼0.60). Non-infectious complications decreased significantly,
from 0.79 to 0.56 events per 100 patient-days (P<0.001), and from 39.4 to 35.4 events per
1000 catheter-days (P¼0.23). Indicated catheters increased from 74.5% to 90.0%
(P<0.001). Re-evaluations increased from 168 to 624 per 1000 catheter-days (P<0.001).
Conclusion: A straightforward bundle of three evidence-based measures reduced catheter
utilization and non-infectious complications, whereas the proportion of indicated urinary
catheters and daily evaluations increased. The CAUTI rate remained unchanged, albeit at
a very low level.
A. Schweiger et al. / Journal of Hospital Infection 106 (2020) 364e371
365 Table I The bundle was implemented on participating wards as well as
units with a central function for the hospital (e.g. emergency
room, operating room and similar). The intervention bundle
consisted of an evidence-based indication list, the instruction
to evaluate this indication daily, and training HCWs in the
correct, non-traumatic and aseptic insertion of urinary cathe-
ters. The indication list (Table I) consisted of six major indi-
cations: (1) urinary retention; (2) monitoring of fluid input and
output; (3) surgery; (4) pressure ulcers and urinary incon-
tinence; (5) prolonged immobilization; and (6) palliative care
and comfort for patients. A group of national experts including
physicians and nurses developed the indications specifically for
this programme based on the Ann Arbor Criteria for Appro-
priate Urinary Catheter Use in Hospitalized Medical Patients
[26]. The list included practical examples, specific situations in
which urinary catheterization is unnecessary, and alternative
methods for urinary management. Daily evaluation of the
catheter indication was an explicit goal, and unnecessary
catheters were expected to be removed on the same day. Finally, theoretical and practical training of HCWs ensured that
catheter insertion was only performed by adequately trained
personnel. The education had to include a ‘refresher course’ hospitalization data (day of hospital admission, discharge
date), catheter-specific data (time and duration of catheter-
ization, type of catheter, re-insertion of further catheters),
process parameters (indication for catheterization, frequency
of evaluation) and complications (CAUTI, urethral bleeding,
gross haematuria, paraphimosis, incorrect positioning, re-
insertion
within
24
h,
unintentional
removal,
catheter
obstruction). Process parameters were only considered to be
executed if documented in the patient’s chart. The study group
trained local surveillance teams in workshops and at site visits
prior to the surveillance. In addition, audits were performed on
site in the postintervention surveillance to ensure the quality
of data. Table I Table I
List of indications for urinary catheter insertion per the Swiss ‘Progress! Safe urinary catheterization’ programme
Indication
Specification
Examples
Urinary retention
Acute urinary retention regardless of aetiology
Symptomatic chronic outlet obstruction plus >300 mL
residual urine
Benign hyperplasia of prostate gland, urethral
strictures, bladderstones
Drug
induced
(anticholinergics,
opioids,
antidepressants)
Measurement of
urine volume/
fluid balance
At regular intervals (hourly or as defined by hospitals) plus
direct consequence on treatment of patients
Fluid balance if patient weight not measurable on a daily
basis
Haemodynamic
instability,
severe
rhabdomyolysis
Coma, sedated and ventilated patient
Surgery
Long surgery (>4 h)
Peri-interventional:
need
for
empty
bladder
during
surgery, removal of catheter after surgery necessary if
no other indication present
Surgery in urogenital or pelvic floor region
Epidural/peridural anaesthesia
Pressure ulcers
plus urinary
incontinence
Stage III or IV pressure ulcers or skin transplants in sacral/
perineal region plus urinary incontinence after exhaustion
of alternative strategies for urinary management
Prolonged
immobilization
Immobilization for medical reasons, especially for pain
management, after exhaustion of alternative strategies
for urinary management
Acute fractures with severe pain due to patient
movement (pelvic fractures, fracture of the
neck of the femur)
Haemodynamic instability possibly caused by
movement of the patient
Transient
immobility
after
specific
interventions
Palliative care plus
comfort
Palliative care plus abnormal bladder function plus/or
inability for regular voiding after exhaustion of
alternative strategies for urinary management
High burden of suffering plus wish of informed patient (or
relatives) Acute fractures with severe pain due to patient
movement (pelvic fractures, fracture of the
neck of the femur)
Haemodynamic instability possibly caused by
movement of the patient
Transient
immobility
after
specific
interventions Haemodynamic instability possibly caused by
movement of the patient Transient
immobility
after
specific
interventions Palliative care plus
comfort
Palliative care plus abnormal bladder function plus/or
inability for regular voiding after exhaustion of
alternative strategies for urinary management
High burden of suffering plus wish of informed patient (or
relatives) the primary components of the intervention bundle. Except for
one larger hospital that participated as a whole, each hospital
was required to capture 100e200 of their surgical beds for
surveillance purposes (i.e. either the whole hospital in the case
of a smaller institution or selected wards in a larger hospital). Surveillance Surveillance was performed for catheterization, and infec-
tious and non-infectious complications prior to (AugusteOc-
tober 2016; baseline surveillance) and after (AugusteOctober
2017; postintervention surveillance) implementation of the
intervention. All patients hospitalized in participating hospi-
tals, irrespective of the location of catheter placement (i.e. emergency room, operating room or other), during the baseline
and postintervention periods were included in the analysis. Refusal of general consent to use of data for scientific research
led to patient exclusion. Data were entered into an online
database provided by the Clinical Trials Unit of the University
of Bern, Switzerland using Secutrial (Interactive Systems
GmbH, Berlin, Germany). Data consisted of demographic data, A. Schweiger et al. / Journal of Hospital Infection 106 (2020) 364e371 366 Statistical analysis Patients from one centre were excluded from the analyses
of non-infectious complications and indicated catheters, as
these items had not been recorded during baseline surveil-
lance. Patients from three centres were excluded from evalu-
ation of the daily re-evaluation rate as this information was not
collected during baseline surveillance. For binary endpoints,
the proportion with a 95% Wilson confidence interval (CI) in
each phase is shown. For count endpoints, rates (per 100 or per
1000 days) with a 95% exact CI are presented. y )
p
Mixed-effects, generalized, linear models were used for
comparison of the before/after surveillance periods. A bino-
mial distribution with logit-link was used for binary endpoints. A negative binomial distribution with log-link fitted with a
square increase of variance was used for count endpoints. A
random intercept for hospitals was integrated in the models to
account for interhospital heterogeneity. In models for count
endpoints, the number of patient- or catheter-days was con-
sidered as exposure. They were also corrected for zero infla-
tion. Due to the small number of CAUTIs, it was not possible to
apply a model with negative binomial distribution. Instead, a
Poisson distribution with a random intercept for hospitals and
patients was used to control for overdispersion. Table II Table II
Definition of symptomatic catheter-associated urinary tract infection [27] Definition of symptomatic catheter-associated urinary tract infection [27]
Criteriona
1. Epidemiological
Patient had an indwelling urinary catheter that had been in place for >2 days on the date of the event and wa
either:
present for any portion of the calendar day on the date of the event or
removed the day before the date of the event
2. Clinical
Patient has at least one of the following signs or symptoms:
fever (>38.0C)
suprapubic tenderness
costovertebral angle pain or tenderness
urinary urgency (only if catheter removed)
urinary frequency (only if catheter removed)
dysuria (only if catheter removed)
3. Microbiological
Patient has a urine culture with no more than two species of organisms identified, at least one of which is
present at 105 colony-forming units/mL
a All three criteria must be met, and have to occur during the window of infection. for all HCWs inserting and taking care of catheters. The course
consisted of general information on urinary catheters and
alternatives for urine diversion, CAUTIs, non-infectious com-
plications, and insertion of and care for catheters. Also, a 10-
min educational video was produced by one institution and
displayed the indication list, the proper insertion technique
and elements of catheter maintenance; this was made avail-
able to all pilot hospitals in the local language. In addition,
certain hospitals offered practical training on dummies. This
intervention bundle was implemented from February 2017,
after a workshop had convened all local project leaders and
representatives for both infection prevention and quality
management from the participating hospitals, and was moni-
tored until October 2017, when the postintervention surveil-
lance ended. Definitions CAUTI was defined in accordance with the 2015 Centers
for Disease Control and Prevention National Healthcare
Safety Network criteria, as described in Table II [27]. Only
events of ‘symptomatic CAUTI’ were included in the analysis. Non-infectious complications due to urinary catheterization
are defined less succinctly in the literature; in this study,
urethral bleeding was defined as frank hemorrhage from the
urogenital tract, gross haematuria was defined as blood-
tinged urine, paraphimosis was defined as constriction of
the prepuce, catheter obstruction was defined as absence of
urine flow, incorrect positioning (false passage, malplace-
ment) was defined as the need to reposition a recently
placed catheter, and unintentional catheter removal was
defined as removal not ordered by the medical team. The
need for catheter re-insertion within 24 h was also consid-
ered a complication. For analyses, these complications were
grouped into medical (the first four) and procedural (the last
three) complications. Differences between the two periods are depicted as
adjusted odds ratios (aOR) or rate ratios (aRR) for binary and
count endpoints, respectively, with a 95% CI. The ratios were
adjusted for age, sex, organizational unit and provenance of
the patient. All provenances except for ‘hospital admission
from home’ were merged due to low patient numbers. With
regard to organizational units, internal medicine, gynaecology
and intensive care units (ICUs) were also merged for adjust-
ment due to low patient numbers in gynaecology and intensive
care. All analyses were performed using the statistical package R
(R-project 3.2). The following terms were used to calculate complication
rates: Intervention Seven pilot hospitals in Switzerland were included in the
intervention, including university hospitals, and tertiary, sec-
ondary and primary care centres. The three major language
regions (German, French and Italian) were represented. Each
hospital formed a project group with a dedicated leader. Education in the form of a project handbook, workshops on the
use of urinary catheters, content of the intervention, surveil-
lance methodology and site visits was provided prior to and
during the project. To accommodate local differences, strict
instructions were not provided; rather, hospitals were free to
design how to deliver the intervention as long as they followed A. Schweiger et al. / Journal of Hospital Infection 106 (2020) 364e371 367 Results Data on 25,880 patients were analysed, with 13,171 patients
included at baseline (AugusteOctober 2016) and 12,709
patients included post intervention (AugusteOctober 2017) A. Schweiger et al. / Journal of Hospital Infection 106 (2020) 364e371 368 Table III
Overview of outcome data before (baseline) and after an intervention aimed at reducing unnecessary urinary catheterization
Baseline surveillance
Postintervention surveillance
Number of patients
13,171
12,709
Proportion or rate
(95% CI)
Proportion or rate
(95% CI)
Crude
Adjusteda
P-
value
Odds or rate ratio
(95% CI)
Odds or rate ratio
(95% CI)
Catheter utilization
Patients with catheters/patients
overall (%)
23.69 (22.97e24.42)
21.02 (20.32e21.73)
0.83 (0.79e0.89)
0.90 (0.84e0.96)
0.001
Catheter-days/100 patient-days
overall
17.40 (17.14e17.67)
13.53 (13.29e13.78)
0.84 (0.78e0.89)
0.96 (0.90e1.02)
0.167
Symptomatic CAUTIs
Infections/100 patient-days
overall
0.02 (0.01e0.03)
0.02 (0.01e0.03)
1.00 (0.51e1.99)
1.01 (0.51e2.00)
0.983
Infections/1000 catheter-days
1.02 (0.60e1.64)
1.33 (0.76e2.17)
1.23 (0.62e2.44)
1.20 (0.60e2.39)
0.603
Non-infectious complications
Complications/100 patient-days
overall
0.79 (0.72e0.86)
0.56 (0.51e0.63)
0.75 (0.63e0.90)
0.73 (0.61e0.88)
<0.001
Complications/1000 catheter-
days
39.43 (36.16e42.93)
35.36 (31.69e39.35)
0.93 (0.79e1.10)
0.90 (0.77e1.07)
0.232
Process parameters
Indicated catheters/all catheters
(%)
74.49 (72.80e76.11)
90.03 (88.72e91.20)
3.70 (3.06e4.47)
4.08 (3.35e4.95)
<0.001
Re-evaluations/1000 catheter-
days
167.66 (159.50e176.13) 623.92 (604.99e643.29)
3.08 (2.87e3.31)
3.13 (2.92e3.36)
<0.001
CAUTI, catheter-associated urinary tract infection; CI, confidence interval. a Adjusted for age, sex, organizational unit and provenance of patients. fore (baseline) and after an intervention aimed at reducing unnecessary urinary catheterization Table III
Overview of outcome data before (baseline) and after an intervention aimed at reducing unnecessary CAUTI, catheter-associated urinary tract infection; CI, confidence interval. a Adjusted for age, sex, organizational unit and provenance of patients. common non-infectious complications were gross haematuria
(235 cases out of 17,296 patient episodes; 1.4%), uninten-
tional catheter removal (72/17,296; 0.4%) and urethral
bleeding (56/17,296; 0.3%). When grouping non-infectious
complications into ‘medical complications’ and ‘procedural
complications’, medical complications decreased from 188/
8887 (2.1%) to 80/8409 (0.95%) (aOR 0.46, 95% CI 0.35e0.60;
P<0.001), without a significant decrease in procedural com-
plications [i.e. 222/8887 (2.5%) vs 183/8409 (2.2%); aOR 0.87,
95% CI 0.71e1.06; P¼0.161). Except for paraphimosis, all
types of medical complications decreased significantly [ure-
thral bleeding: 38/8887 (0.4%) vs 18/8409 (0.2%); aOR 0.54,
95% CI 0.31e0.95; P¼0.031; macrohaematuria: 164/8887
(1.9%) vs 71/8409 (0.8%); aOR 0.47, 95% CI 0.36e0.63;
P<0.001; removal of an obstructed catheter: 21/8887 (0.2%)
vs 7/8409 (0.1%); aOR 0.36, 95% CI 0.15e0.83; P¼0.016]. Indicated catheters increased from 74.5% to 90.0% (aOR 4.08,
95% CI 3.35e4.95; P<0.001). Discussion Among the best evidence currently available is a study
by Saint et al., who selected the three core elements ‘daily
assessment of the need for a catheter’, ‘avoiding catheter use
by considering alternatives’ and ‘emphasizing the importance
of aseptic insertion technique’ to form their intervention in 926
ICU and non-ICU settings in North America, and succeeded in
lowering CAUTI rates [28]. The majority of intervention studies
published to date, however, are single-centre preepost studies
that used different combinations of measures in their inter-
vention bundles [29e34]. It therefore remains to be deter-
mined which combination of measures provides the best
balance between required effort and expected effect. As a sizable proportion of inpatients were expected to have
an indwelling urinary catheter in the baseline surveillance,
catheter utilization was chosen as the primary endpoint, and
the sample size calculation was based on this variable. The
results indicate that catheter utilization decreased, but an
effect on CAUTIs could not be corroborated. Consequently,
focusing on the reduction of catheter utilization may be a
more cost-effective approach to surveillance than focusing on
complications
of
catheterization,
particularly
in
settings
where the CAUTI rate is very low. Similarly, Fakih et al. pro-
posed the ‘device utilization ratio’ as a measure when
undertaking device stewardship or quality improvements
efforts [41]. While this study selected a three-component intervention
bundle and promoted it as such, the programme also included a
broad awareness campaign, during which media releases were
issued. Moreover, the HCWs of the participating pilot hospitals
received a detailed questionnaire on their knowledge about
urinary catheterization, their attitudes and their practices,
both at baseline [24] and post intervention [25]. The results
suggest that changes occurred over the course of the study. As
such, the questionnaire can be viewed as an additional com-
ponent which helped raise awareness and self-reflection con-
cerning local practices. The present study attempted to replicate the success of
the studies cited above. For this purpose, a relatively
straightforward bundle was assembled, of which two elements
appear to be the most relevant drivers to reduce unnecessary
catheter use: (1) offering an evidence-based indication list for
catheter placement; and (2) prompting HCWs to evaluate the
need for ongoing catheterization [19]. Discussion This multi-centre intervention study is the first to shed light
on
urinary
catheter
utilization,
its
infectious
and
non-
infectious consequences, and the impact of an evidence-
based bundle of prevention measures on these outcome
measures. In the seven pilot hospitals, almost every fourth
inpatient was catheterized, and these patients experienced
many more non-infectious complications than CAUTIs at base-
line. A straightforward three-fold intervention e consisting of
distributing an indication list for placing a catheter, promoting
daily evaluation for ongoing need of a urinary catheter, and
ensuring adequate education in catheter handling e managed
to decrease catheter utilization and non-infectious complica-
tions, and increase the proportion of documented justified
catheterization and the frequency of documented daily cath-
eter evaluations. The authors were unable to determine if this
increase was due to raised awareness because of the study or
general improvements in documentation, or if it is misleading
in the sense that practices before the intervention were cor-
rect but simply not documented properly. In summary, this
pilot study has set the stage for planning a national surveillance
and disseminating the successfully piloted intervention tools. p
p
In contrast to previous studies, this collaboration of infec-
tion prevention and patient safety experts also meant that e in
addition to the primary endpoint (urinary catheter utilization)
e both infectious and non-infectious complications were tar-
geted. Although the importance of non-infectious complica-
tions of urinary catheters was highlighted in a recent multi-
centre study [7], to the best of the authors’ knowledge, no
trials have been launched with these complications in mind. It
is worth noting that this study found that non-infectious com-
plications (both medical and procedural) were more common
than CAUTI events e this may change how preventable harm is
viewed in future surveillance surrounding urinary catheters. Considering these results, policy makers may opt to forego
CAUTI surveillance in favour of surveillance of non-infectious
complications. g
y p
While certain countries have set up CAUTI surveillances, this
has not been the case for Switzerland, where urinary catheters
are the first medical device to come under scrutiny. Elsewhere,
regional [16] and national [28] studies have demonstrated that
device use can be reduced (along with the corresponding
complications from unnecessary and incorrect device place-
ment). A. Schweiger et al. / Journal of Hospital Infection 106 (2020) 364e371 evidence of the other bundle components, yet is a prominent
feature in recent CAUTI prevention guidelines [39] and was
therefore included in the bundle [35,40]. The authors inten-
tionally left the participating hospitals to decide how they
planned to deliver the intervention bundle. This permitted
electronic capturing of the catheterization indication in one
of the pilot hospitals, whereas other hospitals relied on simply
disseminating the indication list among their workforce. Despite the flexibility of this approach, the authors are fully
aware of the corresponding heterogeneity, which was evi-
denced during site visits by the study team. The issue of
implementation challenges, however, is not new to CAUTI
prevention efforts and should even be expected [19]. It is felt
that the intervention bundle assembled for this study is
intuitive and easy to memorize and convey. Given that the
optimal bundle is yet to be determined, it seems sensible to
keep the number of bundle components small. complications and compliance with process parameters. Data
are provided in Table S1 (see online supplementary material). Results Documented re-evaluations
increased from 168 to 624 per 1000 catheter-days (aRR 3.13,
95% CI 2.92e3.36; P<0.001). (Table III). The average age was 61 [standard deviation (SD)
20.13] years and 53% were female. The minimum number of
patients accrued from a single hospital during the study period
was 1903 and the maximum was 8584. Most patients were
either from general medical or surgical wards, with the relative
contributions varying between the participating hospitals. In
total, 3494 catheters were placed during the baseline period
and 2929 were placed during the postintervention period. The
mean duration of catheterization decreased from 4.8 (SD 5.82)
days to 4.1 (SD 4.16) days between the two periods. Informa-
tion on the catheter material for all catheters (2956 at baseline
and 2375 post intervention) was not received; however, the mix
of known materials was 84% silicone, 10% latex and 6% others
(coated and irrigation catheters) (baseline), and 70% silicone,
24% latex and 6% others (post intervention). (p
)
The proportion of catheterized inpatients decreased from
23.7% to 21.0% (aOR 0.90, 95% CI 0.84e0.96; P¼0.001), and in
terms of catheter-days per 100 patient-days decreased from
17.4 to 13.5 (aRR 0.96, 95% CI 0.90e1.02; P¼0.167). The
CAUTI rate remained stable at a low level with 0.02 infections
per 100 patient-days (baseline) and 0.02 infections per 100
patient-days (post intervention) (aRR 1.01, 95% CI 0.51e2.00;
P¼0.98). In an alternative approach, measuring infections per
1000 catheter-days, the rate was 1.02 (baseline) and 1.33
(post intervention) [aRR 1.20, 95% CI 0.60e2.39; P¼0.6). Non-
infectious complications decreased significantly, from 0.79 to
0.56 events per 100 patient-days (aRR 0.73, 95% CI 0.61e0.88;
P<0.001), and from 39.4 to 35.4 events per 1000 catheter-
days (aRR 0.90, 95% CI 0.77e1.07; P¼0.23). The most The most common reasons for placing the first urinary
catheter were surgery (61% at baseline and 57% post inter-
vention), urine monitoring (16% at baseline and post inter-
vention) and urinary retention (13% at baseline and 17% post
intervention). Re-insertions
were
mainly
due
to
urinary
retention (60% vs 66%), surgery (21% vs 18%) and urine mon-
itoring (14% vs 10%). Age, sex, organizational unit and origin of the patient had an
impact
on
catheter
utilization,
rate
of
non-infectious A. Schweiger et al. / Journal of Hospital Infection 106 (2020) 364e371 369 References major confounder when assessing the effects of administered
measures. The participating hospitals e being ‘volunteers’ and
possibly top performers e may have exhibited lower levels of
urinary catheterization and its infectious and non-infectious
complications than other Swiss hospitals, and therefore may
not be representative for the country. Also, thorough infor-
mation on past CAUTI prevention activities in the study hospi-
tals was not collected. Finally, the mix of catheter materials
may have influenced the complication rates. Unfortunately, it
was not possible to determine the catheter material employed
in all cases, and therefore it is not possible to draw a robust
conclusion. [1] Magill SS, Edwards JR, Bamberg W, Beldavs ZG, Dumyati G,
Kainer MA, et al. Multistate point-prevalence survey of health
care-associated infections. N Engl J Med 2014;370:1198e208. [2] Uckay I, Sax H, Gayet-Ageron A, Ruef C, Muhlemann K,
Troillet N, et al. High proportion of healthcare-associated uri-
nary tract infection in the absence of prior exposure to urinary
catheter: a cross-sectional study. Antimicrob Resist Infect
Control 2013;2:5. [3] Zarb P, Coignard B, Griskeviciene J, Muller A, Vankerckhoven V,
Weist K, et al. The European Centre for Disease Prevention and
Control (ECDC) pilot point prevalence survey of healthcare-
associated infections and antimicrobial use. Eurosurveillance
2012;17:1e16. In conclusion, in this before/after intervention study of
urinary catheter utilization, a straightforward bundle of three
evidence-based measures (provision of a catheter indication
list, promotion of daily catheter evaluation and teaching state-
of-the-art catheter insertion) reduced catheter utilization and
led to increases in indicated urinary catheters and daily eval-
uations. The intervention had an impact on non-infectious
complications, whereas the CAUTI rate remained at a low
level, The next step is planning the national roll-out of both the
surveillance module and the intervention bundle, the compo-
nents of which have been made available to the public. [4] Lo E, Nicolle LE, Coffin SE, Gould C, Maragakis LL, Meddings J,
et al. Strategies to prevent catheter-associated urinary tract
infections in acute care hospitals. Infect Control Hosp Epidemiol
2014;35:S32e47. [5] Jansen IA, Hopmans TE, Wille JC, van den Broek PJ, van der
Kooi TI, van Benthem BH. Appropriate use of indwelling urethra
catheters in hospitalized patients: results of a multicentre prev-
alence study. BMC Urol 2012;12:25. [6] Aaronson DS, Wu AK, Blaschko SD, McAninch JW, Garcia M. National incidence and impact of noninfectious urethral catheter
related complications on the Surgical Care Improvement Project. J Urol 2011;185:1756e60. Funding source The ‘Progress! Safe urinary catheterization’ pilot pro-
gramme was funded by the Swiss Federal Office of Public
Health (SFOPH) and run by the Swiss Patient Safety Foun-
dation, which in turn subcontracted the National Center for
Infection Control (Swissnoso) to design and conduct the
surveillance component of the study. SFOPH (15.011083)
had no role in the study’s design, conduct and reporting. [15] Fakih MG, George C, Edson BS, Goeschel CA, Saint S. Imple-
menting a national program to reduce catheter-associated uri-
nary tract infection: a quality improvement collaboration of state
hospital associations, academic medical centers, professional
societies, and governmental agencies. Infect Control Hosp Epi-
demiol 2013;34:1048e54. [16] Fakih MG, Watson SR, Greene MT, Kennedy EH, Olmsted RN,
Krein SL, et al. Reducing inappropriate urinary catheter use: a
statewide effort. Arch Intern Med 2012;172:255e60. References [7] Saint S, Trautner BW, Fowler KE, Colozzi J, Ratz D, Lescinskas E,
et al. A multicenter study of patient-reported infectious and
noninfectious complications associated with indwelling urethral
catheters. JAMA Intern Med 2018;178:1078e85. Discussion A concise list of indi-
cations for catheterization, along with a description of sit-
uations that do not warrant catheterization, is the primary
step towards the reduction of unnecessary catheterization
[14], and several evidence-based indication lists are available
in the literature [4,26,35,36]. The use of reminders or stop
orders facilitates the daily re-evaluation of a catheter’s indi-
cation and potential for removal, thus reducing complications
[14,19,37,38]. The third element, ensuring that HCWs are
trained properly in the aseptic insertion of urinary catheters
and subsequent maintenance, lacks the level of supporting This was not a randomized study, and it is possible that
certain differences between the baseline and postintervention
populations were not accounted for. The uptake of the inter-
vention was only partially monitored (i.e. with respect to the
daily re-evaluation); it is known that subtle differences in the
implementation of infection prevention interventions are a A. Schweiger et al. / Journal of Hospital Infection 106 (2020) 364e371 370 Conflict of interest statement
None declared. Conflict of interest statement
None declared. [14] Saint S, Olmsted RN, Fakih MG, Kowalski CP, Watson SR, Sales AE,
et al. Translating health care-associated urinary tract infection
prevention research into practice via the bladder bundle. Jt
Comm J Qual Patient Saf 2009;35:449e55. Acknowledgements The authors wish to thank all the participating hospitals
and, more specifically, participating departments as well as
their local project leaders. [8] Hollingsworth JM, Rogers MA, Krein SL, Hickner A, Kuhn L,
Cheng A, et al. Determining the noninfectious complications of
indwelling urethral catheters: a systematic review and meta-
analysis. Ann Intern Med 2013;159:401e10. The ‘Progress! Safe urinary catheterization’ collaboration
group members and their respective institutions are: Adriana
Degiorgi (Ente Ospedaliero Cantonale, Lugano Civico), Thomas
Bregenzer (Hospital of Lachen), Stefan P. Kuster (University
and University Hospital Zurich), Hugo Sax (University and Uni-
versity Hospital Zurich), Marianne Laguardia (Inselspital, Bern
University Hospital and University of Bern), Isabelle Montavon-
Blondet (Hoˆpital Neuchaˆtelois), Jasmin Perret (Winterthur
Cantonal Hospital), Vineeta Bansal (Winterthur Cantonal Hos-
pital), Anja Waltenspu¨l (Lucerne Cantonal Hospital) and Sonja
Bertschy (Lucerne Cantonal Hospital). [9] Leuck AM, Wright D, Ellingson L, Kraemer L, Kuskowski MA,
Johnson JR. Complications of Foley catheters e is infection the
greatest risk? J Urol 2012;187:1662e6. [10] Munasinghe RL, Yazdani H, Siddique M, Hafeez W. Appropriate-
ness of use of indwelling urinary catheters in patients admitted to
the
medical
service. Infect
Control
Hosp
Epidemiol
2001;22:647e9. [11] Jain P, Parada JP, David A, Smith LG. Overuse of the indwelling
urinary tract catheter in hospitalized medical patients. Arch
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appropriateness in U.S. emergency departments 1995e2010. Acad Emerg Med 2014;21:292e300. The results of this study were presented, in part, at IDweek
2018, San Francisco, CA, USA on 6th October 2018. [13] Tiwari MM, Charlton ME, Anderson JR, Hermsen ED, Rupp ME. Inappropriate use of urinary catheters: a prospective observa-
tional study. Am J Infect Control 2012;40:51e4. A. Schweiger et al. / Journal of Hospital Infection 106 (2020) 364e371 371 catheter-associated urinary tract infection. Int J Qual Health
Care 2017;29:564e70. catheter-associated urinary tract infection. Int J Qual Health
Care 2017;29:564e70. [18] Titsworth WL, Hester J, Correia T, Reed R, Williams M, Guin P,
et al. Reduction of catheter-associated urinary tract infections
among patients in a neurological intensive care unit: a single
institution’s success. J Neurosurg 2012;116:911e20. [30] Knoll
BM,
Wright
D,
Ellingson
L,
Kraemer
L,
Patire
R,
Kuskowski MA, et al. Reduction of inappropriate urinary catheter
use at a veterans affairs hospital through a multifaceted quality
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grative review. BMJ Qual Saf 2014;23:277e89. p
g
mprovement project. Clin Infect Dis 2011;52:1283e90. [31] Parry MF, Grant B, Sestovic M. Successful reduction in catheter-
associated urinary tract infections: focus on nurse-directed
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agement with empowerment of nursing staff to reduce urinary
catheter use. Antimicrob Resist Infect Control 2015;4:P217. [32] Norman RE, Ramsden R, Ginty L, Sinha SK. Effect of a multimodal
educational intervention on use of urinary catheters in hospi-
talized individuals. J Am Geriat Soc 2017;65:2679e84. [21] Egger M, Balmer F, Friedli-Wu¨thrich H, Mu¨hlemann K. Reduc-
tion of urinary catheter use and prescription of antibiotics for
asymptomatic bacteriuria in hospitalised patients in internal
medicine. Swiss Med Wkly 2012;143:1e5. [33] Miller BL, Krein SL, Fowler KE, Belanger K, Zawol D, Lyons A,
et al. A multimodal intervention to reduce urinary catheter use
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Pittet D. Reduction of urinary tract infection and antibiotic use
after surgery: a controlled, prospective, beforeeafter inter-
vention study. Clin Infect Dis 2006;42:1544e51. [34] Wooller KR, Backman C, Gupta S, Jennings A, Hasimja-Saraqini D,
Forster AJ. A pre and post intervention study to reduce unnec-
essary urinary catheter use on general internal medicine wards of
a large academic health science center. BMC Health Serv Res
2018;18:642. [23] Harris AD, Bradham DD, Baumgarten M, Zuckerman IH, Fink JC,
Perencevich
EN. The
use
and
interpretation
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quasi-
experimental studies in infectious diseases. Clin Infect Dis
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prevention of catheter-associated urinary tract infections 2009. Infect Control Hosp Epidemiol 2010;31:319e26. [24] Niederhauser A, Zullig S, Marschall J, Schwappach DL. Nurses’
and physicians’ perceptions of indwelling urinary catheter prac-
tices
and
culture
in
their
institutions. J
Patient
Saf
2020;16:e82e9. [36] Harrod M, Kowalski CP, Saint S, Forman J, Krein S. Variations in
risk perceptions: a qualitative study of why unnecessary urinary
catheter use continues to be problematic. BMC Health Serv Res
2013;13:151. [25] Niederhauser A, Zullig S, Marschall J, Schweiger A, John G,
Kuster SP, et al. Safe urinary catheterization collaboration. Change in staff perspectives on indwelling urinary catheter use
after implementation of an intervention bundle in seven Swiss
acute care hospitals: results of a before/after survey study. BMJ
Open 2019;9:e028740. [37] Kachare SD, Sanders C, Myatt K, Fitzgerald TL, Zervos EE. Toward
eliminating catheter-associated urinary tract infections in an
academic health center. J Surg Res 2014;192:280e5. [38] Meddings J, Rogers MA, Macy M, Saint S. Systematic review and
meta-analysis: reminder systems to reduce catheter-associated
urinary tract infections and urinary catheter use in hospitalized
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hospitalized medical patients: results obtained by using the
RAND/UCLA
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method. Ann
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2015;162:S1e34. [39] Lo E, Nicolle LE, Coffin SE, Gould C, Maragakis LL, Meddings J,
et al. Strategies to prevent catheter-associated urinary tract
infections in acute care hospitals: 2014 update. Infect Control
Hosp Epidemiol 2014;35:464e79. [27] National Healthcare Safety Network. Urinary tract infection
(catheter-associated urinary tract infection [CAUTI] and non-
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nary system infection [USI]). Atlanta, GA: Centers for Disease
Control and Prevention; 2015. [40] Manojlovich M, Saint S, Meddings J, Ratz D, Havey R, Bickmann J,
et al. Indwelling urinary catheter insertion practices in the
emergency department: an observational study. Infect Control
Hosp Epidemiol 2016;37:117e9. [28] Saint S, Greene MT, Krein SL, Rogers MA, Ratz D, Fowler KE, et al. A program to prevent catheter-associated urinary tract infection
in acute care. N Engl J Med 2016;374:2111e9. [41] Fakih MG, Gould CV, Trautner BW, Meddings J, Olmsted RN,
Krein SL, et al. Beyond infection: device utilization ratio as a
performance measure for urinary catheter harm. Appendix A. Supplementary data [17] Jain M, Miller L, Belt D, King D, Berwick DM. Decline in ICU
adverse events, nosocomial infections and cost through a quality
improvement
initiative
focusing
on
teamwork
and
culture
change. Qual Saf Health Care 2006;15:235e9. Supplementary data to this article can be found online at
https://doi.org/10.1016/j.jhin.2020.07.002. A. Schweiger et al. / Journal of Hospital Infection 106 (2020) 364e371 A. Schweiger et al. / Journal of Hospital Infection 106 (2020) 364e371 Infect Control
Hosp Epidemiol 2016;37:327e33. [29] Theobald CN, Resnick MJ, Spain T, Dittus RS, Roumie CL. A multifaceted quality improvement strategy reduces the risk of
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Generative Embedding for Model-Based Classification of fMRI Data
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PLOS computational biology/PLoS computational biology
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cc-by
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ETH Library Abstract Decoding models, such as those underlying multivariate classification algorithms, have been increasingly used to infer
cognitive or clinical brain states from measures of brain activity obtained by functional magnetic resonance imaging (fMRI). The practicality of current classifiers, however, is restricted by two major challenges. First, due to the high data
dimensionality and low sample size, algorithms struggle to separate informative from uninformative features, resulting in
poor generalization performance. Second, popular discriminative methods such as support vector machines (SVMs) rarely
afford mechanistic interpretability. In this paper, we address these issues by proposing a novel generative-embedding
approach that incorporates neurobiologically interpretable generative models into discriminative classifiers. Our approach
extends previous work on trial-by-trial classification for electrophysiological recordings to subject-by-subject classification
for fMRI and offers two key advantages over conventional methods: it may provide more accurate predictions by exploiting
discriminative information encoded in ‘hidden’ physiological quantities such as synaptic connection strengths; and it affords
mechanistic interpretability of clinical classifications. Here, we introduce generative embedding for fMRI using a
combination of dynamic causal models (DCMs) and SVMs. We propose a general procedure of DCM-based generative
embedding for subject-wise classification, provide a concrete implementation, and suggest good-practice guidelines for
unbiased application of generative embedding in the context of fMRI. We illustrate the utility of our approach by a clinical
example in which we classify moderately aphasic patients and healthy controls using a DCM of thalamo-temporal regions
during speech processing. Generative embedding achieves a near-perfect balanced classification accuracy of 98% and
significantly outperforms conventional activation-based and correlation-based methods. This example demonstrates how
disease states can be detected with very high accuracy and, at the same time, be interpreted mechanistically in terms of
abnormalities in connectivity. We envisage that future applications of generative embedding may provide crucial advances
in dissecting spectrum disorders into physiologically more well-defined subgroups. Citation: Brodersen KH, Schofield TM, Leff AP, Ong CS, Lomakina EI, et al. (2011) Generative Embedding for Model-Based Classification of fMRI Data. PLoS Comput
Biol 7(6): e1002079. doi:10.1371/journal.pcbi.1002079 Editor: Olaf Sporns, Indiana University, United States of America Received January 7, 2011; Accepted April 20, 2011; Published June 23, 2011 Received January 7, 2011; Accepted April 20, 2011; Published June 23, 2011 Copyright: 2011 Brodersen 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. Abstract Funding: This study was funded by the University Research Priority Program ‘Foundations of Human Social Behaviour’ at the University of Zurich (KHB, KES), the
SystemsX.ch project NEUROCHOICE (KHB, KES; http://www.systemsx.ch/), the NCCR ‘Neural Plasticity’ (KES; http://www.nccr-neuro.uzh.ch/), the Wellcome Trust
(APL; grant ME033459MES), and the NIHR CBRC at University College Hospitals London (APL). 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: kay.brodersen@inf.ethz.ch Kay H. Brodersen1,2*, Thomas M. Schofield3, Alexander P. Leff3, Cheng Soon Ong1, Ekaterina I.
Lomakina1,2, Joachim M. Buhmann1, Klaas E. Stephan2,3 1 Department of Computer Science, ETH Zurich, Zurich, Switzerland, 2 Laboratory for Social and Neural Systems Research, Department of Economics, University of Zurich,
Zurich, Switzerland, 3 Wellcome Trust Centre for Neuroimaging, University College London, London, United Kingdom Journal Article Originally published in:
PLoS Computational Biology 7(6), https://doi.org/10.1371/journal.pcbi.1002079 This page was generated automatically upon download from the ETH Zurich Research Collection. For more information, please consult the Terms of use. This page was generated automatically upon download from the ETH Zurich Research Collection. For more information, please consult the Terms of use. PLoS Computational Biology | www.ploscompbiol.org Generative Embedding for Model-Based Classification of
fMRI Data Kay H. Brodersen1,2*, Thomas M. Schofield3, Alexander P. Leff3, Cheng Soon Ong1, Ekaterina I. Lomakina1,2, Joachim M. Buhmann1, Klaas E. Stephan2,3 June 2011 | Volume 7 | Issue 6 | e1002079 Citation: Brodersen KH, Schofield TM, Leff AP, Ong CS, Lomakina EI, et al. (2011) Generative Embedding for Model-Based Classification of fMRI Data. PLoS Comput
Biol 7(6): e1002079. doi:10.1371/journal.pcbi.1002079 Challenges for current classification approaches Challenges for current classification approaches
Despite their increasing popularity, two challenges critically
limit the practical applicability of current classification methods for
functional neuroimaging data. First, classifying subjects directly in
voxel space is often a prohibitively difficult task. This is because
functional neuroimaging datasets (i) typically exhibit a low signal-
to-noise ratio, (ii) are obtained in an extremely high-dimensional
measurement space (a conventional fMRI scan contains more than
100,000 voxels), and (iii) are characterized by a striking mismatch
between the large number of voxels and the small number of
available subjects. As a result, even the most carefully designed
algorithms have great difficulties in reliably finding jointly
informative voxels while ignoring uninformative sources of noise. Popular strategies include: preselecting voxels based on an
anatomical mask [18,19] or a separate functional localizer
[20,21]; spatial subsampling [22]; finding informative voxels using
univariate models [3,11,12] or locally multivariate searchlight
methods [23,24]; and unsupervised dimensionality reduction
[4,25]. Other recently proposed strategies attempt to account for
the inherent spatial structure of the feature space [23,26,27] or use
voxel-wise models to infer a particular stimulus identity [28–30]. Finally, those submissions that performed best in the Pittsburgh
Brain Activity Interpretation Competition (PBAIC 2007) high-
lighted the utility of kernel ridge regression [31] and relevance
vector regression [31,32]. The common assumption underlying all
of these approaches is that interesting variations of the data with
regard to the class variable are confined to a manifold that
populates a latent space of much lower dimensionality than the
measurement space. Generative embedding represents a special case of using
generative kernels for classification, such as the P-kernel [52] or the
Fisher kernel [53]. Generative kernels have been fruitfully
exploited in a range of applications [54–66] and define an active
area of research [67–70]. In the special case of generative
embedding, a generative kernel is used to construct a generative score
space. This is a model-based feature space in which the original
observations have been replaced by statistical representations that
potentially yield better class separability when fed into a
discriminative classifier. Thus, an unsupervised embedding step
is followed by a supervised classification step. In previous work, we
suggested a concrete implementation of this approach for the trial-
by-trial classification of electrophysiological recordings [61]. Author Summary Author Summary Neurological
and
psychiatric
spectrum
disorders
are
typically defined in terms of particular symptom sets,
despite increasing evidence that the same symptom may
be caused by very different pathologies. Pathophysiolog-
ical classification and effective treatment of such disorders
will increasingly require a mechanistic understanding of
inter-individual differences and clinical tools for making
accurate
diagnostic
inference
in
individual
patients. Previous classification studies have shown that functional
magnetic resonance imaging (fMRI) can be used to
differentiate between healthy controls and neurological
or psychiatric patients. However, these studies are typically
based on descriptive patterns and indirect measures of
neural activity, and they rarely afford mechanistic insights
into the underlying condition. In this paper, we address
this challenge by proposing a classification approach that
rests on a model of brain function and exploits the rich
discriminative information encoded in directed interre-
gional connection strengths. Based on an fMRI dataset
acquired from moderately aphasic patients and healthy
controls, we illustrate that our approach enables more
accurate classification and deeper mechanistic insights
about disease processes than conventional classification
methods. Introduction differences in brain activity across clinical groups, they are less well
suited for clinical decision making, where the challenge is to
predict the disease state of an individual subject from measured
brain activity [3–5]. Recent years have seen a substantial increase in the use of
functional neuroimaging data for investigating healthy brain
function and detecting abnormalities. The most popular type of
analysis is statistical parametric mapping (SPM), a mass-univariate
encoding model of fMRI data in which the statistical relationship
between experimental (or clinical) variables and haemodynamic
measurements of neural activity is examined independently for
every voxel in the brain [1]. While this approach has led to many
insights
about
functional
abnormalities
in
psychiatric
and
neurological disorders, it suffers from two limitations. First, since
univariate models are insensitive to spatially distributed patterns of
neural activity, they may fail to detect subtle, distributed
differences between patients and healthy controls that are not
expressed as local peaks or clusters of activity [2]. Second, while
encoding models such as SPM are excellent for describing regional An alternative approach is provided by multivariate decoding
methods, in particular classification algorithms. Unlike mass-
univariate encoding models, these methods predict an experimen-
tal variable (e.g., a trial-specific condition or subject-specific
disease state) from the activity pattern across voxels (see [6–10] for
reviews). Using multivariate decoding models instead of mass-
univariate encoding models has interesting potential for clinical
practice, particularly for diseases that are difficult to diagnose. Consequently,
much
work
is
currently
being
invested
in
constructing classifiers that can predict the diagnosis of individual
subjects from structural or functional brain data [11,3,12,13,4,14–
16]. Historically, these efforts date back to positron emission
tomography (PET) studies in the early 1990s [8]. Today, attempts PLoS Computational Biology | www.ploscompbiol.org 1 June 2011 | Volume 7 | Issue 6 | e1002079 June 2011 | Volume 7 | Issue 6 | e1002079 Generative Embedding for fMRI support discriminative decisions, but they are blind to the neuronal
mechanisms (such as effective connectivity or synaptic plasticity) that
underlie discriminability of brain or disease states. In other words:
while some conventional classification studies have achieved
impressive diagnostic accuracy [14], their results have not improved
our mechanistic understanding of disease processes. Generative embedding Generative embedding for model-based classification may
provide a solution to the challenges outlined above. It is based
on the idea that both the performance and interpretability of
conventional approaches could be improved by taking into
account available prior knowledge about the process generating
the observed data (see [44] for an overview). (The term generative
embedding is sometimes used to denote a particular model-induced
feature space, or so-called generative score space, in which case
the associated line of research is said to be concerned with generative
embeddings. Here, we will use the term in singular form to denote
the process of using a generative model to project the data into a
generative score space, rather than using the term to denote the
space itself.) Generative embedding rests on two components: a
generative model for principled selection of mechanistically
interpretable features and a discriminative method for classifica-
tion (see Figure 1). Generative models have proven powerful in explaining how
observed data are caused by the underlying (neuronal) system. Unlike
their
discriminative
counterparts,
generative
models
capture the joint probability of the observed data and the class
labels, governed by a set of parameters of a postulated generative
process. One example in neuroimaging is dynamic causal modelling
(DCM) [45]. DCM enables statistical inference on physiological
quantities that are not directly observable with current methods,
such as directed interregional coupling strengths and their
modulation, e.g., by synaptic gating [46]. (We use the term
DCM to refer both to a specific dynamic causal model and to
dynamic causal modelling as a method.) From a pathophysiolog-
ical perspective, disturbances of synaptic plasticity and neuromo-
dulation are at the heart of psychiatric spectrum diseases such as
schizophrenia [47] or depression [48]. It is therefore likely that
classification of disease states could benefit from exploiting
estimates of these quantities. While DCM is a natural (and
presently the only) candidate for obtaining model-based estimates
of synaptic plasticity (cf. [46,49]), the most widely used approach
to classification relies on discriminative methods, such as support
vector machines (SVMs) [50,51]. Together, DCM and SVM
methods thus represent natural building blocks for classification of
disease states. of using multivariate classifiers for subject-by-subject diagnosis
largely focus on MRI and fMRI data [11,3,12,17]. PLoS Computational Biology | www.ploscompbiol.org Challenges for current classification approaches Conceptual overview of generative embedding for fMRI. This schematic illustrates the key principles by which generative
embedding enables model-based classification for functional magnetic resonance imaging (fMRI). Initially, each subject is represented by a measure
of blood oxygen level dependent (BOLD) activity with one temporal and three spatial dimensions. In the first analysis step (model inversion), these
subject-specific data are used to estimate the parameters of a generative model, which represents a mapping of the data x[X onto a probability
distribution p(hjx,m) in a parametric family MH (see Sections ‘DCM for fMRI’ and ‘Model inversion’). In the second step (kernel construction), a kernel
function kM : MH|MH?R is defined that represents a similarity metric between any two fitted models mi and mj. This step can be split up into an
initial mapping MH?Rd followed by a conventional kernel k : Rd|Rd?R. The kernel implies a generative score space (or model-based feature
space; see Section ‘Kernel construction’), which provides a comprehensive statistical representation of every subject. In this illustrative participant, the
influence of region A on region B as well as the self-connection of region B were particularly strong. In the third step, a classifier is used to find a
separating hyperplane between groups of subjects, based exclusively on their model-based representations (see Section ‘Classification’). When using
a linear kernel, each feature corresponds to the coupling strength between two regions, which, in the fourth step, enables a mechanistic
interpretation of feature weights in the context of the underlying model (see Section ‘Interpretation of the feature space’). Here, the influence of A on
B and C were jointly most informative in distinguishing between groups. For a concrete implementation of this procedure, see Figure 2. doi:10.1371/journal.pcbi.1002079.g001 using generative embedding: the higher the temporal and spatial
resolution of the fMRI data, the more precise the estimation of the
parameters of the generative model, leading to better discrimina-
bility. Third, our approach can be used to compare alternative
generative model architectures in situations where evidence-based
approaches, such as Bayesian model selection, are not applicable. We will deal with these three points in more detail in the
Discussion. DCM [45] views the brain as a nonlinear dynamical system of
interconnected neuronal populations whose directed connection
strengths are modulated by external perturbations (i.e., experi-
mental conditions) or endogenous activity. Here, we will use DCM
to
replace
high-dimensional
fMRI
time
series
by
a
low-
dimensional vector of parameter estimates. Challenges for current classification approaches The discriminative
part of our approach will be based on an SVM with a linear
kernel. This algorithm learns to discriminate between two groups
of subjects by estimating a separating hyperplane in their feature
space. Since this paper brings together techniques from different
statistical domains that tend to be used by different communities,
we have tried to adopt a tutorial-like style and introduce basic
concepts of either approach in the Methods section. Challenges for current classification approaches In this
paper, we propose a DCM-based generative-embedding approach
for subject-by-subject classification of fMRI data, demonstrate its
performance using a clinical data set, and highlight potential
methodological pitfalls (and how to avoid them). The second challenge for classification methods concerns the
interpretation of their results. Most classification studies to date draw
conclusions from overall prediction accuracies [33,11], the spatial
deployment of informative voxels [19,34,18,35–39], the temporal
evolution of discriminative information [40,37,41,42,26], or patterns
of undirected regional correlations [43]. These approaches may PLoS Computational Biology | www.ploscompbiol.org June 2011 | Volume 7 | Issue 6 | e1002079 June 2011 | Volume 7 | Issue 6 | e1002079 2 Generative Embedding for fMRI Figure 1. Conceptual overview of generative embedding for fMRI. This schematic illustrates the key principles by which generative
embedding enables model-based classification for functional magnetic resonance imaging (fMRI). Initially, each subject is represented by a measure
of blood oxygen level dependent (BOLD) activity with one temporal and three spatial dimensions. In the first analysis step (model inversion), these
subject-specific data are used to estimate the parameters of a generative model, which represents a mapping of the data x[X onto a probability
distribution p(hjx,m) in a parametric family MH (see Sections ‘DCM for fMRI’ and ‘Model inversion’). In the second step (kernel construction), a kernel
function kM : MH|MH?R is defined that represents a similarity metric between any two fitted models mi and mj. This step can be split up into an
initial mapping MH?Rd followed by a conventional kernel k : Rd|Rd?R. The kernel implies a generative score space (or model-based feature
space; see Section ‘Kernel construction’), which provides a comprehensive statistical representation of every subject. In this illustrative participant, the
influence of region A on region B as well as the self-connection of region B were particularly strong. In the third step, a classifier is used to find a
separating hyperplane between groups of subjects, based exclusively on their model-based representations (see Section ‘Classification’). When using
a linear kernel, each feature corresponds to the coupling strength between two regions, which, in the fourth step, enables a mechanistic
interpretation of feature weights in the context of the underlying model (see Section ‘Interpretation of the feature space’). Here, the influence of A on
B and C were jointly most informative in distinguishing between groups. For a concrete implementation of this procedure, see Figure 2. doi:10.1371/journal.pcbi.1002079.g001 Figure 1. PLoS Computational Biology | www.ploscompbiol.org June 2011 | Volume 7 | Issue 6 | e1002079 Combining generative models and discriminative
methods This product can be replaced
by the evaluation k(xi,xj) of a kernel function k : Rd|Rd?R, which
implicitly computes the inner product between the examples in a
new feature space, Sw(xi),w(xj)T. The specific implementation for fMRI data proposed in this
paper consists of four conceptual steps which are summarized in
Figure 1 and described in the following subsections. First, a
mapping X?MH is designed that projects an example x[X from
data space onto a multivariate probability distribution in a
parametric family MH. In our case, we use the fMRI data from
each subject to estimate the posterior density of the parameters of
a DCM (Sections ‘DCM for fMRI’ and ‘Model inversion’). Second, a probability kernel kM : MH|MH is constructed that
represents a similarity measure between two inverted DCMs. Here, we use a simple linear kernel on the maximum a posteriori
(MAP) estimates of the model parameters (Sections ‘Strategies for
unbiased model specification and inversion’ and ‘Kernel construc-
tion’). Third, this kernel is used for training and testing a
discriminative classifier (Section ‘Classification’). Here, we employ
a linear SVM to distinguish between patients and healthy controls. Fourth, the constructed feature space can be investigated to find
out
which
model
parameters
jointly
contributed
most
to
distinguishing the two groups (Section ‘Interpretation of the
feature space’). We will conclude with an example in which we
distinguish between patients with moderate aphasia and healthy
controls (Sections ‘Experimental design, data acquisition, and
preprocessing,’ ‘Implementation of generative embedding,’ and
‘Comparative analyses’). j
The ‘2-norm SVM is a natural choice when the goal is maximal
prediction accuracy. However, it usually leads to a dense solution
(as opposed to a sparse solution) in which almost all features are
used for classification. This is suboptimal when one wishes to
understand which model parameters contribute most to distin-
guishing
groups,
which
will
be
the
focus
in
the
Section
‘Interpretation of the feature space.’ In this case, an SVM that
enforces feature sparsity may be more useful. One simple way of
inducing sparsity is to penalize the number of non-zero coefficients
by using an ‘0-regularizer. Unlike other regularizers, the ‘0-norm
(also known as the counting norm) reduces the feature-selection bias
inherent in unbounded regularizers such as the ‘1- or ‘2-norm. The computational cost of optimizing an ‘0-SVM objective
function is prohibitive, because the number of subsets of d items
which are of size k is exponential in k. Structure of this paper The remainder of this paper is structured as follows. First, we
summarize the general ideas of generative embedding and the
specific generative and discriminative components used here, i.e.,
DCM and SVM. We then inspect different procedures of how
generative embedding could be implemented practically while
distinguishing between approaches with and without bias. Third,
we illustrate the utility of our approach, using empirical data
obtained during speech processing in healthy volunteers and
patients with moderate aphasia. These data have been explored in
a previous study, in which DCM and Bayesian model selection
(BMS) were applied to investigate the effective connectivity among
cortical areas activated by intelligible speech [71]. In a subsequent
study, we extended this analysis to patients with aphasia (Schofield
et al., in preparation). In the present paper, we ask whether subject-
specific directed connection strengths among cortical regions Generative embedding for fMRI may offer three substantial
advantages over conventional classification methods. First, because
the approach aims to fuse the strengths of generative models with
those of discriminative methods, it may outperform conventional
voxel-based schemes, especially in those cases where crucial
discriminative information is encoded in ‘hidden’ quantities such
as directed (synaptic) connection strengths. Second, the construc-
tion of the feature space is governed and constrained by a
biologically motivated systems model. As a result, feature weights
can be interpreted mechanistically in the context of this model. Incidentally, the curse of dimensionality faced by many conven-
tional feature-extraction methods may turn into a blessing when June 2011 | Volume 7 | Issue 6 | e1002079 June 2011 | Volume 7 | Issue 6 | e1002079 3 Generative Embedding for fMRI efficient difference-of-convex-functions algorithm (Ong & Thi,
under review). involved in speech processing contain sufficiently rich discrimina-
tive information to enable accurate predictions of the diagnostic
category (healthy or aphasic) of a previously unseen individual. In
brief, we found that (i) generative embedding yielded a near-
perfect classification accuracy, (ii) significantly outperformed
conventional ‘gold standard’ activation-based and correlation-
based classification schemes, and (iii) afforded a novel mechanistic
interpretation of the differences between aphasic patients and
healthy controls during processing of speech and speech-like
sounds. In summary, we will use two types of SVM. For the purpose of
classification (Section ‘Classification’), we aim to maximize the
potential for highly accurate predictions by using an ‘2-norm
SVM. Combining generative models and discriminative
methods Most methods for classification attempt to find a linear function
that separates examples as accurately as possible in a space of
features (e.g., voxel-wise measurements). Such discriminative classi-
fication methods differ from generative methods in two ways. First,
rather than trying to estimate the joint density of observations and
class labels, which is not needed for classification, or trying to
estimate class-conditional probability densities, which can be
difficult, discriminative classifiers directly model the class an
example belongs to. Second, many discriminative methods do not
operate on examples themselves but are based on the similarity
between any two examples, expressed as the inner product
between their feature vectors. This provides an elegant way of
transforming a linear classifier into a more powerful nonlinear one. (Note that the term discriminative methods is used here to collectively
describe the class of learning algorithms that find a discriminant
function for mapping an example x onto a class label c, typically
without invoking probability theory. This is in contrast to
discriminative models, which model the conditional probability
p(cjx), and generative models, which first model the full joint
probability p(x,c) and then derive p(cjx).) Generative kernels are functions that define a similarity metric
for observed examples using a generative model. In the case of a
dynamic causal model (DCM), for example, the observed time
series are modelled by a system of parameterized differential
equations with Gaussian observation noise. Generative embedding
defines a generative kernel by transferring the models into a
vectorial feature space in which an appropriate similarity metric is
defined (see Figure 1). This feature space, which we will refer to as
a generative score space, embodies a model-guided dimensionality
reduction of the observed data. The kernel defined in this space
could be a simple inner product of feature vectors, or it could be
based on any other higher-order function, as long as it is positive
definite
[76]. In
conclusion,
model-based
classification
via
generative
embedding
is
a
hybrid
generative-discriminative
approach: it merges the explanatory abilities of generative models
with the classification power of discriminative methods. The most popular classification algorithm of the above kind is the
‘2-norm soft-margin support vector machine (SVM) [50,51,72,73]. The only way in which examples xi[Rd(i~1,:::,n) enter an SVM is
in terms of an inner product Sxi,xjT. Ethics statement The study was approved by the local research ethics committee
at UCL, and all participants gave informed consent. Structure of this paper For the purpose of feature selection and interpretation
(Section ‘Interpretation of the feature space’), we will focus on
feature sparsity by using an approximation to an ‘0-norm SVM,
which will highlight those DCM parameters jointly deemed most
informative in distinguishing between groups. Most
current
applications
of
classification
algorithms
in
neuroimaging begin by embedding the measured recordings of
each subject in a d-dimensional Euclidean space Rd. In fMRI, for
example, a subject can be represented by a vector of d features,
each of which corresponds to the signal measured in a particular
voxel at a particular point in time. This approach makes it possible
to use any learning algorithm that expects vectorial input, such as
an SVM; but it ignores the spatio-temporal structure of the data as
well as the process that generated them. This limitation has
motivated the search for kernel methods that provide a more
natural way of measuring the similarity between the functional
datasets of two subjects, for example by incorporating prior
knowledge about how the data were generated, which has led to
the idea of generative kernels, as described below. PLoS Computational Biology | www.ploscompbiol.org Model inversion Depending on the measurement modality, this can
be a set of nonlinear differential equations (as for fMRI [83]) or a
simple linear equation (as for EEG [84]). While the forward model
plays a critical role in model inversion, it is the parameters of the
neuronal model that are typically of primary scientific interest. In this paper, we will use the classical bilinear DCM for fMRI
[45] as implemented in the software package SPM8/DCM10, dz(t)
dt ~f (z(t),hn,u(t))~(Az
X
j uj(t)B(j))z(t)zCu(t)
ð1Þ Model specification and selection is an important theme in
DCM [87]. In this paper we are not concerned with the question
of which of several alternative DCMs may be optimal for
explaining the data or for classifying subjects; these issues can be
addressed using Bayesian evidence methods [88,89] or by applying
cross-validation to the classifications suggested by each of the
models, respectively (see [61] for an example). However, an
important issue is that model specification cannot be treated in
isolation from its subsequent use for classification. Specifically,
some procedures for selecting time series can lead to biased
estimation of classification accuracy. In the next section, we
therefore provide a detailed assessment of different strategies for
time series selection in DCM-based generative embedding and
highlight those procedures which safeguard against obtaining
optimistic estimates of classification performance. x(t)~g(z(t),hh)ze,
ð2Þ ð2Þ where z(t) represents the neuronal state vector z at time t, A is a
matrix of endogenous connection strengths, B(j) represents the
additive
change
of
these
connection
strengths
induced
by
modulatory input mj, and C denotes the strengths of direct (driving)
inputs. These neuronal parameters hn~(A,B(1),:::,B(J),C) are rate
constants with units s{1. The haemodynamic forward model is given by the function
g(z(t),hh), a nonlinear operator that links a neuronal state z(t) to a
predicted blood oxygen level dependent (BOLD) signal via
changes in vasodilation, blood flow, blood volume, and deox-
yhaemoglobin content (see [83] for details). This forward model
has haemodynamic parameters hh and Gaussian measurement
error e. The haemodynamic parameters primarily serve to account
for variations in neurovascular coupling across regions and
subjects and are typically not of primary scientific interest. In
addition, the haemodynamic parameters exhibit strong inter-
dependencies and thus high posterior covariances and low
precision [83], which makes it difficult to establish the distinct
contribution afforded by each parameter. Model inversion Bayesian inversion of a given dynamic causal model m defines a
map X?MH that projects a given example x[X (i.e., data from a
single subject) onto a multivariate probability distribution p(hjx,m)
in a parametric family MH. The model architecture m specifies the
neuronal populations (regions) of interest, experimentally con-
trolled inputs u, synaptic connections, and a prior distribution over
the parameters p(hjm). Given the model m and subject-specific
data x, model inversion proceeds in an unsupervised and subject-
by-subject fashion, i.e., in ignorance of the subject label that will
later be used in the context of classification. (The literature on
DCM has adopted the convention of denoting the hidden states by
x and the data by y. Here, in order to keep the notation consistent
with the literature on classification, we use x for the data and c for
the labels. A distinct symbol for the hidden states is not required
here.) DCM uses a fully Bayesian approach to parameter
estimation, with empirical priors for the haemodynamic param-
eters and conservative shrinkage priors for the coupling param-
eters [85,45]. Combining the prior density over the parameters
p(hjm) with the likelihood function p(xjh,m) yields the posterior
density p(hjx,m). This inversion can be carried out efficiently by
maximizing a variational free-energy bound to the log model
evidence, ln p(xjm), under Gaussian assumptions about the
posterior (the Laplace assumption; see [86] for details). Given d
parameters, model inversion thus yields a subject-specific proba-
bility density p(hjx,m) that can be fully described in terms of a
vector of posterior means ^m[Rd and a covariance matrix ^S[Rd|d. DCMs consist of two hierarchical layers [82]. The first layer is a
neuronal model of the dynamics of interacting neuronal populations
in the context of experimental perturbations. Critically, its
parameters are neurobiologically interpretable, representing, for
example, synaptic weights and their context-specific modulation;
electrophysiological DCMs describe even more fine-grained
processes such as spike-frequency adaptation or conduction delays. Experimental manipulations u enter the model in two different
ways: they can elicit responses through direct influences on specific
regions (e.g., sensory inputs), or they can modulate the strength of
coupling among regions (e.g., task demands or learning). The
second layer of a DCM is a biophysically motivated forward model
that describes how a given neuronal state translates into a
measurement. DCM for fMRI dynamics in an interconnected network of neuronal populations
and its experimentally induced perturbations. Inverting such a
model (see next section) means to infer the posterior distribution of
the parameters of both the neuronal and the forward model from
observed responses of a specific subject. Its mechanistic interpret-
ability and applicability to single-subject data makes DCM an
attractive candidate for generative embedding of fMRI data. DCM regards the brain as a nonlinear dynamic system of
interconnected nodes, and an experiment as a designed pertur-
bation of the system’s dynamics [45]. Its goal is to provide a
mechanistic model for explaining experimental measures of brain
activity. While the mathematical formulation of DCMs varies
across measurement types, common mechanisms modelled by all
DCMs include synaptic connection strengths and experimentally
induced modulation thereof [46,77–80]. Generally, DCMs strive
for neurobiological interpretability of their parameters; this is one
core feature distinguishing them from alternative approaches, such
as multivariate autoregressive models [81] which characterize
inter-regional connectivity in a phenomenological fashion. Generative Embedding for fMRI Generative Embedding for fMRI Model inversion For these reasons,
the model-induced feature spaces in this paper will be based
exclusively on the neuronal parameters hn. PLoS Computational Biology | www.ploscompbiol.org Combining generative models and discriminative
methods We therefore replace the ‘0-
norm by a capped ‘1-regularizer which has very similar properties
[74]. One way of solving the resulting optimization problem is to
use a bilinear programming approach [75]. Here, we use a more PLoS Computational Biology | www.ploscompbiol.org June 2011 | Volume 7 | Issue 6 | e1002079 4 Strategies for unbiased model specification and inversion Strategies for unbiased model specification and inversion
For conventional fMRI classification procedures, good-practice
guidelines have been suggested for avoiding an optimistic bias in
assessing classification performance [8,10]. Generally, to obtain an
unbiased estimate of generalization accuracy, a classifier must be
applied to test data that have not been used during training. In
generative embedding, this principle implies that the specification
of the generative model cannot be treated in isolation from its use
for classification. In this section, we structure different strategies in
terms of a decision tree and evaluate the degree of bias they invoke
(see Figure 2). The first distinction is based on whether the regions of interest
(ROIs) underlying the DCM are defined anatomically or functionally. In summary, DCM provides a mechanistic model for explaining
measured time series of brain activity as the outcome of hidden June 2011 | Volume 7 | Issue 6 | e1002079 June 2011 | Volume 7 | Issue 6 | e1002079 5 Generative Embedding for fMRI Generative Embedding for fMRI Figure 2. Strategies for unbiased DCM-based generative embedding. This figure illustrates how generative embedding can be implemented
using dynamic causal modelling. Depending on whether regions of interest are defined anatomically, based on across-subjects functional contrasts,
or based on between-group contrasts, there are several possible practical procedures. Some of these procedures may lead to biased estimates of
classification accuracy (grey boxes). Procedures a, c, and f avoid this bias, and are therefore recommended (green boxes). The analysis of the
illustrative dataset described in this paper follows procedure c. doi:10.1371/journal.pcbi.1002079.g002 Figure 2. Strategies for unbiased DCM-based generative embedding. This figure illustrates how generative embedding can be implemented
using dynamic causal modelling. Depending on whether regions of interest are defined anatomically, based on across-subjects functional contrasts,
or based on between-group contrasts, there are several possible practical procedures. Some of these procedures may lead to biased estimates of
classification accuracy (grey boxes). Procedures a, c, and f avoid this bias, and are therefore recommended (green boxes). The analysis of the
illustrative dataset described in this paper follows procedure c. doi:10.1371/journal.pcbi.1002079.g002 cross-validation, a situation referred to as peeking [10]. In
consequence, the resulting generalization estimate may exhibit
an optimistic bias. To avoid this bias, model specification must be
integrated into cross-validation (Figure 2c). Specifically, in each
fold, we leave out one subject as a test subject and compute an
across-subjects group contrast from the remaining n{1 subjects. Strategies for unbiased model specification and inversion The resulting choice of voxels is then used for specifying time series
in each subject and the resulting model is inverted separately for
each subject, including the left-out test subject. This procedure is
repeated n times, each time leaving out a different subject. In total,
the model will be inverted n2 times. In this way, within each cross-
validation fold, the selection of voxels is exclusively based on the
training data, and no peeking is involved. This is the strategy
adopted for the dataset analysed in this paper, as detailed in the
Section ‘Implementation of generative embedding’. When ROIs are defined exclusively on the basis of anatomical
masks (Figure 2a), the selection of voxels is independent of the
functional data. Using time series from these regions, the model is
inverted separately for each subject. Thus, given n subjects, a
single initial model-specification step is followed by n subject-wise
model inversions. The resulting parameter estimates can be safely
submitted to a cross-validation procedure to obtain an unbiased
estimate of classification performance. When ROIs are defined exclusively on the basis of anatomical
masks (Figure 2a), the selection of voxels is independent of the
functional data. Using time series from these regions, the model is
inverted separately for each subject. Thus, given n subjects, a
single initial model-specification step is followed by n subject-wise
model inversions. The resulting parameter estimates can be safely
submitted to a cross-validation procedure to obtain an unbiased
estimate of classification performance. Whenever functional contrasts have played a role in defining
ROIs, subsequent classification may no longer be unbiased. This is
because a functional contrast introduces statistics of the data into
voxel selection, which usually generates a bias. In this case, we ask
whether contrasts are defined in an across-subjects or a between-groups
fashion. In the case of an across-subjects contrast (which does not
take into account group membership), one might be tempted to
follow the same logic as in the case of anatomical ROI definitions:
a single across-subjects contrast, computed for all subjects, guides
the selection of voxels, and the resulting DCM is inverted
separately for each subject (Figure 2b). Unfortunately, this
procedure is problematic. When using the resulting parameter
estimates in a leave-one-out cross-validation scheme, in every
repetition the features would be based on a model with regions
determined by a group contrast that was based on the data from
all subjects, including the left-out test subject. PLoS Computational Biology | www.ploscompbiol.org June 2011 | Volume 7 | Issue 6 | e1002079 Kernel construction If, however,
the classifier has taken advantage of an imbalanced dataset, then
the ordinary accuracy will be inflated, whereas the balanced
accuracy will drop to chance (50%), as desired. The balanced
accuracy thus removes the bias from estimates of generalizability
that may arise in the presence of imbalanced datasets. A
probability
interval
can
be
computed
by
considering
the
convolution
of
two
Beta-distributed
random
variables
that
correspond to the true accuracies on positive and negative
examples, respectively. A p-value can then be obtained by
computing the posterior probability of the accuracy being below
chance [92]. Once
a
generative
score
space
has
been
created,
any
conventional kernel k : Rd|Rd?R can be used to compare
two inverted models. The simplest one is the linear kernel
k(xi,xj)~Sxi,xjT, representing the inner product between two
vectors xi and xj. Nonlinear kernels, such as quadratic, polynomial
or radial basis function kernels, transform the generative score
space, which makes it possible to consider quadratic (or higher-
order) class boundaries and therefore account for possible
interactions between features. Nonlinear kernels, however, have
several disadvantages for generative embedding. As the complexity
of the kernel increases, so does the risk of overfitting. Furthermore,
feature weights are easiest to interpret in relation to the underlying
model when they do not undergo further transformation; then, the
contribution of a particular feature (i.e., model parameter) to the
success of the classifier can be understood as the degree to which
the neuronal mechanism represented by that parameter aids
classification. A simple linear kernel will therefore be our preferred
choice. d Strategies for unbiased model specification and inversion This means that
training the classifier would no longer be independent of the test
data, which violates the independence assumption underlying When functional contrasts are not defined across all subjects but
between groups, the effect of peeking may become particularly
severe. Using a between-groups contrast to define regions of
interest on the basis of all available data, and using these regions to
invert the model for each subject (Figure 2d) would introduce
information about group membership into the process of voxel
selection. Thus, feature selection for both training and test data
would be influenced by both the data and the label of the left-out
test subject. One way of decreasing the resulting bias is to integrate
model specification into cross-validation (Figure 2e). In this
procedure, the between-groups contrast is computed separately PLoS Computational Biology | www.ploscompbiol.org June 2011 | Volume 7 | Issue 6 | e1002079 6 Generative Embedding for fMRI these two steps define a probability kernel kM : MH|MH?R that
represents a similarity metric between two inverted models and
allows for mechanistic interpretations of how group membership
of different subjects is encoded by spatiotemporal fMRI data. for each training set (i.e., based on n{1 subjects), and the resulting
regions are used to invert the model for the test subject. This
means that the class label of the test subject is no longer involved in
selecting features for the test subject. However, the test label
continues to influence the features of the training set, since these
are based on contrasts defined for a group that included the test
subject. This bias can only be removed by adopting the same
laborious procedure as with across-subjects contrasts: by using a
between-groups contrast involving n{1 subjects, inverting the
resulting model separately for each subject, and repeating this
procedure n times (Figure 2f). This procedure guarantees that
neither the training procedure nor the features selected for the test
subject were influenced by the data or the label of the test subject. Classification While a kernel describes how two subjects can be compared
using a generative model of their fMRI data, it does not specify
how such a comparison could be used for making predictions. This
gap is filled by discriminative classification methods. As described
in the Section ‘Combining generative models and discriminative
methods’, a natural choice is the ‘2-norm soft-margin support
vector machine (SVM), which currently represents the most widely
used kernel method for classification [72]. In summary, the above analysis shows that there are three
practical strategies for the implementation of generative embed-
ding that yield an unbiased cross-validated accuracy estimate. If
regions are defined anatomically, the model is inverted separately
for each subject, and the resulting parameter estimates can be
safely used in cross-validation (Figure 2a). Otherwise, if regions are
defined by a functional contrast, both the definition of ROIs and
model inversion for all subjects need to be carried out separately
for each cross-validation fold (Figure 2c,f). An estimate of classification performance with minimal variance
can be obtained by leave-one-out cross-validation. In each fold,
the classifier is trained on n{1 subjects and tested on the left-out
one. Using the training set only, the SVM can be fine-tuned by
carrying out a simple line search over the regularization
hyperparameter C (Eqn. 1), a procedure known as nested cross-
validation [90,91]. There are many ways of assessing the generalization perfor-
mance of a classifier. Here, we are primarily interested in the
balanced accuracy, that is, the mean accuracy obtained on either
class, Kernel construction Given a set of inverted subject-specific generative models, the
kernel defines the similarity metric under which these models are
assessed within a discriminative classifier. In generative embed-
ding, the choice of an appropriate kernel depends on the definition
of the generative score space. A straightforward way to create a
Euclidean vector space from an inverted DCM is to consider the
posterior means or maximum a posteriori (MAP) estimates of model
parameters
of
interest
(e.g.,
parameters
encoding
synaptic
connection strengths). More formally, we can define a mapping
MH?Rd that extracts a subset of MAP estimates ^mMAP from the
posterior distribution p(hjx,m). This simple d-dimensional vector
space
expresses
discriminative
information
encoded
in
the
connection strengths between regions, as opposed to activity levels
within these regions. Alternatively, one could also incorporate
elements of the posterior covariance matrix into the vector space. This would be beneficial if class differences were revealed by the
precision with which connection strengths can be estimated from
the data. 1
2
TP
TPzFN z
TN
TNzFP
,
ð3Þ ð3Þ where TP, FP, TN, and FN represent the number of true
positives, false positives, true negatives, and false negatives,
respectively [92]. The balanced accuracy represents the arithmetic
mean between sensitivity and specificity. If the classifier performs
equally well on either class, it reduces to the ordinary accuracy
(i.e., the ratio of correct predictions to all predictions). If, however,
the classifier has taken advantage of an imbalanced dataset, then
the ordinary accuracy will be inflated, whereas the balanced
accuracy will drop to chance (50%), as desired. The balanced
accuracy thus removes the bias from estimates of generalizability
that may arise in the presence of imbalanced datasets. A
probability
interval
can
be
computed
by
considering
the
convolution
of
two
Beta-distributed
random
variables
that
correspond to the true accuracies on positive and negative
examples, respectively. A p-value can then be obtained by
computing the posterior probability of the accuracy being below
chance [92]. where TP, FP, TN, and FN represent the number of true
positives, false positives, true negatives, and false negatives,
respectively [92]. The balanced accuracy represents the arithmetic
mean between sensitivity and specificity. If the classifier performs
equally well on either class, it reduces to the ordinary accuracy
(i.e., the ratio of correct predictions to all predictions). PLoS Computational Biology | www.ploscompbiol.org June 2011 | Volume 7 | Issue 6 | e1002079 Experimental design, data acquisition, and preprocessing In order to illustrate the utility of generative embedding for
fMRI, we used data from two groups of participants (patients with
moderate aphasia vs. healthy controls) engaged in a simple speech-
processing task. The conventional SPM and DCM analyses of
these data are published elsewhere; we refer to [71] and Schofield
et al. (in preparation) for detailed descriptions of all experimental
procedures. The two groups of subjects consisted of 26 right-handed healthy
participants with normal hearing, English as their first language,
and no history of neurological disease (12 female; mean age 54.1
years; range 26–72 years); and 11 patients diagnosed with
moderate aphasia due to stroke (1 female; mean age 66.1; range
45–90 years). The patients’ aphasia profile was characterized using
the Comprehensive Aphasia Test [93]. As a group, they had scores
in the aphasic range for: spoken and written word comprehension
(single word and sentence level), single word repetition and object
naming. It is important to emphasize that the lesions did not affect
any of the temporal regions which we included in our model
described below (see Schofield et al., in preparation, for detailed
information on lesion localization). Subjects were presented with two types of auditory stimulus: (i)
normal speech; and (ii) time-reversed speech, which is unintelli-
gible but retains both speaker identity and the spectral complexity
of normal speech. Subjects were given an incidental task, to make
a gender judgment on each auditory stimulus, which they
indicated with a button press. p
Functional T2*-weighted echo-planar images (EPI) with BOLD
contrast were acquired using a Siemens Sonata 1.5 T scanner (in-
plane resolution 3 mm63 mm; slice thickness 2 mm; inter-slice
gap 1 mm; TR 3.15 s). In total, 122 volumes were recorded in
each of 4 consecutive sessions. In addition, a T1-weighted
anatomical image was acquired. Following realignment and
unwarping of the functional images, the mean functional image
of each subject was coregistered to its high-resolution structural
image. This image was spatially normalized to standard Montreal
Neurological Institute (MNI152) space, and the resulting defor-
mation field was applied to the functional data. These data were
then spatially smoothed using an isotropic Gaussian kernel
(FWHM 8 mm). In previous work, these data have been analysed
using a conventional general linear model (GLM) and DCM; the
results are described in Schofield et al. (in preparation). Generative Embedding for fMRI Generative Embedding for fMRI contrast based on the first regressor (i.e., a contrast between
auditory stimuli and background scanner noise), designed to find
early auditory regions required for the perception of any broad-
band stimulus, whether it is speech or speech-like. understand which model parameters, and thus mechanisms,
contribute most to distinguishing groups. Therefore, for the
purposes of interpreting the model-induced feature space, we use
an ‘0-regularizer. This approach allows us to characterize the
feature space by counting how often a particular feature has been
selected in leave-one-out cross-validation. Second-level (group) analysis. The second level analysis
served to select regions whose voxels entered the subject-specific
DCMs (in terms of the first eigenvariate of their time series). In the
previous study of these data (Schofield et al., in preparation), we had
compared a set of 512 alternative DCMs that embodied competing
hypotheses about the architecture of the thalamo-temporal network
processing speech-like stimuli per se. Here, we focus on the model
which was found to have the highest evidence in our previous study,
i.e., the model providing the best trade-off between accuracy and
complexity in explaining the data [94,83,88]. Note that this
selection procedure is ignorant of subject labels, which prevents
test labels from influencing the training procedure. (An alternative,
computationally more expensive approach would be to select the
model that affords the best classification accuracy, and integrate this
selection step into an overall cross-validation scheme. See [61] for
an example.) In addition, the selection of time series remains
independent of the test data. The DCM we used contains 6 regions
(medial geniculate body, MGB; Heschl’s gyrus, HG; planum
temporale, PT), three in each hemisphere, and 14 interregional
connections (see Figure 3). Note that this model concerned
processing of acoustic stimuli with speech-like spectral properties
per se, not differentiating between normal and time-reversed speech;
therefore, it did not contain modulatory inputs (corresponding to an
empty B matrix, see Eqn. 4). Critically, instead of identifying regions
functionally by a group contrast, we pre-defined large anatomical
masks (16 mm616 mm616 mm) that specified only the rough
location of the 6 regions of interest (see Table 1 and Supplementary
Material). These masks served to guide the selection of time series,
using a leave-one-out approach to feature selection as described
below. Experimental design, data acquisition, and preprocessing Here, we re-
examined the dataset using the procedure shown in Figure 2c, as
described in detail in the next subsection. Model specification. To specify the exact location and
extent of our 6 regions of interest, and thus the exact time series
that would be modelled by DCM, we used a leave-one-out
approach to feature selection. For this purpose, we carried out n
separate second-level analyses, each time leaving out one subject,
and then used a conventional summary-statistic approach [95]
across the remaining n{1 subjects to find voxels that survived a
one-sample ‘all auditory events’ t-test with a statistical threshold of
p~0:001 (uncorrected), across all subjects, within the anatomical
masks described above. Note that this contrast is agnostic about
diagnostic status (corresponding to Figure 2c). (With the cross-
validation scheme used here, a between-group contrast could have
been used as well without risking bias; see Section ‘Strategies for
unbiased model specification and inversion’. This case would
correspond to Figure 2f.) Within each leave-one-out repetition, our
procedure yielded 6 voxel sets, one for each region of interest. We
used the first eigenvariate over voxels as a representative time
series for each region in DCM. Interpretation of the feature space Most classification algorithms can not only be used for making
predictions and obtaining an estimate of their generalization error;
they can also be used to quantify how much each feature has
contributed to classification performance. Such feature weights can
sometimes be of greater interest than the classification accuracy
itself. In the case of a generative score space, as defined above,
each feature is associated with a neurobiologically interpretable
model parameter. Provided there are no complex transformations
of feature weights (see above), they can be interpreted in the
context of the underlying model. As described in the Section ‘Combining generative models and
discriminative methods’, the ‘2-norm soft-margin SVM is a
natural choice when the goal is maximal prediction accuracy. However, its solution usually implies that almost all features are
used for classification. This is suboptimal when one wishes to In summary, in this paper, we define a mapping MH?Rd from
a subject-specific posterior distribution of model parameters
p(hjx,m) to a feature vector ^mMAP. We then use a linear kernel
k : Rd|Rd?R for this model-based feature space. Together, PLoS Computational Biology | www.ploscompbiol.org June 2011 | Volume 7 | Issue 6 | e1002079 7 Implementation of generative embedding Only three parameters were selected in all cross-validation folds and are thus particularly meaningful for classification (bold red
arrows); these refer to connections mediating information transfer from the right to the left hemisphere, converging on left PT, which is a key
structure in speech processing. doi:10.1371/journal.pcbi.1002079.g003
Generative Embedding for fMRI Generative Embedding for fMRI Generative Embedding for fMRI Figure 3. Dynamic causal model of speech processing. The diagram illustrates the specific dynamic causal model (DCM) that was used for the
illustrative application of generative embedding in this study. It consists of 6 regions (circles), 15 interregional connections (straight arrows between
regions), 6 self-connections (circular arrows), and 2 stimulus inputs (straight arrows at the bottom). The specific set of connections shown here is the
result of Bayesian model selection that was carried out on the basis of a large set of competing connectivity layouts (for details, see Schofield et al., in
preparation). A sparse set of 9 out of 23 connectivity and input parameters (see Figure 10) was found to be sufficiently informative to distinguish
between aphasic patients and healthy controls with near-perfect accuracy (see Figure 5). The connections corresponding to these 9 parameters are
highlighted in red. Only three parameters were selected in all cross-validation folds and are thus particularly meaningful for classification (bold red
arrows); these refer to connections mediating information transfer from the right to the left hemisphere, converging on left PT, which is a key
structure in speech processing. doi:10 1371/journal pcbi 1002079 g003 doi:10.1371/journal.pcbi.1002079.g003 modulatory parameters (as the B matrix was empty in the DCM
we used), and 2 input parameters (C matrix). All feature vectors
were normalized to unit length. To minimize the risk of overfitting
and enable a clear interpretation of feature weights, we used a
linear kernel. Consequently, the similarity between two subjects
was defined as the inner product between the normalized vectors
of the posterior means of their model parameters. from subject j (using the same voxels as the rest of the group). Crucially, in this way, test data and test labels were neither used for
model specification nor for classifier training, preventing optimistic
estimates of classification performance. The principles of this
unbiased procedure are summarized in Figure 4. Implementation of generative embedding Model inversion. Inversion of the DCM was carried out
independently for each subject, and separately for each cross-
validation fold (i.e., each group contrast). With regions (and thus
modelled time series) differing each time depending on the current
set of n{1 subjects, this procedure resulted in a total of n2~1 369
fitted DCMs. We emphasize once more that model inversion was
carried out in an unsupervised fashion, i.e., without reference to
the subjects’ diagnostic status. First-level analysis. The first level of our statistical analysis
employed a mass-univariate analysis in each subject. Each
auditory stimulus was modelled as a separate delta function, and
the resulting trains of auditory events were convolved with a
canonical haemodynamic response function. The first regressor in
the design matrix contained all auditory events (i.e., normal and
time-reversed speech stimuli); the second regressor modelled
intelligibility (normal vs. time-reversed speech) as a parametric
modulation. Beta coefficients were estimated for all brain voxels
using the general linear model [1]. To identify regions responding
to auditory stimulation per se, we used an ‘all auditory events’ Kernel
construction. A
generative
score
space
was
constructed on the basis of the MAP estimates of the neuronal
model parameters (hn in Eqn. 5). The rzzesulting space contained
22 features: 20 interregional connection strengths (A matrix), no PLoS Computational Biology | www.ploscompbiol.org June 2011 | Volume 7 | Issue 6 | e1002079 8 Figure 3. Dynamic causal model of speech processing. The diagram illustrates the specific dynamic causal model (DCM) that was used for the
illustrative application of generative embedding in this study. It consists of 6 regions (circles), 15 interregional connections (straight arrows between
regions), 6 self-connections (circular arrows), and 2 stimulus inputs (straight arrows at the bottom). The specific set of connections shown here is the
result of Bayesian model selection that was carried out on the basis of a large set of competing connectivity layouts (for details, see Schofield et al., in
preparation). A sparse set of 9 out of 23 connectivity and input parameters (see Figure 10) was found to be sufficiently informative to distinguish
between aphasic patients and healthy controls with near-perfect accuracy (see Figure 5). The connections corresponding to these 9 parameters are
highlighted in red. Implementation of generative embedding Within each fold, the complexity penalty C of the SVM was
selected by a line search in log2 space, to minimize an estimate of
the generalization error on the training set (nested cross-
validation). To discourage the classifier from acquiring a bias in
favour of the majority class, the training set was balanced using a
stochastic oversampling strategy. We assessed the generalization
performance of the classifier by comparing its n predictions to the
n true subject labels (‘patient’ or ‘healthy control’), resulting in a
262 confusion matrix that forms the basis of various common
performance measures, such as the accuracy or the area under the
receiver-operator characteristic (ROC) curve. Classification. An ‘2-norm soft-margin linear support vector
machine (SVM) was trained and tested using leave-one-out cross-
validation. Specifically, in each fold j~1,:::,n, the classifier was
trained on all subjects except j, on the basis of the DCM parameter
estimates obtained from fitting the voxel time series selected by the
group analysis based on all subjects except j. The classifier was then
tested by applying it to DCM parameter estimates for the time series Table 1. Regions of interest. Region
MNI coordinates
L.MGB
left medial geniculate body
223 mm, 223 mm, 21 mm
L.HG
left Heschl’s gyrus (A1)
247 mm, 226 mm, 7 mm
L.PT
left planum temporale
264 mm, 223 mm, 8 mm
R.MGB
right medial geniculate body
22 mm, 221 mm, 21 mm
R.HG
right Heschl’s gyrus (A1)
48 mm, 224 mm, 6 mm
R.PT
right planum temporale
65 mm, 222 mm, 3 mm
Speech processing can be modelled using a dynamic causal model (DCM) with
6 regions. The table lists the central coordinates of these regions in MNI152
space. These coordinates define the centre of the rough anatomical masks
(16 mm616 mm616 mm) that guided the specification of the exact location
and extent of the regions of interest underlying model inversion (see Section
‘Implementation of generative embedding’). For an illustration of these masks,
see Figure S1 in the Supplementary Material. doi:10.1371/journal.pcbi.1002079.t001 Table 1. Regions of interest. June 2011 | Volume 7 | Issue 6 | e1002079 Comparative analyses This figure summarizes the three core steps involved in the practical
implementation of generative embedding proposed in this paper. This procedure integrates the inversion of a generative model into cross-validation. In step 1, within a given repetition j~1,:::,n, the model is specified using all subjects except j. This yields a set of time series fxi[Xg for each subject
i~1,:::,n. In step 2, the model is inverted independently for each subject, giving rise to a set of subject-specific posterior parameter means f^mig. In step 3, these parameter estimates are used to train a classifier on all subjects except j and test it on subject j, which yields a prediction about the
class label of subject j. After having repeated these three steps for all j~1,:::,n, the set of predicted labels can be compared with the true labels,
which allows us to estimate the algorithm’s generalization performance. In addition, parameters that proved jointly discriminative can be interpreted
in the context of the underlying generative model. The sequence of steps shown here corresponds to the procedure shown in Figure 2c and 2f,
where it is contrasted with alternative procedures that are simpler but risk an optimistic bias in estimating generalization performance. doi:10.1371/journal.pcbi.1002079.g004 balanced by oversampling. We will refer to this method as contrast feature
selection. eigenvariate. Thus, in this approach, the exact same data was used
to estimate functional connectivity as was used by DCM to infer
effective connectivity. Finally, as suggested in [43], we created yet
another feature space by transforming the correlation coefficients
on eigenvariates into z-scores using the Fisher transformation [96]. The third activation-based method employed a locally multi-
variate ‘searchlight’ strategy for feature selection. Specifically, in
each cross-validation fold, a searchlight sphere (radius 4 mm) was
passed across all voxels contained in the anatomical masks
described above [23]. Using the training set only, a nested leave-
one-out
cross-validation
scheme
was
used
to
estimate
the
generalization performance of each sphere using a linear SVM
with a fixed regularization hyperparameter (C~1). Next, all
spheres with an accuracy greater than 75% were used to form the
feature space for the current outer cross-validation fold, which
corresponds to selecting all voxels whose local neighbourhoods
allowed for a significant discrimination between patients and
healthy controls at a~0:01. Both outer and inner training sets
were balanced by oversampling. We will refer to this method as
searchlight feature selection. Comparative analyses We compared the performance of generative embedding to a
range of alternative approaches. To begin with, we examined
several conventional activation-based classification schemes. The
first method was based on a feature space composed of all voxels
within the predefined anatomical masks used for guiding the
specification of the DCMs. As above, we used a linear SVM, and
all training sets were balanced by oversampling. We will refer to
this approach as anatomical feature selection. The second method, in contrast to the first one, was not only based
on the same classifier as in generative embedding but also used exactly
the same voxels. Specifically, voxels were selected on the basis of the
same ‘all auditory events’ contrast as above, which is a common
approach to defining a voxel-based feature space in subject-by-subject
classification [11,12,10]. In every cross-validation fold, only those
voxels entered the classifier that survived a t-test (a~0:001,
uncorrected) in the current set of n{1 subjects. Training sets were PLoS Computational Biology | www.ploscompbiol.org June 2011 | Volume 7 | Issue 6 | e1002079 9 Generative Embedding for fMRI Figure 4. Practical implementation of generative embedding for fMRI. This figure summarizes the three core steps involved in the practical
implementation of generative embedding proposed in this paper. This procedure integrates the inversion of a generative model into cross-validation. In step 1, within a given repetition j~1,:::,n, the model is specified using all subjects except j. This yields a set of time series fxi[Xg for each subject
i~1,:::,n. In step 2, the model is inverted independently for each subject, giving rise to a set of subject-specific posterior parameter means f^mig. In step 3, these parameter estimates are used to train a classifier on all subjects except j and test it on subject j, which yields a prediction about the
class label of subject j. After having repeated these three steps for all j~1,:::,n, the set of predicted labels can be compared with the true labels,
which allows us to estimate the algorithm’s generalization performance. In addition, parameters that proved jointly discriminative can be interpreted
in the context of the underlying generative model. The sequence of steps shown here corresponds to the procedure shown in Figure 2c and 2f,
where it is contrasted with alternative procedures that are simpler but risk an optimistic bias in estimating generalization performance. doi:10.1371/journal.pcbi.1002079.g004 Figure 4. Practical implementation of generative embedding for fMRI. Comparative analyses To illustrate the location of the most
informative voxels, we carried out an additional searchlight
analysis, based on the entire dataset as opposed to a subset of
size n{1, and used the results to generate a discriminative map
(see Figure S1 in the Supplementary Material). In addition to conventional activation- and correlation-based
approaches, we also investigated the dependence of generative
embedding on the structure of the underlying model. Specifically,
we repeated our original analysis on the basis of three alternative
models. For the first model, we constructed a feedforward system by
depriving the original model of all feedback and interhemispheric
connections (Figure 5a); while this model could still, in principle,
explain neuronal dynamics throughout the system of interest, it
was neurobiologically less plausible. For the second and third
model, we kept all connections from the original model but
modelled either only the left hemisphere (Figure 5b) or only the right
hemisphere (Figure 5c). In summary, we compared the primary approach proposed in
this
paper
to
4
conventional
activation-based
methods,
3
conventional correlation-based methods, and 3 generative-embed-
ding analyses using reduced and biologically less plausible models. The fourth conventional method was based on a principal
component analysis (PCA) to reduce the dimensionality of the
feature space constructed from all voxels in the anatomical masks
described
above. Unlike
generative
embedding,
PCA-based
dimensionality reduction finds a linear manifold in the data
without a mechanistic view of how those data might have been
generated. We sorted all principal components in decreasing order
of explained variance. By retaining the 22 top components, the
resulting dimensionality matched the dimensionality of the feature
space used in generative embedding. PLoS Computational Biology | www.ploscompbiol.org Classification performance Regarding generative embedding itself, when
replacing the original model shown in Figure 3 by a biologically less
plausible feedforward model (Figure 5a) or by a model that captured
the left hemisphere only (Figure 5b), we observed a significant
decrease in performance, from 98% down to 77% (p<0.002) and
81% (p<0.008), respectively, although both accuracies remained
significantly above chance (p<0.001 and p,0.001). By contrast,
when modelling the right hemisphere only (Figure 5c), performance
dropped to a level indistinguishable from chance (59.3%, p<0.134). One way of characterizing the discriminative information
encoded in individual model parameters more directly is to
estimate class-conditional univariate feature densities (see Figure 9). Here, densities were estimated in a nonparametric way using a
Gaussian kernel with an automatically selected bandwidth, making
no assumptions about the distributions other than smoothness
[99]. While most densities are heavily overlapping, a two-sample
t-test
revealed
significant
group
differences
in
four
model
parameters (denoted by stars in Figure 9): the self-connection of
L.HG (parameter 4); the influence that L.HG exerts over L.PT
(parameter 5); the influence R.MGB on R.PT (parameter 13); and
the influence of R.HG on L.HG (parameter 14). All of these were
significant at the 0.001 level while no other parameter survived
p = 0.05. An extended plot of all bivariate feature distributions,
illustrating how well any two features jointly discriminated
between patients and healthy controls, can be found in the
Supplementary Material (Figure S2). In order to provide a better intuition as to how the generative
model shown in Figure 3 created a score space in which examples
were much better separated than in the original voxel-based
feature space, we produced two scatter plots of the data (see
Figure 7). The first plot is based on the peak voxels of the three
most
discriminative clusters
among all
regions of
interest,
evaluated by a searchlight classification analysis. The second plot,
by analogy, is based on the three most discriminative model
parameters, as measured by two-sample t-tests in the (normalized)
generative score space. This illustration shows how the voxel-based
projection (left) leads to classes that still overlap considerably,
whereas the model-based projection (right) provides an almost
perfectly linear separation of patients and controls. Classification performance In order to better understand which DCM parameters jointly
enabled the distinction between patients and controls, we
examined the frequency with which features were selected in
leave-one-out cross-validation when using an SVM with a sparsity-
inducing regularizer [75,74] (see Figure 10). We found that the
classifier favoured a highly consistent and sparse set of 9 (out of 22)
model parameters; the corresponding synaptic connections are
highlighted in red in Figure 3. Notably, this 9-dimensional feature
space, when used with the original ‘2-norm SVM, yielded the
same balanced classification accuracy (98%) as the full 22-
dimensional feature space, despite discarding more than two
thirds of its dimensions. Classification performance The classification performance of generative embedding was
evaluated using the procedure described in Figure 2c. This
procedure was compared to several conventional activation-based
and correlation-based approaches. As an additional control,
generative embedding was carried out on the basis of three
biologically ill-informed models. In all cases, a leave-one-subject-
out cross-validation scheme was used to obtain the posterior
distribution of the balanced accuracy [92] as well as smooth
estimates
of the
underlying
receiver-operating
characteristic
(ROC) and precision-recall (PC) curves [97]. Results are presented
in Table 2 and Figure 6. In
addition
to
the
above
activation-based
methods,
we
compared generative embedding to several approaches based on
undirected regional correlations. We began by averaging the
activity within each region of interest to obtain region-specific
representative time series. We then computed pairwise correlation
coefficients to obtain a 15-dimensional feature space of functional
connectivity. Next, instead of computing spatial averages, we
summarized the activity within each region in terms of the first The strongest classification performance was obtained when
using generative embedding with the full model shown in Figure 3. The approach correctly associated 36 out of 37 subjects with their PLoS Computational Biology | www.ploscompbiol.org June 2011 | Volume 7 | Issue 6 | e1002079 June 2011 | Volume 7 | Issue 6 | e1002079 10 Generative Embedding for fMRI Figure 5. Biologically unlikely alternative models. To illustrate the specificity of generative embedding, the analysis described in the main text
was repeated on the basis of three biologically less plausible models. In contrast to the full model shown in Figure 3, these alternative models either
(a) contained no feedback or interhemispheric connections, (b) accounted for activity in the left hemisphere only, or (c) focussed exclusively on the
right hemisphere. For results, see Table 2 and Figure 6. doi:10.1371/journal.pcbi.1002079.g005 Figure 5. Biologically unlikely alternative models. To illustrate the specificity of generative embedding, the analysis described in the main text
was repeated on the basis of three biologically less plausible models. In contrast to the full model shown in Figure 3, these alternative models either
(a) contained no feedback or interhemispheric connections, (b) accounted for activity in the left hemisphere only, or (c) focussed exclusively on the
right hemisphere. For results, see Table 2 and Figure 6. doi:10.1371/journal.pcbi.1002079.g005 true disease state, corresponding to a balanced accuracy of 98%. Classification performance Regarding conventional activation-based methods, classification
based on anatomical feature selection did not perform significantly
above chance (balanced accuracy 62%, p<0.089). Contrast feature
selection (75%, p<0.003), searchlight feature selection (73%,
p<0.006),
and
PCA-based
dimensionality
reduction
(80%,
p,0.001) did perform significantly above chance; however, all
methods were outperformed significantly by generative embedding
(p<0.003, p<0.001, and p<0.045, paired-sample Wald test). Regarding conventional correlation-based methods, all three
approaches performed above chance, whether based on correlations
amongst the means (70%, p<0.011), correlations amongst eigen-
variates (83%, p,0.001), or z-transformed correlations amongst
eigenvariates
(74%,
p<0.002). Critically,
however,
all
were
significantly outperformed by generative embedding (p,0.001,
p<0.045, p<0.006). Regarding generative embedding itself, when
replacing the original model shown in Figure 3 by a biologically less
plausible feedforward model (Figure 5a) or by a model that captured
the left hemisphere only (Figure 5b), we observed a significant
decrease in performance, from 98% down to 77% (p<0.002) and
81% (p<0.008), respectively, although both accuracies remained
significantly above chance (p<0.001 and p,0.001). By contrast,
when modelling the right hemisphere only (Figure 5c), performance
dropped to a level indistinguishable from chance (59.3%, p<0.134). where each axis corresponds to a particular model parameter
(see Figure 8). When using parameters that represent directed
connection strengths, this form of visualization is reminiscent of
the
notion
of
‘connectional
fingerprints’
for
characterizing
individual cortical regions [98]. In our case, there is no
immediately obvious visual difference in fingerprints between
aphasic patients and healthy controls. On the contrary, the plot
gives an impression of the large variability across subjects and
suggests that differences might be subtle and possibly jointly
encoded in multiple parameters. true disease state, corresponding to a balanced accuracy of 98%. Regarding conventional activation-based methods, classification
based on anatomical feature selection did not perform significantly
above chance (balanced accuracy 62%, p<0.089). Contrast feature
selection (75%, p<0.003), searchlight feature selection (73%,
p<0.006),
and
PCA-based
dimensionality
reduction
(80%,
p,0.001) did perform significantly above chance; however, all
methods were outperformed significantly by generative embedding
(p<0.003, p<0.001, and p<0.045, paired-sample Wald test). Regarding conventional correlation-based methods, all three
approaches performed above chance, whether based on correlations
amongst the means (70%, p<0.011), correlations amongst eigen-
variates (83%, p,0.001), or z-transformed correlations amongst
eigenvariates
(74%,
p<0.002). Critically,
however,
all
were
significantly outperformed by generative embedding (p,0.001,
p<0.045, p<0.006). Characterization of the feature space The above representation disclosed interesting potential mech-
anisms. For example, discriminative parameters were restricted to The low dimensionality of the model-based feature space makes
it possible to visualize each example in a radial coordinate system, PLoS Computational Biology | www.ploscompbiol.org June 2011 | Volume 7 | Issue 6 | e1002079 11 Generative Embedding for fMRI Table 2. Classification results. Measure (n = 37)
Anatomical feature
selection
Contrast feature
selection
Searchlight feature
selection
PCA-based dimensionality
reduction
(1) Accuracy
0.649
0.757
0.730
0.865
(2) Balanced accuracy
0.619
0.748
0.729
0.799
(3) Significantly above chance
p<0.089
p<0.003
p<0.006
p,0.001
(4) True positive rate (TPR;
sensitivity; recall)
0.545
0.727
0.727
0.636
(5) True negative rate (TNR;
specificity)
0.692
0.769
0.731
0.962
(6) Positive predictive
value (PPV; precision)
0.429
0.571
0.533
0.875
(7) Negative predictive
value (NPV)
0.783
0.870
0.864
0.862
(8) Area under the ROC curve
(AUC)
0.657
0.829
0.794
0.846
(9) Area under the PR curve
(average precision)
0.756
0.854
0.842
0.885
…
Region-means
correlations
Eigenvariates
correlations
Eigenvariates
z-correlations
0.730
0.865
0.784
0.703
0.825
0.741
p<0.011
p,0.001
p<0.002
0.636
0.727
0.636
0.769
0.923
0.846
0.538
0.800
0.636
0.833
0.889
0.846
0.804
0.958
0.857
0.873
0.945
0.914
…
Generative
embedding (full model)
Generative embedding
(feedforward model)
Generative embedding
(left hemisphere)
Generative embedding (right
hemisphere)
0.973
0.784
0.838
0.649
0.981
0.767
0.806
0.593
p,0.001
p<0.001
p,0.001
p<0.134
1.000
0.727
0.727
0.455
0.962
0.808
0.885
0.731
0.917
0.615
0.727
0.417
1.000
0.875
0.885
0.760
0.990
0.867
0.923
0.706
0.957
0.916
0.934
0.803
This table contrasts the classification results obtained through generative embedding with those afforded by three conventional methods. As described in the main
text, the underlying dataset serves illustrative purposes, and so, due to its small sample size (n = 37), all numbers are associated with considerable uncertainty. The
measure of primary interest is the balanced accuracy (2). Its uncertainty can be captured by computing a posterior probability interval (as shown in Figure 6a), or by
computing a p-value (3), which represents the probability with which the observed performance would have been obtained by chance. doi:10.1371/journal.pcbi.1002079.t002 This table contrasts the classification results obtained through generative embedding with those afforded by three conventional methods. As described in the main
text, the underlying dataset serves illustrative purposes, and so, due to its small sample size (n = 37), all numbers are associated with considerable uncertainty. The
measure of primary interest is the balanced accuracy (2). PLoS Computational Biology | www.ploscompbiol.org Characterization of the feature space Its uncertainty can be captured by computing a posterior probability interval (as shown in Figure 6a), or by
computing a p-value (3), which represents the probability with which the observed performance would have been obtained by chance. doi:10.1371/journal.pcbi.1002079.t002 analyses of group-wise DCM parameters in the study by Schofield et
al. (in preparation). Furthermore, all of the connections from the right
to the left hemisphere were informative for group membership, but
none of the connections in the reverse direction. This pattern is
interesting given the known specialization of the left hemisphere in
language and speech processing and previous findings that
language-relevant
information
is
transferred
from
the
right
hemisphere to the left, but not vice versa [100]. It implies that
aphasia leads to specific changes in connectivity, even in non-
lesioned parts of the language network, with a particular effect on analyses of group-wise DCM parameters in the study by Schofield et
al. (in preparation). Furthermore, all of the connections from the right
to the left hemisphere were informative for group membership, but
none of the connections in the reverse direction. This pattern is
interesting given the known specialization of the left hemisphere in
language and speech processing and previous findings that
language-relevant
information
is
transferred
from
the
right
hemisphere to the left, but not vice versa [100]. It implies that
aphasia leads to specific changes in connectivity, even in non-
lesioned parts of the language network, with a particular effect on cortico-cortical and thalamo-cortical connection strengths, whereas
parameters representing auditory inputs to thalamic nuclei did not
contribute to the distinction between patients and healthy controls. This finding implies that, as one would expect, low-level processing
of auditory stimuli, from brain stem to thalamus, is unimpaired in
aphasia and that processing deficiencies are restricted to thalamo-
cortical and cortico-cortical networks. In particular, the discrimi-
native connections included the top-down connections from planum
temporale to Heschl’s gyrus bilaterally; the importance of these
connections had also been highlighted by the previous univariate PLoS Computational Biology | www.ploscompbiol.org June 2011 | Volume 7 | Issue 6 | e1002079 12 Generative Embedding for fMRI Figure 6. Classification performance. Characterization of the feature space Classification based on generative embedding using the model shown in Figure 3 was compared to ten
alternative methods: anatomical feature selection, contrast feature selection, searchlight feature selection, PCA-based dimensionality reduction, regional
correlations based on region means, regional correlations based on eigenvariates, regional z-transformed correlations based on eigenvariates, as well as
generative embedding using three biologically unlikely alternative models (see inset legends for abbreviations). (a) The balanced accuracy and its central
95% posterior probability interval show that all methods performed significantly better than chance (50%) with the exception of classification with
anatomical feature selection and generative embedding using a nonsensical model. Differences between activation-based methods (light grey) and
correlation-based methods (dark grey) were largely statistically indistinguishable. By contrast, using the full model shown in Figure 3, generative
embedding (blue) significantly outperformed all other methods, except when used with biologically unlikely models (Figure 5). (b) Receiver-operating
characteristic (ROC) curves of the eleven methods illustrate the trade-off between true positive rate (sensitivity) and false positive rate (1 – specificity)
across the entire range of detection thresholds. A larger area under the curve is better. (c) Precision-recall (PR) curves illustrate the trade-off between
positive prediction value (precision) and true positive rate (recall). A larger area under the curve is better. Smooth ROC and PR curves were obtained
using a binormal assumption on the underlying decision values [97]. For a numerical summary of all results, see Table 2. doi:10.1371/journal.pcbi.1002079.g006 Figure 6. Classification performance. Classification based on generative embedding using the model shown in Figure 3 was compared to ten
alternative methods: anatomical feature selection, contrast feature selection, searchlight feature selection, PCA-based dimensionality reduction, regional
correlations based on region means, regional correlations based on eigenvariates, regional z-transformed correlations based on eigenvariates, as well as
generative embedding using three biologically unlikely alternative models (see inset legends for abbreviations). (a) The balanced accuracy and its central
95% posterior probability interval show that all methods performed significantly better than chance (50%) with the exception of classification with
anatomical feature selection and generative embedding using a nonsensical model. Differences between activation-based methods (light grey) and
correlation-based methods (dark grey) were largely statistically indistinguishable. By contrast, using the full model shown in Figure 3, generative
embedding (blue) significantly outperformed all other methods, except when used with biologically unlikely models (Figure 5). Characterization of the feature space (b) Receiver-operating
characteristic (ROC) curves of the eleven methods illustrate the trade-off between true positive rate (sensitivity) and false positive rate (1 – specificity)
across the entire range of detection thresholds. A larger area under the curve is better. (c) Precision-recall (PR) curves illustrate the trade-off between
positive prediction value (precision) and true positive rate (recall). A larger area under the curve is better. Smooth ROC and PR curves were obtained
using a binormal assumption on the underlying decision values [97]. For a numerical summary of all results, see Table 2. doi:10.1371/journal.pcbi.1002079.g006 cross-validation folds) and are thus particularly meaningful for
classification (bold red arrows in Figure 3). The associated
connections mediate information transfer from the right to the left inter-hemispheric transfer of speech information. This specificity is
seen even more clearly when considering only those three
parameters which were selected 100% of the time (i.e., in all Figure 7. Induction of a generative score space. This figure provides an intuition of how a generative model transforms the data from a voxel-
based feature space into a generative score space (or model-based feature space), in which classes become more separable. The left plot shows how
aphasic patients (red) and healthy controls (grey) are represented in voxel space, based on t-scores from a simple ‘all auditory events’ contrast (see main
text). The three axes represent the peaks of those three clusters that showed the strongest discriminability between patients and controls, based on a
locally multivariate searchlight classification analysis. They are located in L.PT, L.HG, and R.PT, respectively (cf. Table 1). The right plot shows the three
individually most discriminative parameters (two-sample t-test) in the (normalized) generative score space induced by a dynamic causal model of speech
processing (see Figure 3). The plot illustrates how aphasic patients and healthy controls become almost perfectly linearly separable in the new space. Note that this figure is based on normalized examples (as used by the classifier), which means the marginal densities are not the same as those shown in
Figure 9 but are exactly those seen by the classifier. A stereogram of the generative score space can be found in the Supplementary Material (Figure S4). doi:10.1371/journal.pcbi.1002079.g007 Figure 7. Induction of a generative score space. PLoS Computational Biology | www.ploscompbiol.org Perspectives for generative embedding of fMRI data Perspectives for generative embedding of fMRI data
Generative
embedding
for
subject-by-subject
classification
provides three potential advantages over conventional voxel-based
methods. The first advantage is that it combines the explanatory
strengths of generative models with the classification power of
discriminative methods. Thus, in contrast to purely discriminative
or purely generative methods, generative embedding is a hybrid
approach. It fuses a feature space that captures both the data and
their generative process with a classifier that finds the maximum-
margin boundary for class separation. Intuitively, this exploits the
idea that differences in the generative process between two
examples (observations) might provide optimal discriminative
information required to enable accurate predictions. In the case
of DCM for fMRI, this rationale should pay off whenever the
directed connection strengths between brain regions contain more
information about a disease state than regional activations or
undirected correlations. Indeed, this is what we found in our
analyses (cf. Figure 6). Using a DCM-informed data representation
might prove particularly relevant in psychiatric disorders, such as
schizophrenia or depression, where aberrant effective connectivity
and synaptic plasticity are central to the disease process [48,47]. Figure 8. Connectional fingerprints. Given the low dimensionality
of the model-induced feature space, subjects can be visualized in terms
of ‘connectional fingerprints’ [98] that are based on a simple radial
coordinate system in which each axis corresponds to the maximum a
posteriori (MAP) estimate of a particular model parameter. The plot
shows that the difference between aphasic patients (red) and healthy
controls (grey) is not immediately obvious, suggesting that it might be
subtle and potentially of a distributed nature. doi:10.1371/journal.pcbi.1002079.g008 Figure 9. Univariate feature densities. Separately for patients (red) and healthy controls (grey), the figure shows nonparametric estimates of the
class-conditional densities of the maximum a posteriori (MAP) estimates of model parameters. The estimates themselves are shown as a rug along the
x-axis. The results of individual (uncorrected) two-sample t-tests, thresholded at p = 0.05, are indicated in the title of each diagram. Three stars (***)
correspond to p,0.001, indicating that the associated model parameter assumes very different values for patients and controls. doi:10.1371/journal.pcbi.1002079.g009 Figure 9. Univariate feature densities. Separately for patients (red) and healthy controls (grey), the figure shows nonparametric estimates of the
class-conditional densities of the maximum a posteriori (MAP) estimates of model parameters. The estimates themselves are shown as a rug along the
x-axis. Characterization of the feature space In summary, all selected features represented connectivity
parameters (as opposed to stimulus input), their selection was both
sparse and highly consistent across resampling repetitions, and their
combination was sufficient to afford the same classification accuracy
as the full feature set. In summary, all selected features represented connectivity
parameters (as opposed to stimulus input), their selection was both
sparse and highly consistent across resampling repetitions, and their
combination was sufficient to afford the same classification accuracy
as the full feature set. Characterization of the feature space This figure provides an intuition of how a generative model transforms the data from a voxel-
based feature space into a generative score space (or model-based feature space), in which classes become more separable. The left plot shows how
aphasic patients (red) and healthy controls (grey) are represented in voxel space, based on t-scores from a simple ‘all auditory events’ contrast (see main
text). The three axes represent the peaks of those three clusters that showed the strongest discriminability between patients and controls, based on a
locally multivariate searchlight classification analysis. They are located in L.PT, L.HG, and R.PT, respectively (cf. Table 1). The right plot shows the three
individually most discriminative parameters (two-sample t-test) in the (normalized) generative score space induced by a dynamic causal model of speech
processing (see Figure 3). The plot illustrates how aphasic patients and healthy controls become almost perfectly linearly separable in the new space. Note that this figure is based on normalized examples (as used by the classifier), which means the marginal densities are not the same as those shown in
Figure 9 but are exactly those seen by the classifier. A stereogram of the generative score space can be found in the Supplementary Material (Figure S4). doi:10.1371/journal.pcbi.1002079.g007 PLoS Computational Biology | www.ploscompbiol.org June 2011 | Volume 7 | Issue 6 | e1002079 13 Generative Embedding for fMRI hemisphere and converge on the left planum temporale which is a
critical structure for processing of language and speech [101,102]. In summary, all selected features represented connectivity
parameters (as opposed to stimulus input), their selection was both
sparse and highly consistent across resampling repetitions, and their
combination was sufficient to afford the same classification accuracy
as the full feature set. Figure 8. Connectional fingerprints. Given the low dimensionality
of the model-induced feature space, subjects can be visualized in terms
of ‘connectional fingerprints’ [98] that are based on a simple radial
coordinate system in which each axis corresponds to the maximum a
posteriori (MAP) estimate of a particular model parameter. The plot
shows that the difference between aphasic patients (red) and healthy
controls (grey) is not immediately obvious, suggesting that it might be
subtle and potentially of a distributed nature. doi:10.1371/journal.pcbi.1002079.g008 hemisphere and converge on the left planum temporale which is a
critical structure for processing of language and speech [101,102]. Perspectives for generative embedding of fMRI data Second, BMS is limited when different groups cannot
be mapped onto different model structures, for example when the
differences in neuronal mechanisms operate at a finer conceptual
scale than can be represented within the chosen modelling
framework. In this case, discriminability of subjects may be
afforded by differences in (combinations of) parameter estimates
under the same model structure (see [106] for a recent example). Figure 10. Discriminative features. A support vector machine with
a sparsity-inducing regularizer (capped ‘1-regularizer) was trained and
tested in a leave-one-out cross-validation scheme, resulting in n subsets
of selected features. The figure summarizes these subsets by visualizing
how often each feature (printed along the y-axis) was selected across
the n repetitions (given as a fraction on the x-axis). Error bars represent
central 95% posterior probability intervals of a Beta distribution with a
flat prior over the interval [0, 1]. A group of 9 features was consistently
found jointly informative for discriminating between aphasic patients
and healthy controls (see main text). An additional figure showing
which features were selected in each cross-validation fold can be found
in the Supplementary Material (Figure S3). Crucially, since each feature
corresponds to a model parameter that describes one particular
interregional connection strength, the group of informative features
can be directly related back to the underlying dynamic causal model
(see highlighted connections in Figure 3). doi:10.1371/journal.pcbi.1002079.g010 In both these scenarios, the approach proposed in this paper
may provide a solution, in that the unsupervised creation of a
generative score space can be viewed as a method for biologically
informed feature extraction, and the performance of the classifier
reflects how much class information is encoded in the model
parameters. This view enables a form of model comparison in
which the best model is the one that enables the highest
classification accuracy. This procedure can be applied even when
(i) the underlying data (e.g., the chosen regional fMRI time series)
are different, or when (ii) the difference between two models lies
exclusively in the pattern of parameter estimates. In this paper, we
have illustrated both ideas: structural model selection to decide
between a full model and two reduced models that disregard one
hemisphere; and dynamic model selection to distinguish between
different groups of subjects under the same model structure. Perspectives for generative embedding of fMRI data The second advantage of generative embedding for fMRI is that
it enables an intuitive and mechanistic interpretation of features
and their weights, an important property not afforded by most
conventional classification methods [103,104]. By using parameter
estimates from a mechanistically interpretable model for con-
structing a feature space, the subsequent classification no longer
yields ‘black box’ results but allows one to assess the relative
importance of different mechanisms for distinguishing groups (e.g.,
whether or not synaptic plasticity alters the strengths of certain
connections in a particular context). Put differently, generative
embedding embodies a shift in perspective: rather than represent-
ing sequential data in terms of high-dimensional and potentially
highly complex trajectories, we are viewing the data in terms of the
coefficients of a well-behaved model of system dynamics. Again,
this may be of particular importance for clinical applications, as
discussed in more detail below. It is also interesting to note that
models like DCM, when used in the context of generative
embedding, turn the curse of dimensionality faced by conventional
classification methods into a blessing: the higher the spatial and
temporal resolution of the underlying fMRI data, the more precise
the resulting DCM parameter estimates; this in turn should lead to
more accurate predictions. In summary, BMS evaluates the goodness of a model with
regard to its generalizability for explaining the data, whereas
generative embedding evaluates a model in relation to an external
criterion, i.e., how well it allows for inference on group
membership of any given subject. This difference is important as
it highlights that the concept of a ‘good’ model can be based on
fundamentally different aspects, and one could imagine scenarios
where BMS and generative embedding arrive at opposing results. If, for example, discriminability of groups relies on a small
subspace of the data, then one model (which provides a good
accuracy-complexity trade-off for most of the data except that
subspace) may have higher evidence, but another model that
describes this subspace particularly well but is generally worse for
the rest of the data may result in better classification performance
(cf. our discussion in [106]). We will examine the relation and
complementary
nature
of
BMS
and
generative-embedding
approaches in future work. As discussed in this paper, there are three valid strategies for the
implementation of generative embedding in fMRI that allow for
an unbiased estimate of classification accuracy (Figure 2). Perspectives for generative embedding of fMRI data Second, BMS is limited when different groups cannot
be mapped onto different model structures, for example when the
differences in neuronal mechanisms operate at a finer conceptual
scale than can be represented within the chosen modelling
framework. In this case, discriminability of subjects may be
afforded by differences in (combinations of) parameter estimates
under the same model structure (see [106] for a recent example). In both these scenarios, the approach proposed in this paper
may provide a solution, in that the unsupervised creation of a
generative score space can be viewed as a method for biologically
informed feature extraction, and the performance of the classifier
reflects how much class information is encoded in the model
parameters. This view enables a form of model comparison in
which the best model is the one that enables the highest
classification accuracy. This procedure can be applied even when
(i) the underlying data (e.g., the chosen regional fMRI time series)
are different, or when (ii) the difference between two models lies
exclusively in the pattern of parameter estimates. In this paper, we
have illustrated both ideas: structural model selection to decide
between a full model and two reduced models that disregard one
hemisphere; and dynamic model selection to distinguish between
different groups of subjects under the same model structure. their parameterization (e.g., priors). Competing models can be
compared using Bayesian model selection (BMS) [89,83,86,88],
where the best model is the one with the highest model evidence,
that is, the highest probability of the data given the model [105]. BMS is a generic approach to distinguish between different models
that is grounded in Bayesian probability theory and, when group-
specific mechanisms can be mapped onto distinct models,
represents a powerful technique for model-based classification in
itself. However, there are two scenarios in which BMS is
problematic and where classification based on generative embed-
ding may represent a useful alternative [61]. First, BMS requires
the data to be identical for all competing models. Thus, in the case
of current implementations of DCM for fMRI, BMS enables
dynamic model selection (concerning the parameterization and
mathematical form of the model equations) but not structural model
selection (concerning which regions or nodes should be included in
the model). PLoS Computational Biology | www.ploscompbiol.org June 2011 | Volume 7 | Issue 6 | e1002079 Perspectives for generative embedding of fMRI data The results of individual (uncorrected) two-sample t-tests, thresholded at p = 0.05, are indicated in the title of each diagram. Three stars (***)
correspond to p,0.001, indicating that the associated model parameter assumes very different values for patients and controls. doi:10.1371/journal.pcbi.1002079.g009 PLoS Computational Biology | www.ploscompbiol.org June 2011 | Volume 7 | Issue 6 | e1002079 PLoS Computational Biology | www.ploscompbiol.org PLoS Computational Biology | www.ploscompbiol.org 14 Generative Embedding for fMRI Figure 10. Discriminative features. A support vector machine with
a sparsity-inducing regularizer (capped ‘1-regularizer) was trained and
tested in a leave-one-out cross-validation scheme, resulting in n subsets
of selected features. The figure summarizes these subsets by visualizing
how often each feature (printed along the y-axis) was selected across
the n repetitions (given as a fraction on the x-axis). Error bars represent
central 95% posterior probability intervals of a Beta distribution with a
flat prior over the interval [0, 1]. A group of 9 features was consistently
found jointly informative for discriminating between aphasic patients
and healthy controls (see main text). An additional figure showing
which features were selected in each cross-validation fold can be found
in the Supplementary Material (Figure S3). Crucially, since each feature
corresponds to a model parameter that describes one particular
interregional connection strength, the group of informative features
can be directly related back to the underlying dynamic causal model
(see highlighted connections in Figure 3). doi:10.1371/journal.pcbi.1002079.g010 their parameterization (e.g., priors). Competing models can be
compared using Bayesian model selection (BMS) [89,83,86,88],
where the best model is the one with the highest model evidence,
that is, the highest probability of the data given the model [105]. BMS is a generic approach to distinguish between different models
that is grounded in Bayesian probability theory and, when group-
specific mechanisms can be mapped onto distinct models,
represents a powerful technique for model-based classification in
itself. However, there are two scenarios in which BMS is
problematic and where classification based on generative embed-
ding may represent a useful alternative [61]. First, BMS requires
the data to be identical for all competing models. Thus, in the case
of current implementations of DCM for fMRI, BMS enables
dynamic model selection (concerning the parameterization and
mathematical form of the model equations) but not structural model
selection (concerning which regions or nodes should be included in
the model). Inference on mechanisms for clinical applications pp
The example described in this paper was chosen to illustrate the
implementation and use of generative embedding for fMRI. It is
important to emphasize that this example does not represent the
sort of clinical application that we envisage in the long term. Clearly,
there are few diagnostic problems when dealing with aphasia and
usually a clinical examination by the physician is sufficient. However, this example is useful for demonstrating the utility of
generative embedding since the diagnostic status of each subject is
known without doubt and the networks involved in speech
processing are well characterized. In the future, we hope that our
approach will be useful for addressing clinical problems of high
practical relevance, for instance for dissecting psychiatric spectrum
disorders, such as schizophrenia, into physiologically defined
subgroups [47], or for predicting the response of individual patients
to specific drugs. While an increasing number of studies have tried
to describe neurobiological markers for psychiatric disorders
[22,107,108,3,109,110,14,15], we argue that these studies should
be complemented by model-based approaches for inferring
biologically plausible mechanisms. Such approaches will be useful
in two domains of application: they can be used to decide between
competing hypotheses (as in traditional applications of DCM and
BMS); and they can harvest the potentially rich discriminative
information encoded in aspects of synaptic plasticity or neuromo-
dulation to build classifiers that distinguish between different
subtypes of a psychiatric disorder on a physiological basis (using
techniques such as generative embedding). y
pp
p
g
Second, since features correspond to model parameters, our
approach allowed us to characterize a subset of features (Figure 10)
that can be interpreted in the context of the underlying model
(Figure 3). This subset showed four remarkable properties. (i) Discriminative parameters were restricted to cortico-cortical
and thalamo-cortical connection strengths. On the contrary,
parameters representing auditory inputs to thalamic nuclei did
not contribute to the distinction between patients and healthy
controls. (ii) We observed a high degree of stability across
resampling folds. That is, the same 9 (out of 22) features were
selected on almost every repetition. (iii) The set of discriminative
parameters was found to be sparse, not just within repetitions
(which is enforced by the underlying regularizer) but also across
repetitions (which is not enforced by the regularizer; see Figure S3
in the Supplementary Material). At the same time, the set was
considerably larger than what would be expected from univariate
feature-wise t-tests (Figure 9). Summary of our findings In order to demonstrate the utility of generative embedding for
fMRI, we acquired and analysed a dataset consisting of 11 aphasic
patients
and
26
healthy
controls. During
the
experiment,
participants were listening to a series of speech and speech-like
stimuli. In an initial analysis (Schofield et al., in preparation), we
designed a dynamic causal model to explain observed activations
in 6 auditory regions of interest. Here, we extended this analysis by
examining whether patients and healthy controls could be
distinguished on the basis of differences in subject-specific
generative models. Specifically, we trained and tested a linear
support vector machine on subject-wise estimates of connection
strengths. This approach delivered two sets of results. It is worthwhile briefly commenting on how the present findings
relate to those of the original DCM study by Schofield et al. (in
preparation). Two crucial differences are that the previous study (i)
applied Bayesian model averaging to a set of 512 models and (ii)
statistically examined each of the resulting average connection
strengths in a univariate fashion. They found group differences for
most connections, highlighting in particular the top-down connec-
tions from planum temporale to primary auditory cortex bilaterally. In our multivariate analysis, these two connections were also
amongst the most informative ones for distinguishing patients from
controls (Figure 3). Schofield et al. also found group differences for
interhemispheric connection strengths between left and right
Heschl’s gyrus, but their univariate approach did not demonstrate
any asymmetries. In contrast, our multivariate approach yielded a
sparser set of discriminative connections, highlighting the asymme-
tries of interhemispheric connections described above (Figure 3). First, we found strong evidence in favour of the hypothesis that
aphasic patients and healthy controls may be distinguished on the
basis of differences in the parameters of a generative model alone. Generative embedding did not only yield a near-perfect balanced
classification accuracy (98%). It also significantly outperformed
conventional activation-based methods, whether they were based
on anatomical (62%), contrast (75%), searchlight feature selection
(73%), or on a PCA-based dimensionality reduction (80%). Similarly, our approach outperformed conventional correlation-
based methods, whether they were based on regional means (70%)
or regional eigenvariates (83% and 74%). Furthermore, it is
interesting
to observe that group
separability was reduced
considerably when using a less plausible feedforward model
(77%). Perspectives for generative embedding of fMRI data If
regions (and thus time series) are defined anatomically, the model
is inverted separately for each subject, and the resulting parameter
estimates can be safely used in cross-validation. If regions are
defined by a functional contrast, both time series selection and
model inversion for all subjects need to be carried out separately
for each cross-validation fold. These procedures clearly have The third advantage provided by generative embedding is
related to model comparison. For any given dataset, there is an
infinite number of possible dynamic causal models, differing in the
number and location of nodes, in connectivity structure, and in 15 June 2011 | Volume 7 | Issue 6 | e1002079 Generative Embedding for fMRI speech and speech-like sounds between aphasic patients and
healthy controls. In particular, all of the connections from the right
to the left hemisphere were informative with regard to group
membership, but none of the connections in the reverse direction. This asymmetry resonates with previous findings that language-
relevant information is transferred from the right hemisphere to
the left, but not vice versa [100], and suggests that in aphasia
connectivity changes in non-lesioned parts of the language
network have particularly pronounced effects on inter-hemispheric
transfer of speech information from the (non-dominant) right
hemisphere to the (dominant) left hemisphere. higher computational demands than conventional classification
techniques, but the subject-wise nature of model inversion means
that generative embedding for fMRI can exploit methods for
distributed computing and can thus be implemented even for
larger numbers of subjects. Summary of our findings Finally, performance decreased significantly when modelling
only the left hemisphere (81%), and it dropped to chance when
considering the right hemisphere by itself (60%), which is precisely
what one would expect under the view that the left hemisphere is
predominantly,
but
not
exclusively,
implicated
in
language
processing. Taken together, our findings provide strong support
for the central idea of this paper: that critical differences between
groups of subjects may be expressed in a highly nonlinear manifold
which remains inaccessible by methods relying on activations or
undirected correlations, but which can be unlocked by the nonlinear
transformation embodied by an appropriate generative model. June 2011 | Volume 7 | Issue 6 | e1002079 PLoS Computational Biology | www.ploscompbiol.org Figure S4 Stereogram of the generative score space. Based on the
generative score space illustrated in the paper (see right plot in
Figure 7), we here show the same plot from two slightly different
angles. Readers are invited to try and focus an imaginary point
behind the two plots, or use a stereoscope, to recover a fully three-
dimensional impression. Figure S2
Further characterization of the generative
score space. By analogy with the univariate feature densities
shown in Figure 9, the discriminative information encoded in
simple combinations of model parameters can be illustrated using
bivariate scatter plots. The figure indicates how well any two
features jointly discriminated between patients and healthy
controls. Note that the matrix is symmetric. (TIF) References 14. Klo¨ppel S, Stonnington CM, Chu C, Draganski B, Scahill RI, et al. (2008)
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parameters proved sufficient to yield the same balanced classifi-
cation accuracy (98%) as the full set. These results are consistent
with the notion that a distinct mechanism, and thus few
parameters, are sufficient to explain differences in processing of In the case of the illustrative dataset analysed in this paper,
generative embedding yielded stronger classification performance
than conventional methods, whether they were based on activations
or regional correlations. One might think that this superior ability to
accurately classify individual subjects determines the clinical value
of the approach. Instead, we wish to argue that its clinical value will
ultimately depend on whether patients that share the same
symptoms can be differentially treated according to the underlying
pathophysiology of the disorder. Generative embedding, using PLoS Computational Biology | www.ploscompbiol.org June 2011 | Volume 7 | Issue 6 | e1002079 16 Generative Embedding for fMRI biologically plausible and mechanistically interpretable models, may
prove critical in establishing diagnostic classification schemes that
distinguish
between
pathophysiologically
distinct subtypes
of
spectrum diseases and allow for predicting individualized behav-
ioural and pharmacological therapy. Figure S3
Feature selection using a sparse SVM. A
support vector machine with a sparsity-inducing regularizer [75]
was used to investigate, based on leave-one-out cross validation,
which features were typically selected across the underlying n folds. (a) The left figure shows in detail which features were selected in
each repetition. For example, when based on all subjects but the
first, the classifier selected exactly those 9 features that were
selected most of the time; when based on all subjects but the last, a
slightly different group of 10 features was favoured. The figure
shows that the set of selected features is both sparse and highly
consistent across resampling repetitions. As described in the paper,
it afforded the same classification accuracy as the full set. (b) The
right figure shows the posterior variance of each model parameter,
separately for selected and discarded parameters. The data
provide no evidence that the algorithm simply selected those
parameters that were easier to fit, as would be indicated by a lower
posterior variance (two-tailed t-test, p<0.640). (TIF) Author Contributions Conceived and designed the experiments: TMS APL KES. Performed the
experiments: TMS APL. Analyzed the data: KHB TMS APL CSO EIL
JMB KES. Wrote the paper: KHB. Supporting Information Figure S1
Further characterization of the voxel-based
feature space. (1) Regions of interest. In order to illustrate
generative embedding for fMRI, a dynamic causal model was
constructed on the basis of 6 anatomical regions of interest. As
described in the paper, the exact location of these regions was
determined on the basis of an n{1 group contrast and hence varied
between
cross-validation
folds. Regions
were
defined
by
16 mm616 mm616 mm cubes centred on the group maxima
(see Table 1 in the paper). The figure shows the location and extent
of the anatomical masks (green) that were used to define fold-specific
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Faulty Feeder Identification Based on Data Analysis and Similarity Comparison for Flexible Grounding System in Electric Distribution Networks
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Citation: Liu, K.; Zhang, S.; Li, B.;
Zhang, C.; Liu, B.; Jin, H.; Zhao, J. Faulty Feeder Identification Based on
Data Analysis and Similarity
Comparison for Flexible Grounding
System in Electric Distribution
Networks. Sensors 2021, 21, 154. https://doi.org/10.3390/s21010154 Keywords: faulty feeder identification; data analysis; similarity comparison; flexible grounding
system; grey T-type correlation degree; wavelet packet transform; electric distribution networks Faulty Feeder Identification Based on Data Analysis and
Similarity Comparison for Flexible Grounding System in
Electric Distribution Networks Liu 1,2,*, Sen Zhang 1, Baorun Li 1,3, Chi Zhang 1, Biyang Liu 1, Hao Jin 1 and Jianfeng Zhao 1 1
School of Electrical Engineering, Southeast University, Nanjing 210096, China; sen_zhang@seu.edu.cn (S.Z.);
220172670@seu.edu.cn (B.L.); 220192759@seu.edu.cn (C.Z.); 230179573@seu.edu.cn (B.L.);
230169162@seu.edu.cn (H.J.); jianfeng_zhao@seu.edu.cn (J.Z.)
2
Jiangsu Provincial Key Laboratory of Smart Grid Technology and Equipment, Nanjing 210096, China
3
State Grid Suqian Power Supply Company, Suqian 223800, China
*
Correspondence: kangli@seu.edu.cn 1
School of Electrical Engineering, Southeast University, Nanjing 210096, China; sen_zhang@seu.edu.cn (S.Z.);
220172670@seu.edu.cn (B.L.); 220192759@seu.edu.cn (C.Z.); 230179573@seu.edu.cn (B.L.);
230169162@seu.edu.cn (H.J.); jianfeng_zhao@seu.edu.cn (J.Z.)
2
Jiangsu Provincial Key Laboratory of Smart Grid Technology and Equipment, Nanjing 210096, China
3
State Grid Suqian Power Supply Company, Suqian 223800, China
*
Correspondence: kangli@seu.edu.cn Abstract: Reliability and safety are the most important indicators in the electric system. When a
ground fault occurs, the electrical equipment and personnel will be greatly threatened. Due to the
zero-sequence voltage/current sensor networks applied in the system, the fault identification and
diagnosis technology are developing rapidly, including the application of ground fault suppression. A flexible grounding system (FGS) is a new technology applied to arc extinguishing in medium and
high voltage electric distribution networks. Its characteristic is that when the single-phase ground
fault occurs, the power-electronic-based device is put into the electric system to compensate and
suppress the ground point current to be close to zero in a very short time. In order to implement the
above process, the corresponding faulty feeder identification method needs to meet the requirements
of rapidity and accuracy. In this article, based on the real-time sampled data from the zero-sequence
current/voltage sensors, an improved faulty feeder identification method combining wavelet packet
transform (WPT) and grey T-type correlation degree is proposed, which features both accuracy
and rapidity. The former is used to reconstruct the transient characteristic signal, and the latter is
responsible for calculating and comparing the similarity of relative variation trend. Simulation results
verify the rationality and effectiveness of the proposed method and analysis. Citation: Liu, K.; Zhang, S.; Li, B.;
Zhang, C.; Liu, B.; Jin, H.; Zhao, J.
Faulty Feeder Identification Based on
Data Analysis and Similarity
Comparison for Flexible Grounding
System in Electric Distribution
Networks. Sensors 2021, 21, 154.
https://doi.org/10.3390/s21010154 sensors sensors 1. Introduction Fire potentially caused by the ground fault in electric distribution networks. g
p
y
y
g
At present, the faulty feeder identification methods for the grounding system can b
oughly divided into four categories [14–23], and they are all based on the transient signal
nalysis: (1) First half-wave method; (2) energy method; (3) time-frequency domain ana
sis method; (4) similarity comparison method. The first half-wave method compares th
nitial polarity of the bus transient zero-sequence voltage of the bus and the polarity of th
ransient zero-sequence current of each feeder. The line with the opposite polarity of th
ransient zero-sequence voltage of the bus is the faulty feeder [24,25]. However, thi
method needs to collect the waveform of the first half period after the fault, which require
igh speed and data synchronization, and its reliability is poor. The energy method use
he transient zero-sequence voltage and current of the non-faulty/faulty feeder first to ob
ain the energy of the transient process and then realizes the fault line selection by com
aring the difference in energy magnitude and direction [26]. However, this method ig
ores large amounts of high-frequency signals, which can easily lead to false selection
The time-frequency domain analysis method mainly includes the wavelet transform (WT
nd wavelet packet transform (WPT) [27]. In addition, there are some other time-fre
uency domain analysis methods that are utilized to extract the transient feature, such a
he Fourier transform (The time-domain signal is transformed into the frequency-domai
ignal) [28], short-time Fourier transform (STFT, and the window function is added) [29
mpirical mode decomposition (EMD) [30], Hilbert–Huang Transform (HHT) [31,32
Wigner–Ville distribution (WVD) [33], and so on. WPT improves high-frequency resolu
ion on the basis of WT, thus it is more conducive to fault signal processing [34–36]. It firs
ecomposes the fault transient signal in each frequency band, then determines the fre
uency band that can reflect the fault characteristics most obviously, and finally uses th
At present, the faulty feeder identification methods for the grounding system can
be roughly divided into four categories [14–23], and they are all based on the transient
signals analysis: (1) First half-wave method; (2) energy method; (3) time-frequency domain
analysis method; (4) similarity comparison method. The first half-wave method compares
the initial polarity of the bus transient zero-sequence voltage of the bus and the polarity of
the transient zero-sequence current of each feeder. 1. Introduction Received: 2 December 2020
Accepted: 24 December 2020
Published: 29 December 2020 As an important part of the electric system, the medium and high voltage electric
distribution networks are closely related to all industrial and residential users. Its safety
and reliability have always been a research hotspot that has been widely discussed. Among
many fault types of distribution networks, the single-phase ground fault is more frequent in
practice. When a ground fault occurs in the system, the safety of electrical equipment and
personnel will be greatly threatened. For example, fires potentially caused by ground faults
have occurred in many countries, as shown in Figure 1. Therefore, the implementation of
reliable and effective grounding is an important premise to ensure the safety of distribution
networks. In recent years, with the continuous development of the distribution network
topology and the increasing number of cable lines, the resonant grounding method (arc
suppression coil (ASC) or Peterson coil) has been difficult to meet the requirement of rapid
arc extinction. In order to solve this problem, the power-electronics-devices-based flexible
grounding system (FGS) has been proposed in recent years [1–6]. Flexible grounding
means that on the basis of traditional resonant grounding, when a single-phase ground
fault occurs, power-electronic-based devices will be put into the distribution network, and
the fault current at the ground point is suppressed to be close to zero in a very short period Publisher’s Note: MDPI stays neu-
tral with regard to jurisdictional claims
in published maps and institutional
affiliations. Publisher’s Note: MDPI stays neu-
tral with regard to jurisdictional claims
in published maps and institutional
affiliations. Copyright: © 2020 by the authors. Li-
censee 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/). https://www.mdpi.com/journal/sensors Sensors 2021, 21, 154. https://doi.org/10.3390/s21010154 Sensors 2021, 21, 154 2 of 16
be clos
l
ti of time. In addition, the fault arc will be completely extinguished. The existing flexible
grounding schemes include open-loop and closed-loop compensation schemes [7,8]. For
the former, it is estimated to obtain the compensation current, and the active inverter is
equivalent to the current source. The working principle of the inverter in this scheme is
similar to an active power filter (APF). For the latter, it feeds back the voltage at the fault
point, and the active inverter is equivalent to the voltage source. 1. Introduction The output voltage forces
the fault point voltage to be equal to zero or below the safety threshold. However, no matter
which kind of flexible grounding scheme is adopted, higher requirements are put forward
for the faulty feeder selection technology: (1) Accuracy: The faulty feeder identification
results determine the parameter values calculation in the compensation process, which will
directly affect the reliability of FGS. Especially, when a high-resistance ground fault occurs,
smaller fault characteristics will make the feeder identification process more difficult; (2)
rapidity: FGS needs to realize arc extinguishing in a very short time (usually several power
frequency cycles), which makes the feeder identification process need to be completed in a
very short time. In addition to the faulty feeder identification, fault detection and location
are also very important in the electric system, which are not the focus of this paper. Some
effective methods have been proposed [9–13]. ompensation schemes [7,8]. For the former, it is estimated to obtain the compensatio
urrent, and the active inverter is equivalent to the current source. The working principl
f the inverter in this scheme is similar to an active power filter (APF). For the latter,
eeds back the voltage at the fault point, and the active inverter is equivalent to the voltag
ource. The output voltage forces the fault point voltage to be equal to zero or below th
afety threshold. However, no matter which kind of flexible grounding scheme i
dopted, higher requirements are put forward for the faulty feeder selection technology
1) Accuracy: The faulty feeder identification results determine the parameter values ca
ulation in the compensation process, which will directly affect the reliability of FGS. Es
ecially, when a high-resistance ground fault occurs, smaller fault characteristics wi
make the feeder identification process more difficult; (2) rapidity: FGS needs to realize ar
xtinguishing in a very short time (usually several power frequency cycles), which make
he feeder identification process need to be completed in a very short time. In addition t
he faulty feeder identification, fault detection and location are also very important in th
lectric system, which are not the focus of this paper. Some effective methods have bee
roposed [9–13]. i
1 Fi
t
ti ll
d b
th
d f
lt i
l
t i di t ib ti
t
k
Figure 1. Fire potentially caused by the ground fault in electric distribution networks. Figure 1. 1. Introduction The other methods, such as EMD, HHT, and Wigner–Ville
distribution, are more widely used in mechanical fault diagnosis, power quality assessment,
and other fields. The similarity comparison method determines the similarity between the
zero-sequence current waveforms of the feeder by finding the correlation coefficient or
grey correlation degree and finds the feeder with the most different from other lines as the
fault feeder [37–43]. This method has higher line selection accuracy than other methods
mentioned above but requires a longer period of the waveform to extract the transient
components contained in the fault waveform. In [40], in order to extract the transient
characteristic quantity, the zero-sequence current waveform after 10 cycles is subtracted
from the zero-sequence current when the fault occurs. Hence, its dynamic performance
will be affected. In summary, none of the above four methods can meet the requirements
of accuracy and rapidity at the same time, and, therefore, it is not suitable for a flexible
grounding scheme. An improved faulty feeder identification method for FGS is required. Due to the large number of zero-sequence voltage and current sensors in the electric
distribution network, the accurate and rapid fault identification result can be achieved. In addition, the key to faulty feeder identification is to separate fault information from a
large number of real-time sampled data. Figures 2 and 3 show the schematic diagrams of
FGS and its neutral-point voltage compensation strategy. The hardware mainly includes
an active inverter, impedance grounding system (ASC or low-resistance), and sensor
networks. When a ground fault occurs, the fault-point voltage exceeds the safety threshold,
and the fault arc is thus formed. Since the sum of the fault-phase voltage (UA, UB, UC)
and neutral-point voltage (UNO) is equal to the fault-point voltage, in order to suppress
the fault-point voltage and make it below the safety threshold, the active inverter can be
used to compensate the neutral-point voltage, which can offset the fault-phase voltage. U′NAset, U′NBset and U′NCset are the compensated neutral-point voltages. In Figure 3, the
blue areas represent the safe range of the fault-phase voltage value. That is, when the fault-
phase voltage is offset to this range, the fault point can not arcing, and the ground fault
is effectively suppressed. Additionally, according to different application scenarios, the
topology structure of the active inverter can be single-phase H-bridge, cascaded H-bridge,
modular multilevel converter (MMC), and multi-material devices [44–53]. 1. Introduction The line with the opposite polarity of
the transient zero-sequence voltage of the bus is the faulty feeder [24,25]. However, this
method needs to collect the waveform of the first half period after the fault, which requires
high speed and data synchronization, and its reliability is poor. The energy method uses
the transient zero-sequence voltage and current of the non-faulty/faulty feeder first to
obtain the energy of the transient process and then realizes the fault line selection by
comparing the difference in energy magnitude and direction [26]. However, this method
ignores large amounts of high-frequency signals, which can easily lead to false selection. The time-frequency domain analysis method mainly includes the wavelet transform (WT)
and wavelet packet transform (WPT) [27]. In addition, there are some other time-frequency
domain analysis methods that are utilized to extract the transient feature, such as the Fourier
transform (The time-domain signal is transformed into the frequency-domain signal) [28],
short-time Fourier transform (STFT, and the window function is added) [29], empirical
mode decomposition (EMD) [30], Hilbert–Huang Transform (HHT) [31,32], Wigner–Ville
distribution (WVD) [33], and so on. WPT improves high-frequency resolution on the basis
of WT, thus it is more conducive to fault signal processing [34–36]. It first decomposes
the fault transient signal in each frequency band, then determines the frequency band
that can reflect the fault characteristics most obviously, and finally uses the amplitude,
polarity and phase, entropy value, and other characteristic quantities of the signal in the
band to select the fault line. The WPT method has strong signal extraction ability and
can realize the transient signals processing in a short time. However, the accuracy of
the method is affected by the sampling frequency and the complexity of the distribution Sensors 2021, 21, 154 3 of 16 network. Compared with the Fourier transform, the signal with WPT can be processed
within one or two fundamental current cycles, which means the signal processing has a
better real-time performance. Besides that, WPT is more suitable for time domain and
frequency domain analysis at the same time. In addition, for the Fourier transform, as the
basis functions are sine or cosine functions, it is more suitable for the analysis of stationary
signals. To solve this, the window function is added to the Fourier transform (STFT). However, the selection of the window function will seriously affect the time-frequency
resolution of signal analysis. 1. Introduction In order to
achieve the high voltage output of the active inverter, the multilevel topology or cascaded
H-bridge can be adopted [44–49]. If the output voltage level of the active inverter is lower,
an isolation booster transformer is necessary. In addition, in order to achieve the tradeoff
of power density, performance, and cost, multi-material devices can be applied [50–53]. The whole software process includes fault diagnosis, fault feeder identification (mainly
discussed in this article), and suppression. Firstly, the controller of the flexible grounding
system samples the signal data of zero-sequence voltage and current transformer, then
performs necessary data processing and analysis, and finally sends driving signals to power
electronic devices, thus that the voltage or current at fault point will be suppressed to a
safe range. 4 of 16
of 16
4 of 16 Sensors 2021, 21, 154
nsors 2021, 21, x FOR PE
Sensors 2021, 21, x FOR SM2
SM3
SMn
SM1
Multi-Material-Devices
Based Power Conversion
WBG
Traditional
SMn-1
A
D
B
C
Single Phase H-bridge
Cascaded H-bridge
MMC
out
out
out
out
L
C
Isolated
Transformer
L
L1
L2
Cdc
CH1
CH2
CHn
Module 1
Module 2
Module n
Common DC Link
Cdc
Ln
Grid
110/10kV Transformer
FGS
ASC
S
I01
I02
I0n
PT
U0
CT
Zero-sequence Current
DSP+FPGA
based
Controller
DI/DO, u, i
Sensor Networks
Figure 2. Schematic diagram of the flexible grounding system (FGS)-based electric distribution
network with zero-sequence current/voltage sensors. And the topologies of the active inverter can
be: (A) single-phase H-bridge; (B) cascaded H-bridge; (C) modular multilevel converter (MMC);
(D) multi-material devices-based topology. Figure 2. Schematic diagram of the flexible grounding system (FGS)-based electric distribution
network with zero-sequence current/voltage sensors. And the topologies of the active inverter can
be: (A) single-phase H-bridge; (B) cascaded H-bridge; (C) modular multilevel converter (MMC); (D)
multi-material devices-based topology. SM2
SM3
SMn
SM1
Multi-Material-Devices
Based Power Conversion
WBG
Traditional
SMn-1
A
D
B
C
Single Phase H-bridge
Cascaded H-bridge
MMC
out
out
out
out
L
C
Isolated
Transformer
L
L1
L2
Cdc
CH1
CH2
CHn
Module 1
Module 2
Module n
Common DC Link
Cdc
Ln
Grid
110/10kV Transformer
FGS
ASC
S
I01
I02
I0n
PT
U0
CT
Zero-sequence Current
DSP+FPGA
based
Controller
DI/DO, u, i
Sensor Networks
Figure 2. Schematic diagram of the flexible grounding system (FGS)-based electric distribution
network with zero-sequence current/voltage sensors. 1. Introduction And the topologies of the active inverter can
be: (A) single-phase H-bridge; (B) cascaded H-bridge; (C) modular multilevel converter (MMC);
(D) multi-material devices-based topology. Ln
Grid
110/10kV Transformer
FGS
ASC
S
I01
I02
I0n
PT
U0
CT
Zero-sequence Current
DSP+FPGA
based
Controller
DI/DO, u, i
S
N t
k
Ln
Grid
110/10kV Transformer
FGS
ASC
S
I01
I02
I0n
PT
U0
CT
Zero-sequence Current
DSP+FPGA
based
Controller
DI/DO, u, i Zero-sequence Current
Zero-sequence Curre 110/10kV Transformer
110/10kV Transform SM2
SM3
SMn
SM1
SMn-1
C
MMC
out
L1
L2
Common DC Link
SM2
SM3
SMn
SM1
SMn-1
C
e
MMC
out
out
L1
L2
Common DC Link
rks A
Single Phase H-bridge
out
L
C
Isolated
Transformer
Cdc
A
Single Phase H-bridge
L
C
Isolated
Transform
Cdc B
Cascaded H-bridge
out
L
CH1
CH2
CHn
Module 1
Module 2
Module n
B
Cascaded H-bridg
L
CH1
CH2
CHn
Module 1
Module 2
Module n
Sensor Netwo B Cascaded H-bridge
Module n
Cascaded H-brid
Module n gure 2. Schematic diagram of the flexible grounding system (FGS)-based electric distribution
twork with zero-sequence current/voltage sensors. And the topologies of the active inverter can
: (A) single-phase H-bridge; (B) cascaded H-bridge; (C) modular multilevel converter (MMC);
) multi-material devices-based topology. Figure 2. Schematic diagram of the flexible grounding system (FGS)-based electric distribution
network with zero-sequence current/voltage sensors. And the topologies of the active inverter can
be: (A) single-phase H-bridge; (B) cascaded H-bridge; (C) modular multilevel converter (MMC); (D)
multi-material devices-based topology. Figure 2. Schematic diagram of the flexible grounding system (FGS)-based electric distribution
network with zero-sequence current/voltage sensors. And the topologies of the active inverter can
be: (A) single-phase H-bridge; (B) cascaded H-bridge; (C) modular multilevel converter (MMC);
(D) multi-material devices-based topology. Sampled data：zero-sequence
voltage and current
(a)
Fault diagnosis
Faulty feeder
identification
Fault suppression
❶
❷
❸
A
U
C1
C2
C3
B1
B2
A1
A2
A3
B3
B
U
C
U
set
NB
U′
B
U′
A
U
C1
C2
C3
B1
B2
B3
A1
A2
A3
B
U
C
U
set
NA
U′
set
NB
U′
B
U′
C
U′
set
NC
U′
A
U′
set
NC
U′
C
U′
A
U′
(b)
A
U′
B
U′
AB
U
CA
U
BC
U
AB
U
CA
U
BC
U
0
N
U
0
N
U
set
NA
U′
gure 3. Schematic diagram of neutral-point voltage compensation strategy. 1. Introduction (a) Before the compensation; (b) after the
ompensation. Sampled data：zero-sequence
voltage and current
(a)
Fault diagnosis
Faulty feeder
identification
Fault suppression
❶
❷
❸
A
U
C1
C2
C3
B1
B2
A1
A2
A3
B3
B
U
C
U
set
NB
U′
B
U′
A
U
C1
C2
C3
B1
B2
B3
A1
A2
A3
B
U
C
U
set
NA
U′
set
NB
U′
B
U′
C
U′
set
NC
U′
A
U′
set
NC
U′
C
U′
A
U′
(b)
A
U′
B
U′
AB
U
CA
U
BC
U
AB
U
CA
U
BC
U
0
N
U
0
N
U
set
NA
U′
Figure 3. Schematic diagram of neutral-point voltage compensation strategy. (a) Before the compensation; (b) after the
compensation. Figure 3. Schematic diagram of neutral-point voltage compensation strategy. (a) Before the compensation; (b) after the
compensation. ❶❶ a)(a)
BC
U b)(b)
BC
U gure 3. Schematic diagram of neutral-point voltage compensation strategy. (a) Before the compensation; (b) after the
mpensation. Figure 3. Schematic diagram of neutral-point voltage compensation strategy. (a) Before the compensation; (b) after the
compensation. Figure 3. Schematic diagram of neutral-point voltage compensation strategy. (a) Before the compensation; (b) after the
compensation. of neutral-point voltage compensation strategy. (a) Before the compensation; (b) after the
m of neutral-point voltage compensation strategy. (a) Before the compensation; (b) after the
am of neutral-point voltage compensation strategy. (a) Before the compensation; (b) after the ure 3. Schematic diagram of neutral-point voltage compensation strategy. (a) Before the compensation; (b) after the
mpensation. Figure 3. Schematic diagram of neutral-point voltage compensation strategy. (a) Before the compensation; (b) after the
compensation. Figure 3. Schematic diagram of neutral-point voltage compensation strategy. (a) Before the compensation; (b) after the
compensation In this article, combining the advantages of wavelet packet transform and the simi-
arity comparison method based on grey T-type correlation degree, a new faulty feeder
dentification method with both excellent accuracy and speed is proposed. The wavelet
In this article, combining the advantages of wavelet packet transform and the simi-
larity comparison method based on grey T-type correlation degree, a new faulty feeder
identification method with both excellent accuracy and speed is proposed. The wavelet
In this article, combining the advantages of wavelet packet transform and the simi-
larity comparison method based on grey T-type correlation degree, a new faulty feeder
identification method with both excellent accuracy and speed is proposed. 2. Wavelet Packet Transformation for FGS
2.1. Principle of Wavelet Packet Transform
2. Wavelet Packet Transformation for FGS
2.1. Principle of Wavelet Packet Transform
W
l t
k t t
f
i
th d Wavelet packet transform is a method of time-frequency domain analysis. Compared
with the Fourier transform, the signal with WPT can be processed within one or two
fundamental current cycles. Compared with wavelet transform, the frequency band can
be divided into multiple layers with WPT, and the high-frequency part of the signal is
further decomposed, thus as to ensure that the signal features are complete and has good
time-frequency resolution. WPT performs high-frequency and low-frequency filtering on
the signal over a period of time. Each transformation separates the low-frequency overview
and high-frequency details of the signal. After decomposing n layers, according to the
above steps, the wavelet packet coefficients in 2n frequency bands are obtained. Figure 4 is
a schematic diagram of the three-layer wavelet packet decomposition example. The time
and frequency domains are equally divided into eight segments. At a certain period of
time, such as t = 2, the signal characteristics in eight frequency bands can be represented by
wavelet packet coefficients, which reflect the projection value of the signal on each base
component. It is related to the wavelet base, signal sampling frequency, and the number of
decomposition layers. After the wavelet packet coefficients are obtained, the signal can be
reconstructed in each frequency band according to the proportion of each component in
the signal to obtain the waveform of the signal in each frequency band. Wavelet packet transform is a method of time-frequency domain analysis. Compared
with the Fourier transform, the signal with WPT can be processed within one or two fun-
damental current cycles. Compared with wavelet transform, the frequency band can be
divided into multiple layers with WPT, and the high-frequency part of the signal is further
decomposed, thus as to ensure that the signal features are complete and has good time-
frequency resolution. WPT performs high-frequency and low-frequency filtering on the
signal over a period of time. Each transformation separates the low-frequency overview
and high-frequency details of the signal. After decomposing n layers, according to the
above steps, the wavelet packet coefficients in 2n frequency bands are obtained. Figure 4
is a schematic diagram of the three-layer wavelet packet decomposition example. The time
and frequency domains are equally divided into eight segments. 1. Introduction The wavelet Sensors 2021, 21, 154
Sensors 2021, 21, x FO 5 of 16 packet transform is applied to locate the fault moment and extract the transient characteris-
tic quantity in the fault waveform quickly and accurately. In addition, the grey correlation
degree is applied to analyze the similarity of the relative change trend of each feeder wave-
form to achieve faulty feeder identification. The zero-sequence voltage and current sensor
networks provide necessary real-time data support for algorithm implementation. Suffi-
cient simulation results based on MATLAB/Simulink verify the validity and rationality of
the proposed method. istic quantity in the fault waveform quickly and accurately. In addition, the grey correla-
tion degree is applied to analyze the similarity of the relative change trend of each feeder
waveform to achieve faulty feeder identification. The zero-sequence voltage and current
sensor networks provide necessary real-time data support for algorithm implementation. Sufficient simulation results based on MATLAB/Simulink verify the validity and ration-
ality of the proposed method. This paper is organized as follows. The description of wavelet packet transformation p
p
This paper is organized as follows. The description of wavelet packet transformation
is given in Section 2, including the principle and extraction of zero-sequence current tran-
sient characteristics. Additionally, in this section, the processing of transient waveforms
using WPT is introduced. In Section 3, the similarity comparison method based on grey
T-type correlation degree is presented. Especially, the flow chart of the proposed method
combining WPT and grey T-type correlation degree are given. Taking into account sev-
eral typical ground fault cases, the data analysis and simulation results are presented in
Section 4. Finally, Section 5 summarizes the whole work. is given in Section 2, including the principle and extraction of zero-sequence current tran-
sient characteristics. Additionally, in this section, the processing of transient waveforms
using WPT is introduced. In Section 3, the similarity comparison method based on grey
T-type correlation degree is presented. Especially, the flow chart of the proposed method
combining WPT and grey T-type correlation degree are given. Taking into account several
typical ground fault cases, the data analysis and simulation results are presented in Sec-
tion 4. Finally, Section 5 summarizes the whole work. 2. Wavelet Packet Transformation for FGS
2.1. Principle of Wavelet Packet Transform
2. Wavelet Packet Transformation for FGS
2.1. Principle of Wavelet Packet Transform
W
l t
k t t
f
i
th d 2. Wavelet Packet Transformation for FGS
2.1. Principle of Wavelet Packet Transform
2. Wavelet Packet Transformation for FGS
2.1. Principle of Wavelet Packet Transform
W
l t
k t t
f
i
th d At a certain period of
time, such as t = 2, the signal characteristics in eight frequency bands can be represented
by wavelet packet coefficients, which reflect the projection value of the signal on each base
component. It is related to the wavelet base, signal sampling frequency, and the number
of decomposition layers. After the wavelet packet coefficients are obtained, the signal can
be reconstructed in each frequency band according to the proportion of each component
in the signal to obtain the waveform of the signal in each frequency band. Wavelet packet coefficient
Figure 4. Schematic diagram of the three-layer wavelet packet decomposition example. Figure 4. Schematic diagram of the three-layer wavelet packet decomposition example. Figure 4. Schematic diagram of the three-layer wavelet packet decomposition example. Figure 4. Schematic diagram of the three-layer wavelet packet decomposition example. Sensors 2021, 21, 154 6 of 16 2.2. Extraction of Zero-Sequence Current Transient Characteristic 2.2. Extraction of Zero-Sequence Current Transient Characteristic In the normal operating state, since the flexible compensation device is not put into
use, it is the same as the transient equivalent circuit of the single-phase ground fault
in the traditional resonant grounding system. The transient current id at the ground
point [37,40] is: id = iL + iC
= (ICm −ILm) cos(ωt + ϕ) + ILm cos ϕe−t
τL
+ ICm(
ω f
ω sin ϕ sin ω f t −cos ϕ sin ω f t)e−t
τC
(1) (1) where ICm and ILm are the amplitude of the transient capacitor current and inductor where ICm and ILm are the amplitude of the transient capacitor current and inductor
current; ω is the angular frequency of the power grid; ϕ is the initial phase angle of the
phase voltage at the time of fault; ωf is the resonance frequency of the loop; τL and τC are
the time constant of the inductive loop and the capacitive loop. This article applies the wavelet packet transform to the processing of transient wave-
forms, which mainly includes two steps: (1)
Determine the actual fault time—the system continuously collects the bus zero-
sequence voltage and feeder zero-sequence current during normal operation and
saves at least two cycles of data. In order to distinguish between ground fault and
voltage unbalance, the zero-sequence voltage threshold Uset is set to 15% of the maxi-
mum phase voltage, thus as to identify 0–2 kΩground fault. 2. Wavelet Packet Transformation for FGS
2.1. Principle of Wavelet Packet Transform
2. Wavelet Packet Transformation for FGS
2.1. Principle of Wavelet Packet Transform
W
l t
k t t
f
i
th d When the instantaneous
value of the zero-sequence voltage exceeds the threshold, it is judged that a fault has
occurred. However, sometimes it is affected by the grounding resistance or other
conditions, and the instantaneous value of the zero-sequence voltage does not imme-
diately cross the boundary. That is to say, the fault time t1 obtained according to the
above method often lags the actual fault time t0. In this article, the Coiflet5 wavelet
is used to transform the zero-sequence voltage using the wavelet packet singularity
principle to determine the maximum point. At this point, the signal mutation is the
most obvious, corresponding to the actual fault time t0. (2) p
g
0
Transient characteristic waveform extraction—under typical conditions, such as the
broken wire-to-ground fault, the ground medium is the cement floor, weeds and so on. The transition resistance is usually hundreds to thousands of ohms. Therefore, when
this kind of fault occurs, the fault current is mainly composed of the fundamental
current. In addition, due to the existence of nonlinear load, the fault current will also
contain some low-frequency harmonic current components. Thus, in the fault current,
1st, 3rd, and 5th signal amplitude are the largest, and their characteristics are most
obvious when the fault occurs. Using this as the reference waveform for line selection
can reduce the interference of high frequencies and other uncontrollable factors. Select
the sampling frequency as 10 kHz, consider the boundary effect, and take the feeder
zero-sequence current signals of 1/4 and 3/4 cycles before and after the fault time to
perform four-layer wavelet packet decomposition, and the schematic diagram of the
four-layer wavelet packet tree is shown in Figure 5. According to Shannon’s sampling
theorem, the effective frequency bandwidth represented by each node on the fourth
layer = 10,000/2/16 = 312.5 Hz. Therefore, the reconstructed signal at node (4, 0) can
accurately extract the signal of 0–312.5 Hz through only one period of sampling signal. The principle of this method is equivalent to low-pass filtering without time delay. p
g
Transient characteristic waveform extraction—under typical conditions, such as the
broken wire-to-ground fault, the ground medium is the cement floor, weeds and so on. The transition resistance is usually hundreds to thousands of ohms. Therefore, when
this kind of fault occurs, the fault current is mainly composed of the fundamental
current. 2. Wavelet Packet Transformation for FGS
2.1. Principle of Wavelet Packet Transform
2. Wavelet Packet Transformation for FGS
2.1. Principle of Wavelet Packet Transform
W
l t
k t t
f
i
th d , m, p ̸= q
(2) (2) =
=
≠
0 ,
1,2,
,
,
op
p
i
C
p
m p
q
dt
(2)
h
U i th
t
lt
C i th
it
t
d f th f
d
where U0 is the system zero-sequence voltage; Cp is the capacitance to ground of the
feeder p. where U0 is the system zero-sequence voltage; Cp is the capacitance to ground of the feeder
p. It can be found from (2) that iop is obtained by multiplying the 2 parts, and its value
depends on its own capacitance to the ground and the change rate of the zero-sequence
voltage. Different feeders have different capacitances to ground, which resulted in differ-
ent zero-sequence current amplitudes, but different feeder zero-sequence currents con-
tained the same zero-sequence voltage change rate. Therefore, the dynamic change trend
It can be found from (2) that iop is obtained by multiplying the 2 parts, and its value
depends on its own capacitance to the ground and the change rate of the zero-sequence
voltage. Different feeders have different capacitances to ground, which resulted in different
zero-sequence current amplitudes, but different feeder zero-sequence currents contained
the same zero-sequence voltage change rate. Therefore, the dynamic change trend was
consistent, which provides a theoretical basis for extracting feature quantities and judging
the similarity between non-faulty feeders. q
g
g
,
y
was consistent, which provides a theoretical basis for extracting feature q
y
y
The zero-sequence current of the faulty feeder q can be expressed as: en non-faulty feeders. ent of the faulty feeder q can be expressed as:
0
m
du
i
C
i
i
ioq = −
m
∑
p=1,p̸=q
Cp
du0
dt + iR + iL
(3) (3) =
≠
=
+
+
o
1,
q
p
R
L
p
p q
i
C
i
i
dt
where iR and iL are the resistive and inductive components of ioq. where iR and iL are the resistive and inductive components of ioq. It can be found from (3) that if iR and iL are not considered, ioq and the sum of the zero-
sequence current at the outlet of the non-faulty feeder is equal in magnitude, and the di-
rection of change is opposite. 2. Wavelet Packet Transformation for FGS
2.1. Principle of Wavelet Packet Transform
2. Wavelet Packet Transformation for FGS
2.1. Principle of Wavelet Packet Transform
W
l t
k t t
f
i
th d Although iR and iL will affect the amplitude and phase of ioq
to a certain extent it can be found that there is a significant difference in the amplitude
It can be found from (3) that if iR and iL are not considered, ioq and the sum of the
zero-sequence current at the outlet of the non-faulty feeder is equal in magnitude, and the
direction of change is opposite. Although iR and iL will affect the amplitude and phase of
ioq to a certain extent, it can be found that there is a significant difference in the amplitude
and dynamic change trend between the faulty feeder and the non-faulty feeder. 2. Wavelet Packet Transformation for FGS
2.1. Principle of Wavelet Packet Transform
2. Wavelet Packet Transformation for FGS
2.1. Principle of Wavelet Packet Transform
W
l t
k t t
f
i
th d In addition, due to the existence of nonlinear load, the fault current will also
contain some low-frequency harmonic current components. Thus, in the fault current,
1st, 3rd, and 5th signal amplitude are the largest, and their characteristics are most
obvious when the fault occurs. Using this as the reference waveform for line selection
can reduce the interference of high frequencies and other uncontrollable factors. Select
the sampling frequency as 10 kHz, consider the boundary effect, and take the feeder
zero-sequence current signals of 1/4 and 3/4 cycles before and after the fault time to
perform four-layer wavelet packet decomposition, and the schematic diagram of the
four-layer wavelet packet tree is shown in Figure 5. According to Shannon’s sampling
theorem, the effective frequency bandwidth represented by each node on the fourth
layer = 10,000/2/16 = 312.5 Hz. Therefore, the reconstructed signal at node (4, 0) can
accurately extract the signal of 0–312.5 Hz through only one period of sampling signal. The principle of this method is equivalent to low-pass filtering without time delay. 7 of 16
7 of 16 Sensors 2021, 21, 154
Sensors 2021, 21, x FOR (0,0)
(1,0)
(1,1)
(2,0)
(2,1)
(2,2)
(2,3)
(3,0)
(3,1)
(3,2)
(3,3)
(3,4)
(3,5)
(3,6)
(3,7)
(4,0) (4,1) (4,2) (4,3) (4,4) (4,5) (4,6) (4,7)
(4,9) (4,10)(4,11)(4,12)(4,13)(4,14)(4,15)
(4,8)
f=10kHZ
Figure 5 Schematic diagram of four layer wavelet packet tree
Figure 5 Schematic diagram of four layer wavelet packet tree Figure 5. Schematic diagram of four-layer wavelet packet tree. Figure 5. Schematic diagram of four-layer wavelet packet tree. 3. Feeder Identification Method Based on Grey T-type correlation Degree
3. Feeder Identification Method Based on Grey T-Type Correlation Degree eeder Identification Method Based on Grey T-type correlation Degree
Feeder Identification Method Based on Grey T-Type Correlation Degree
Characteristic Analysis of Transient Zero Sequence Current 3. Feeder Identification Method Based on Grey T type correlati
3.1. Characteristic Analysis of Transient Zero-Sequence Current
3.1. Characteristic Analysis of Transient Zero-Sequence Current 3.1. Characteristic Analysis of Transient Zero Sequence Current
When the ground fault occurs, the zero-sequence current of non-faulty feeder p can
b
d
When the ground fault occurs, the zero-sequence current of non-faulty feeder p can be
expressed as: ≠
0
1 2
du
i
C
(2)
iop = Cp
du0
dt , p = 1, 2, . . . g
and dynamic change trend between the faulty feeder and the non-faulty feede
3.2. Principle of Faulty Feeder Identification Using Grey T-Type Correlation Degree , n −1
(5) (5) The average value of the absolute value of the increment of the sequence in the
sampling interval is:
1 di = ∑n−1
t=1
∆Sit0
n −1
(6) (6) The average value of the increment of the sequence Si at the tth
0 sampling point is: The average value of the increment of the sequence Si at the tth
0 sampling point is: Zit0 =
∆Sit0
di
(7) (7) The grey T-type correlation coefficient ρij[Sit, Sjt] of any two signal sequences Si and
Sj at the tth
0 sampling point is defined as: The grey T-type correlation coefficient ρij[Sit, Sjt] of any two signal sequences Si and
Sj at the tth
0 sampling point is defined as: ρij
Sit0, Sjt0
=
sgn(Zit0, Zjt0)
1 + 0.5
Zit0
−
Zjt0
(8)
sgn(Zit0, Zjt0) =
1, Zit0Zjt0 ≥0
−1, Zit0Zjt0 < 0
(9) (8) (9) Then the grey T-type correlation coefficient ρij of Si and Sj in the sampling period can
be expressed as: Then the grey T-type correlation coefficient ρij of Si and Sj in the sampling period can
be expressed as: ρij =
1
n −1
n−1
∑
t0=1
ρij
Sit0, Sjt0
(10) (10) where ρij∈[−1,1], the more similar the dynamic trends of Si and Sj are, the closer ρij is to 1,
otherwise it is closer to −1. . Identification Criterion Based on Grey T-Type Correlation Degree According to the above method, the grey T-type correlation coefficient matrix ρ can
be obtained: ρ =
1
ρ12
. . . ρ1m
ρ21
1
. . . ρ2m
. . . . . . . . . . . . ρm1
ρm2
. . . 1
(11) (11) The integrated grey T-type correlation coefficient of feeder k and other feeders can be
defined as:
1
m The integrated grey T-type correlation coefficient of feeder k and other feeders can be
defined as:
1
m µk =
1
m −1
m
∑
j=1,j̸=i
ρkj
(12) (12) According to the above analysis, the line selection criteria can be determined as follows: µk = min[µ1, µ2, . . . , µm] ≤µset, the kth feeder fault
µk = min[µ1, µ2, . . . , µm] > µset, the bus fault
(13) µk = min[µ1, µ2, . . . , µm] ≤µset, the kth feeder fault
µk = min[µ1, µ2, . . g
and dynamic change trend between the faulty feeder and the non-faulty feede
3.2. Principle of Faulty Feeder Identification Using Grey T-Type Correlation Degree 3.2. Principle of Faulty Feeder Identification Using Grey T-type Correlation Degree
In order to express the similarity of the two curves, the traditional method of feeder
identification based on similarity compares the static shapes of the two curves [39]. How-
ever, because the actual distribution network contains overhead lines and cables, and the
capacitance to ground of the cables is much larger than the transmission lines, it leads to
a large difference in the amplitude of the zero-sequence current. If the static correlation
coefficient continues to be used, it will lead to misjudgment. In order to solve this problem,
this article will use the grey T-type correlation degree. Based on the changing trend of the
In order to express the similarity of the two curves, the traditional method of feeder
identification based on similarity compares the static shapes of the two curves [39]. How-
ever, because the actual distribution network contains overhead lines and cables, and the
capacitance to ground of the cables is much larger than the transmission lines, it leads to
a large difference in the amplitude of the zero-sequence current. If the static correlation
coefficient continues to be used, it will lead to misjudgment. In order to solve this problem,
this article will use the grey T-type correlation degree. Based on the changing trend of the
fault waveform, it can accurately indicate the similarity of the dynamic change trend of the
non-faulted overhead line and cable, and no misjudgment of the line will be made. Sensors 2021, 21, 154 8 of 16 Assuming that the transient signal sequence Sk of the kth feeder contains n data points
in the sampling period, written as a row vector in the form Sk = (Sk1, Sk2, . . . , Skn), then the
transient signal composed of m feeders the matrix S is as follows: S =
S1
S2
. . . Sm
=
S11
S12
. . . S1n
S21
S22
. . . S2n
. . . . . . . . . . . . Sm1
Sm2
. . . Smn
(4) (4) The increment of the sequence Si at the tth
0 sampling point can be expressed as The increment of the sequence Si at the tth
0 sampling point can be expressed as ∆Sit0 = Si(t0+1) −Sit0, t0 = 1, 2, . . . g
and dynamic change trend between the faulty feeder and the non-faulty feede
3.2. Principle of Faulty Feeder Identification Using Grey T-Type Correlation Degree . , µm] > µset, the bus fault
(13) (13) Sensors 2021, 21, 154 9 of 16
(13) where µset is equal to 0.2 here. set
q
Figure 6 is the flow chart Figure 6 is the flow chart of the proposed faulty feeder identification method com-
bining wavelet packet and grey T-type correlation degree. Especially, in order to avoid
misjudgment and distinguish bus faults (point of common connection of all feeders) from
feeder faults, that is, to determine the bus fault as a feeder fault or the opposite condition,
the threshold setting is necessary. A large number of simulation results show that the
line selection accuracy is high when µset is set as 0.2. The threshold setting with too high
or too low value will lead to confusion between feeder faults and bus faults. The steps
can be described as follows: Firstly, by sampling the zero-sequence voltage/current and
comparing them with thresholds, the ground fault is diagnosed; then, through wavelet
packet transformation, the transient characteristic waveform is extracted, and the matrix
S is obtained; after that the grey T-type correlation coefficient is calculated; finally, by
analyzing µset, the faulty feeder can be identified. g
p
p
y
bining wavelet packet and grey T-type correlation degree. Especially, in order to avoid
misjudgment and distinguish bus faults (point of common connection of all feeders) from
feeder faults, that is, to determine the bus fault as a feeder fault or the opposite condition,
the threshold setting is necessary. A large number of simulation results show that the line
selection accuracy is high when
set
μ
is set as 0.2. The threshold setting with too high or
too low value will lead to confusion between feeder faults and bus faults. The steps can
be described as follows: Firstly, by sampling the zero-sequence voltage/current and com-
paring them with thresholds, the ground fault is diagnosed; then, through wavelet packet
transformation, the transient characteristic waveform is extracted, and the matrix S is ob-
tained; after that the grey T-type correlation coefficient is calculated; finally, by analyzing
set
μ
, the faulty feeder can be identified. U0>Uset? g
and dynamic change trend between the faulty feeder and the non-faulty feede
3.2. Principle of Faulty Feeder Identification Using Grey T-Type Correlation Degree Sample U0，Iok (k=1,2…,m)
N
Singularity detection for U0,
and determine actual fault
time t1
Y
Take 1/4 and 3/4 cycles
around t1, and determine line
selection interval
WPT reconstruction for Iok,
and extract transient
characteristic waveform
WPT for Iok, and Obtain
transient characteristic
waveform Matrix S
Calculate grey
T-type correlation
matrix ρ
Calculate grey T-type
correlation coefficient μk
(k=1,2…,n)
min[μk]>μset? Identify faulty
feeder k
Bus fault
N
Y
Initialization
Finish
Flow chart
Grey T-type correlation degree
Wavelet packet transformation
①
②
Figure 6. The flow chart of the proposed method. Figure 6. The flow chart of the proposed method. Flow chart Figure 6. The flow chart of the proposed method. Figure 6. The flow chart of the proposed method. 4. Verification Results and Discussion
4. Verification Results and Discussion As mentioned above, both the flexible grounding system and traditional resonant
grounding system involve fault line selection. Due to its high controllability and excellent
compensation performance, the former is attracting more and more attention. However,
the flexible grounding system needs both fast and accurate line selection process. In order
to verify the proposed faulty feeder identification method, the MATLAB/Simulink-based
As mentioned above, both the flexible grounding system and traditional resonant
grounding system involve fault line selection. Due to its high controllability and excellent
compensation performance, the former is attracting more and more attention. However,
the flexible grounding system needs both fast and accurate line selection process. In
order to verify the proposed faulty feeder identification method, the MATLAB/Simulink-
based model is established, as shown in Figure 7. Especially, limited by the experimental
conditions, and from the perspective of safety, it is a better choice to verify the proposed
method by sufficient simulations. The electric distribution network model contains five
feeders including two main types of lines: L1 is 25 km overhead line, L2 is 10 km overhead
line, L3 is a hybrid line of 5 km overhead line and 10 km cable, L4 is 20 km cable, and L5
is 8 km Cable. The system voltage level is 110 kV, the rated voltage of the transformer is
110/10.5 kV, and the rated capacity is 50 MVA. The no-load current is 1%, the no-load loss
is 35 kW, the short-circuit loss is 250 kW, and the short-circuit voltage ratio is 10%. The Sensors 2021, 21, 154 10 of 16
rmer is
oad loss 10 of 16
rmer is
oad loss ground resistance is Rd. The active load of each feeder is 4 × 106 W, and the reactive load
is 3 × 106 Var. The line uses a distributed parameter model, and the parameter values are
shown in Table 1. According to the mentioned parameters, the total ground capacity of
the system ΣC = 1.26 × 10−5 F can be calculated and obtained. If the over-compensation
degree of the arc suppression coil is 5%, the inductivity value of the arc suppression coil
can be obtained:
1
1
is 3 × 106 Var. The line uses a distributed parameter model, and the parameter values are
shown in Table 1. According to the mentioned parameters, the total ground capacity of
the system ΣC = 1.26 × 10−5 F can be calculated and obtained. 4. Verification Results and Discussion
4. Verification Results and Discussion If the over-compensation
degree of the arc suppression coil is 5%, the inductivity value of the arc suppression coil
can be obtained:
1
1 L =
1
1.05 ∗
1
3ω2ΣC= 0.243H
(14)
2
1
1
=
=0.243H
1.05 3
L
C
ω
∗
Σ
(14) (14)
(14) The internal resistance RL of the arc suppression coil is 10% of the inductive reactance,
and RL = 8 Ω. The internal resistance RL of the arc suppression coil is 10% of the inductive reactance,
and RL = 8 Ω. Grid
110/10kV transformer
ASC
L1
L2
L3
L4
L5
25km
Overhead line
10km
Overhead line
5km
Overhead line
20km
Cable
8km
Cable
10km
Cable
Figure 7. Schematic diagram of the simulation model: Two types of lines are considered. Figure 7. Schematic diagram of the simulation model: Two types of lines are considered. 110/10kV transformer Grid Figure 7. Schematic diagram of the simulation model: Two types of lines are considered. Figure 7. Schematic diagram of the simulation model: Two types of lines are considered. p
Types
Phase Sequence
R0 (Ω/km)
L0 (mH/km)
C0 (nF/km)
Overhead Lines
Positive sequence
0.132
1.258
9.780
Zero sequence
0.389
4.126
7.758
Cable
Positive sequence
0.270
0.255
339
Zero sequence
2.700
1.019
280
4.1. Characteristics of Transient Zero-Sequence Current
Table 1. Line parameters. Types
Phase Sequence
R0 (Ω/km)
L0 (mH/km)
C0 (nF/km)
Overhead Lines
Positive sequence
0.132
1.258
9.780
Zero sequence
0.389
4.126
7.758
Cable
Positive sequence
0.270
0.255
339
Zero sequence
2.700
1.019
280 Figure 8 is the waveform diagram of the transie
gle phase ground fault It can be found that: (1) Wh
4.1. Characteristics of Transient Zero-Sequence Current gle-phase ground fault. It can be found that: (1) When the fault occurs on the feeder, no
matter what time the fault occurs, the variation trend and amplitude of zero sequence
current of the faulty feeder and normal feeder are very different; (2) the zero-sequence
current of the normal feeders have a large difference in amplitude and a small difference
in variation trend; (3) when the fault occurs on the bus, all the feeders can be regarded as
normal feeders, the zero-sequence currents of all the feeders have a similar variation
trend, and have a large difference in amplitude. Figure 8 is the waveform diagram of the transient zero-sequence current during single-
phase ground fault. It can be found that: (1) When the fault occurs on the feeder, no matter
what time the fault occurs, the variation trend and amplitude of zero sequence current of
the faulty feeder and normal feeder are very different; (2) the zero-sequence current of the
normal feeders have a large difference in amplitude and a small difference in variation
trend; (3) when the fault occurs on the bus, all the feeders can be regarded as normal
feeders, the zero-sequence currents of all the feeders have a similar variation trend, and
have a large difference in amplitude. 11 of 16
11 of 16 Sensors 2021, 21, 154
Sensors 2021, 21, x FO (a)
(b)
(c)
(d)
Faulty feeder
Faulty feeder
Faulty feeder
Figure 8. Transient zero-sequence current waveform: (a) Feeder fault, φ = π/2; (b) feeder fault, φ =
Figure 8. Transient zero-sequence current waveform: (a) Feeder fault, ϕ = π/2; (b) feeder fault, (a)
(b)
(c)
(d)
Faulty feeder
Faulty feeder
Faulty feeder
Figure 8. Transient zero-sequence current waveform: (a) Feeder fault, φ = π/2; (b) feeder fault, φ =
π/4; (c) feeder fault, φ = 0; (d) bus fault, φ = π/2. Figure 8. Transient zero-sequence current waveform: (a) Feeder fault, ϕ = π/2; (b) feeder fault,
ϕ = π/4; (c) feeder fault, ϕ = 0; (d) bus fault, ϕ = π/2. (a)
(b)
Faulty feeder
Faulty feeder (c)
(d)
Faulty feeder Figure 8. Transient zero-sequence current waveform: (a) Feeder fault, φ = π/2; (b) feeder fault, φ =
π/4; (c) feeder fault, φ = 0; (d) bus fault, φ = π/2. Figure 8. Figure 8 is the waveform diagram of the transie
gle phase ground fault It can be found that: (1) Wh
4.1. Characteristics of Transient Zero-Sequence Current Transient zero-sequence current waveform: (a) Feeder fault, ϕ = π/2; (b) feeder fault,
ϕ = π/4; (c) feeder fault, ϕ = 0; (d) bus fault, ϕ = π/2. 4.2. Faulty Feeder Identification under Typical Situations
4.2. Faulty Feeder Identification under Typical Situations y
f
yp
The fault waveform is affected by the voltage phase angle φ, the grounding resistance
Rd, the fault location, and the type of fault line when the fault occurs. To verify the validity
and rationality of the above line selection method under different influencing factors,
three typical situations are considered in this article
The fault waveform is affected by the voltage phase angle ϕ, the grounding resistance
Rd, the fault location, and the type of fault line when the fault occurs. To verify the validity
and rationality of the above line selection method under different influencing factors, three
typical situations are considered in this article. three typical situations are considered in this article. Figures 9 and 10 are the transient waveform of the zero-sequence current and the
reconstructed waveform (Rd = 0 Ω) at (4, 0) after extraction when the feeder fault occurs,
respectively. The feeder faults and bus fault can be found in Figure 9a–d, respectively. In
Figure 10, the x-axis represents the data points of the transient signal within the sampling
period. It can be found that, compared with the original transient waveform, the extracted
waveforms are filtered out of high-frequency interference. It smooths the waveform
curve, strengthens the similarity between non-faulty feeders and the difference between
faulty feeders and non-faulty feeders, and enhances the calculation accuracy of the grey
T type o elatio
ele tio
oeffi ie t
yp
Figures 9 and 10 are the transient waveform of the zero-sequence current and the
reconstructed waveform (Rd = 0 Ω) at (4, 0) after extraction when the feeder fault occurs,
respectively. The feeder faults and bus fault can be found in Figure 9a–d, respectively. In
Figure 10, the x-axis represents the data points of the transient signal within the sampling
period. It can be found that, compared with the original transient waveform, the extracted
waveforms are filtered out of high-frequency interference. It smooths the waveform curve,
strengthens the similarity between non-faulty feeders and the difference between faulty
feeders and non-faulty feeders, and enhances the calculation accuracy of the grey T-type
correlation selection coefficient. 4.2. Faulty Feeder Identification under Typical Situations
4.2. Faulty Feeder Identification under Typical Situations 12 of 16
12 of 16
12 of 16 12 of 16
12 of 16
12 of 16 Sensors 2021, 21, 154
Sensors 2021, 21, x FO
Se
o
2021 21
FOR 0
0.002
0.004
0.006
0.008
0.01
0.012
0.014
0.016
0.018
0.02
-50
0
50
100
-50
0
50
100
-20
0
20
40
60
-20
-10
0
10
20
Amplitude (A)
Amplitude (A)
Amplitude (A)
Amplitude (A)
Time (s)
Time (s)
Time (s)
Time (s)
(a)
(b)
(c)
(d)
0
0.002
0.004
0.006
0.008
0.01
0.012
0.014
0.016
0.018
0.02
0
0.002
0.004
0.006
0.008
0.01
0.012
0.014
0.016
0.018
0.02
0
0.002
0.004
0.006
0.008
0.01
0.012
0.014
0.016
0.018
0.02
Figure 9. Transient waveforms when Rd = 0Ω: (a)(b)(c) Feeder faults; (d) bus fault. 0
20
40
60
80
100
120
140
160
180
200
-0.5
0
0.5
1
0
20
40
60
80
100
120
140
160
180
200
-20
0
20
40
0
20
40
60
80
100
120
140
160
180
200
-20
0
20
0
20
40
60
80
100
120
140
160
180
200
-20
0
20
0
20
40
60
80
100
120
140
160
180
200
-5
0
5
10
Amplitude (A)
L1
L2
L3
L4
L5
Date point
Faulty feeder
Figure 10. Transient characteristic waveforms extracted at (4, 0) node when Rd = 0 Ω, φ = π/2. The steps of judgment can be described as follows: Using the waveform afte
packet processing as the original waveform of the line selection; changing the fa
Figure 9. Transient waveforms when Rd = 0 Ω: (a–c) Feeder faults; (d) bus fault. 0
0.002
0.004
0.006
0.008
0.01
0.012
0.014
0.016
0.018
0.02
-50
0
50
100
-50
0
50
100
-20
0
20
40
60
-20
-10
0
10
20
Amplitude (A)
Amplitude (A)
Amplitude (A)
Amplitude (A)
Time (s)
Time (s)
Time (s)
Time (s)
(a)
(b)
(c)
(d)
0
0.002
0.004
0.006
0.008
0.01
0.012
0.014
0.016
0.018
0.02
0
0.002
0.004
0.006
0.008
0.01
0.012
0.014
0.016
0.018
0.02
0
0.002
0.004
0.006
0.008
0.01
0.012
0.014
0.016
0.018
0.02
Figure 9. Transient waveforms when Rd = 0Ω: (a)(b)(c) Feeder faults; (d) bus fault. 4.2. Faulty Feeder Identification under Typical Situations
4.2. Faulty Feeder Identification under Typical Situations The steps of judgment can be described as follows: Using the waveform afte
k
h
l
f
f h l
l
h
h 0
0.002
0.004
0.006
0.008
0.01
0.012
0.014
0.016
0.018
0.02
-50
0
50
100
-50
0
50
100
-20
0
20
40
60
-20
-10
0
10
20
Amplitude (A)
Amplitude (A)
Amplitude (A)
Amplitude (A)
Time (s)
Time (s)
Time (s)
Time (s)
(a)
(b)
(c)
(d)
0
0.002
0.004
0.006
0.008
0.01
0.012
0.014
0.016
0.018
0.02
0
0.002
0.004
0.006
0.008
0.01
0.012
0.014
0.016
0.018
0.02
0
0.002
0.004
0.006
0.008
0.01
0.012
0.014
0.016
0.018
0.02
i
f
h
b
d
f
l
d b
f
0
0.002
0.004
0.006
0.008
0.01
0.012
0.014
0.016
0.018
0.02
-50
0
50
100
-50
0
50
100
-20
0
20
40
60
-20
-10
0
10
20
Amplitude (A)
Amplitude (A)
Amplitude (A)
Amplitude (A)
Time (s)
Time (s)
Time (s)
Time (s)
(a)
(b)
(c)
(d)
0
0.002
0.004
0.006
0.008
0.01
0.012
0.014
0.016
0.018
0.02
0
0.002
0.004
0.006
0.008
0.01
0.012
0.014
0.016
0.018
0.02
0
0.002
0.004
0.006
0.008
0.01
0.012
0.014
0.016
0.018
0.02 0
0.002
0.004
0.006
0.008
0.01
0.012
0.014
0.016
0.018
0.02
-50
0
50
100
-50
0
50
100
-20
0
20
40
60
-20
-10
0
10
20
Amplitude (A)
Amplitude (A)
Amplitude (A)
Amplitude (A)
Time (s)
Time (s)
Time (s)
Time (s)
(a)
(b)
(c)
(d)
0
0.002
0.004
0.006
0.008
0.01
0.012
0.014
0.016
0.018
0.02
0
0.002
0.004
0.006
0.008
0.01
0.012
0.014
0.016
0.018
0.02
0
0.002
0.004
0.006
0.008
0.01
0.012
0.014
0.016
0.018
0.02
0
0.002
0.004
0.006
0.008
0.01
0.012
0.014
0.016
0.018
0.02
-50
0
50
100
-50
0
50
100
-20
0
20
40
60
-20
-10
0
10
20
Amplitude (A)
Amplitude (A)
Amplitude (A)
Amplitude (A)
Time (s)
Time (s)
Time (s)
Time (s)
(a)
(b)
(c)
(d)
0
0.002
0.004
0.006
0.008
0.01
0.012
0.014
0.016
0.018
0.02
0
0.002
0.004
0.006
0.008
0.01
0.012
0.014
0.016
0.018
0.02
0
0.002
0.004
0.006
0.008
0.01
0.012
0.014
0.016
0.018
0.02 Figure 9. Transient waveforms when Rd = 0Ω: (a)(b)(c) Feeder faults; (d) bus faul
Figure 9. Transient waveforms when Rd = 0 Ω: (a–c) Feeder faults; (d) bus fault. ( )
Figure 9. 4.2. Faulty Feeder Identification under Typical Situations
4.2. Faulty Feeder Identification under Typical Situations In this case, the transient
process of the fault waveform is the largest, the characteristic quantity is the most obvious,
and it is most conducive to the judgment of line selection; Table 3 shows the results of line
The steps of judgment can be described as follows: Using the waveform after wavelet
packet processing as the original waveform of the line selection; changing the fault occur-
rence line and location, respectively; calculating [μ1, μ2, μ3, μ4, μ5] according to the grey T-
type correlation degree; and determining the faulty feeder according to (13), finally. Table
2 shows the results of line selection when Rd = 0 Ω and φ = π/2. In this case, the transient
process of the fault waveform is the largest, the characteristic quantity is the most obvious,
and it is most conducive to the judgment of line selection; Table 3 shows the results of line
The steps of judgment can be described as follows: Using the waveform after wavelet
packet processing as the original waveform of the line selection; changing the fault oc-
currence line and location, respectively; calculating [µ1, µ2, µ3, µ4, µ5] according to the
grey T-type correlation degree; and determining the faulty feeder according to (13), finally. Table 2 shows the results of line selection when Rd = 0 Ωand ϕ = π/2. In this case, the
transient process of the fault waveform is the largest, the characteristic quantity is the The steps of judgment can be described as follows: Using the waveform after wavelet
packet processing as the original waveform of the line selection; changing the fault occur-
rence line and location, respectively; calculating [μ1, μ2, μ3, μ4, μ5] according to the grey T-
type correlation degree; and determining the faulty feeder according to (13), finally. Table
2 shows the results of line selection when Rd = 0 Ω and φ = π/2. 4.2. Faulty Feeder Identification under Typical Situations
4.2. Faulty Feeder Identification under Typical Situations The steps of judgment can be described as follows: Using the waveform after wavelet
packet processing as the original waveform of the line selection; changing the fault occur-
rence line and location, respectively; calculating [μ1, μ2, μ3, μ4, μ5] according to the grey T-
type correlation degree; and determining the faulty feeder according to (13), finally. Table
2 shows the results of line selection when Rd = 0 Ω and φ = π/2. In this case, the transient
process of the fault waveform is the largest, the characteristic quantity is the most obvious,
and it is most conducive to the judgment of line selection; Table 3 shows the results of line
The steps of judgment can be described as follows: Using the waveform after wavelet
packet processing as the original waveform of the line selection; changing the fault occur-
rence line and location, respectively; calculating [μ1, μ2, μ3, μ4, μ5] according to the grey T-
type correlation degree; and determining the faulty feeder according to (13), finally. Table
2 shows the results of line selection when Rd = 0 Ω and φ = π/2. In this case, the transient
process of the fault waveform is the largest, the characteristic quantity is the most obvious,
and it is most conduci e to the judgment of line selection Table 3 shows the results of line
The steps of judgment can be described as follows: Using the waveform after wavelet
packet processing as the original waveform of the line selection; changing the fault oc-
currence line and location, respectively; calculating [µ1, µ2, µ3, µ4, µ5] according to the
grey T-type correlation degree; and determining the faulty feeder according to (13), finally. Table 2 shows the results of line selection when Rd = 0 Ωand ϕ = π/2. In this case, the
transient process of the fault waveform is the largest, the characteristic quantity is the The steps of judgment can be described as follows: Using the waveform after wavelet
packet processing as the original waveform of the line selection; changing the fault occur-
rence line and location, respectively; calculating [μ1, μ2, μ3, μ4, μ5] according to the grey T-
type correlation degree; and determining the faulty feeder according to (13), finally. Table
2 shows the results of line selection when Rd = 0 Ω and φ = π/2. 4.2. Faulty Feeder Identification under Typical Situations
4.2. Faulty Feeder Identification under Typical Situations Transient waveforms when Rd = 0Ω: (a)(b)(c) Feeder faults; (d) bus fault Figure 9. Transient waveforms when Rd = 0Ω: (a)(b)(c) Feeder faults; (d) bus faul
Figure 9. Transient waveforms when Rd = 0 Ω: (a–c) Feeder faults; (d) bus fault. ( )
Figure 9. Transient waveforms when Rd = 0Ω: (a)(b)(c) Feeder faults; (d) bus fault Figure 9. Transient waveforms when Rd = 0Ω: (a)(b)(c) Feeder faults; (d) bus faul
Figure 9. Transient waveforms when Rd = 0 Ω: (a–c) Feeder faults; (d) bus fault. ( )
Figure 9. Transient waveforms when Rd = 0Ω: (a)(b)(c) Feeder faults; (d) bus fault 0
20
40
60
80
100
120
140
160
180
200
-0.5
0
0.5
1
0
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180
200
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40
0
20
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20
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-20
0
20
0
20
40
60
80
100
120
140
160
180
200
-5
0
5
10
Amplitude (A)
L1
L2
L3
L4
L5
Date point
Faulty feeder
Figure 10. Transient characteristic waveforms extracted at (4, 0) node when Rd = 0 Ω, φ = π/2. 0
20
40
60
80
100
120
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160
180
200
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0
0.5
1
0
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0
20
0
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120
140
160
180
200
-5
0
5
10
Amplitude (A)
L1
L2
L3
L4
L5
Date point
Faulty feeder
Figure 10. Transient characteristic waveforms extracted at (4, 0) node when Rd = 0 Ω, φ = π/2. Figure 10. Transient characteristic waveforms extracted at (4, 0) node when Rd = 0 Ω, ϕ = π/2. Amplitude (A)
Amplitude (A) Figure 10. Transient characteristic waveforms extracted at (4, 0) node when Rd = 0 Ω, φ = π/2. Figure 10. Transient characteristic waveforms extracted at (4, 0) node when Rd = 0 Ω, φ = π/2. Figure 10. Transient characteristic waveforms extracted at (4, 0) node when Rd = 0 Ω, ϕ = π/2. 4.2. Faulty Feeder Identification under Typical Situations
4.2. Faulty Feeder Identification under Typical Situations 0
20
40
60
80
100
120
140
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180
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0
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1
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0
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0
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140
160
180
200
-5
0
5
10
Amplitude (A)
L1
L2
L3
L4
L5
Date point
Faulty feeder
Figure 10. Transient characteristic waveforms extracted at (4, 0) node when Rd = 0 Ω, φ = π/2. The steps of judgment can be described as follows: Using the waveform afte
Figure 10. Transient characteristic waveforms extracted at (4, 0) node when Rd = 0 Ω, ϕ = π/2. 4.2. Faulty Feeder Identification under Typical Situations
4.2. Faulty Feeder Identification under Typical Situations Fault Line
Fault Location/km
[µ1, µ2, µ3, µ4, µ5]
Min[µ1, µ2, µ3, µ4, µ5]
Line Selection Result
L1
10
[ −0.5644 , 0.5122, 0.4800, 0.4486, 0.5095]
−0.5644
L1
25
[ −0.5193 , 0.4256, 0.4584, 0.4149, 0.4736]
−0.5193
L1
L2
5
[0.4241, −0.6755 , 0.4115, 0.4367, 0.4846]
−0.6755
L2
10
[0.5238, −0.5423 , 0.5134, 0.4282, 0.5119]
−0.5423
L2
L3
5
[0.5963, 0.5845, −0.4122 , 0.4638, 0.5735]
−0.4122
L3
15
[0.6203, 0.6316, −0.2158 , 0.5369, 0.6135]
−0.2158
L3
L4
10
[0.4839, 0.3656, 0.5552, 0.1218 , 0.5569]
0.1218
L4
20
[0.5763, 0.5554, 0.6210, −0.0796 , 0.6216]
−0.0796
L4
L5
4
[0.5087, 0.4212, 0.5307, 0.4979, −0.0084 ]
−0.0084
L5
8
[0.4731, 0.5204, 0.5399, 0.5347, −0.0585 ]
−0.0585
L5
Bus
0
[0.6351, 0.5808, 0.7460, 0.7594, 0.7642]
0.5808
Bus Table 2. The results of feeder identification when Rd = 0 Ωand ϕ = π/2. Table 3. The results of feeder identification when Rd = 20 Ωand ϕ = π/4. Fault Line
Fault Location/km
[µ1, µ2, µ3, µ4, µ5]
Min[µ1, µ2, µ3, µ4, µ5]
Line Selection Result
L1
10
[ −0.2235 , 0.6375, 0.6312, 0.6296, 0.6438]
−0.2235
L1
25
[ −0.0702 , 0.6839, 0.6558, 0.6459, 0.6381]
−0.0702
L1
L2
5
[0.6328, −0.0473 , 0.6381, 0.6258, 0.6479]
−0.0473
L2
10
[0.6996, −0.0059 , 0.6036, 0.6184, 0.6328]
−0.0059
L2
L3
5
[0.6287, 0.6661, −0.0399 , 0.5736, 0.6509]
−0.0399
L3
15
[0.6689, 0.6586, −0.0403 , 0.6254, 0.6564]
−0.0403
L3
L4
10
[0.6167, 0.5677, 0.6366, 0.0679 , 0.6477]
0.0679
L4
20
[0.5834, 0.5537, 0.6446, 0.0116 , 0.6469]
0.0116
L4
L5
4
[0.5618, 0.5766, 0.5865, 0.6022, 0.0343 ]
0.0343
L5
8
[0.5031, 0.5198, 0.5856, 0.5614, −0.0017 ]
−0.0017
L5
Bus
0
[0.7468, 0.7651, 0.8005, 0.8204, 0.8322]
0.7468
Bus Table 3. The results of feeder identification when Rd = 20 Ωand ϕ = π/4. Table 4. The results of feeder identification when Rd = 2000 Ωand ϕ = 0. 4.2. Faulty Feeder Identification under Typical Situations
4.2. Faulty Feeder Identification under Typical Situations In this case, the transient
process of the fault waveform is the largest, the characteristic quantity is the most obvious,
and it is most conducive to the judgment of line selection; Table 3 shows the results of line
The steps of judgment can be described as follows: Using the waveform after wavelet
packet processing as the original waveform of the line selection; changing the fault occur-
rence line and location, respectively; calculating [μ1, μ2, μ3, μ4, μ5] according to the grey T-
type correlation degree; and determining the faulty feeder according to (13), finally. Table
2 shows the results of line selection when Rd = 0 Ω and φ = π/2. In this case, the transient
process of the fault waveform is the largest, the characteristic quantity is the most obvious,
d it i
t
d
i
t th j d
t f li
l
ti
T bl 3 h
th
lt
f li
The steps of judgment can be described as follows: Using the waveform after wavelet
packet processing as the original waveform of the line selection; changing the fault oc-
currence line and location, respectively; calculating [µ1, µ2, µ3, µ4, µ5] according to the
grey T-type correlation degree; and determining the faulty feeder according to (13), finally. Table 2 shows the results of line selection when Rd = 0 Ωand ϕ = π/2. In this case, the
transient process of the fault waveform is the largest, the characteristic quantity is the Sensors 2021, 21, 154 13 of 16 most obvious, and it is most conducive to the judgment of line selection; Table 3 shows
the results of line selection when Rd = 20 Ωand ϕ = π/4. In this case, the characteristic
quantity is more obvious, which can represent the general resistance of low-resistance
grounding of ϕ∈(0, π/2); Table 4 shows the results of line selection when Rd = 2000 Ωand
ϕ = 0. In this case, the high-resistance ground fault occurs. The fault transient process is
the smallest, the feature quantity is the least obvious, the line selection is the most difficult,
and the line selection accuracy is greatly disturbed. The accuracy of line selection under
high-resistance ground fault is an important reference to evaluate the effectiveness of the
line selection method. Table 2. The results of feeder identification when Rd = 0 Ωand ϕ = π/2. 5. Conclusions In this article, based on the zero-sequence voltage/current sensor networks, a new
faulty feeder identification method for the flexible grounding system combining grey T-type
correlation degree and wavelet packet transform is proposed, which features both accuracy
and rapidity. Different from the traditional method, this proposed method first extracts
the transient characteristic waveform of the zero-sequence current of each feeder through
the wavelet packet. In essence, it uses the timeliness of the wavelet packet to perform fast
low-pass filtering on the signal to remove high-frequency interference and amplify the
signal characteristics, which will promote accurate line selection. Then, through defining a
comprehensive grey T-type correlation coefficient to represent the similarity between the
dynamic change trends of the feeder, it can effectively reflect the correlation between the
dynamic change trend of the overhead line and the cable, thereby reducing misjudgment
due to the difference capacitance-to-ground value between the cable and overhead line. The proposed method can be applied in a flexible grounding system, which can meet
the requirements of fast and accurate faulty feeder selection. Sufficient MATLAB-based
simulation results verify the effectiveness and rationality of the proposed method and
analysis. y
Additionally, from the flow chart of the proposed method, it can be found that date
sampling and calculation are the keys to the fault feeder identification. In the simulation
model, the influence from the sampling time, missing data, and computation time can be
ignored. However, when the proposed method is applied in practice, the above factors
will affect the performance of faulty feeder identification to a certain extent. For example,
the digital signal processor will determine the upper limit of the sampling frequency and
the computation time. Besides that, due to the irrationality of the software program, the
sampling data or processing data of intermediate links may be lost, which will influence the
fault identification performance. In addition, when the saturation of the current transformer
occurs, the current sampling error will become very large, which will affect the algorithm
processing. These engineering problems need to be focused on in the future experimental
stage. Author Contributions: Conceptualization, K.L., S.Z., B.L. (Baorun Li), and J.Z.; funding acquisition,
K.L.; methodology, B.L. (Baorun Li), and B.L. (Biyang Liu); software, H.J.; validation, K.L., S.Z., and
H.J.; writing—original draft, K.L., B.L. (Baorun Li), and C.Z.; writing—review and editing, K.L. and
C.Z. All authors have read and agreed to the published version of the manuscript. 4.2. Faulty Feeder Identification under Typical Situations
4.2. Faulty Feeder Identification under Typical Situations Fault Line
Fault Location/km
[µ1, µ2, µ3, µ4, µ5]
Min[µ1, µ2, µ3, µ4, µ5]
Line Selection Result
L1
10
[ −0.6703 , 0.4789, 0.4503, 0.4188, 0.4996]
−0.6703
L1
25
[ −0.7266 , 0.4576, 0.4569, 0.4250, 0.4563]
−0.7266
L1
L2
5
[0.4239, −0.6702 , 0.4241, 0.4264, 0.4461]
−0.6702
L2
10
[0.2697, −0.6931 , 0.3334, 0.3589, 0.3202]
−0.6931
L2
L3
5
[0.4817, 0.4935, −0.6572 , 0.4517, 0.5066]
−0.6572
L3
15
[0.5092, 0.4963, −0.6208 , 0.4759, 0.5183]
−0.6208
L3
L4
10
[0.5100, 0.4516, 0.5161, −0.6454 , 0.5204]
−0.6454
L4
20
[0.5347, 0.5119, 0.5245, −0.6338 , 0.5429]
−0.6338
L4
L5
4
[0.4646, 0.4326, 0.4692, 0.4588, −0.6833 ]
−0.6833
L5
8
[0.4890, 0.4209, 0.4931, 0.4783, −0.6873 ]
−0.6873
L5
Bus
0
[0.7385, 0.8162, 0.7997, 0.7989, 0.7197]
0.7197
Bus Table 4. The results of feeder identification when Rd = 2000 Ωand ϕ = 0. Sensors 2021, 21, 154 14 of 16 Analyzing and comparing the above three typical situations, the following conclusions
can be obtained: (1) No matter what type of fault, the correct rate of feeder identification is
high, especially when a high resistance ground fault occurs; (2) In the non-faulty feeder,
the correlation coefficient of the overhead line is not much different from that of the cable. It proves that the grey T-type correlation degree for feeder identification can effectively
reflect the correlation of the dynamic change trend of overhead lines and cables, thereby
reducing the misjudgment caused by the difference in the capacitance to ground between
the cables and overhead lines; (3) the correlation coefficients between the faulty feeder and
the non-faulty feeder are obviously different, and their change trend of the zero-sequence
current is in the opposite direction; (4) bus fault can be regarded as a special case where
all feeders are non-faulty feeders. In this case, the comprehensive grey T-type correlation
coefficient of the zero-sequence current of each feeder is not much different. References A Novel Traveling-Wave-Based Method Improved by Unsupervis
L
ti
f P
C bl
i Sh
th C
t M
it
i
S
2019 19 2083 [C
R f] [P bM d] 16. Li, M.; Liu, J.; Zhu, T.; Zhou, W.; Zhou, C. A Novel Traveling-Wave-Based Method Improved by Unsupe
Location of Power Cables via Sheath Current Monitoring. Sensors 2019, 19, 2083. [CrossRef] [PubMed] ocation of Power Cables via Sheath Current Monitoring. Sensors 2019, 19, 2083. [CrossRef] [PubMed] 17. Wang, X.; Fu, Z.; Wang, Y.; Liu, R.; Chen, L. A Non-Destructive Testing Method for Fault Detection of Substation Grounding
Grids. Sensors 2019, 19, 2046. [CrossRef] [PubMed] 18. Lowczowski, K.; Lorenc, J.; Zawodniak, J.; Dombek, G. Detection and Location of Earth Fault in MV Feeders Using Screen
Earthing Current Measurements. Energies 2020, 13, 1293. [CrossRef] Lowczowski, K.; Lorenc, J.; Zawodniak, J.; Dombek, G. Detection and Location of Earth Fault in MV F
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The strategic use of the scope of justice in legitimizing intergroup conflict
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The strategic use of the scope of justice in legitimizing intergroup conflict Aline Lima-Nunes1
Kamila Gadelha Farias2 1Universidade Federal de Campina Grande, Campina Grande, PB
2Instituto Superior Múltiplo (IESM), Timon, MA Psico-USF, Bragança Paulista, v. 24, n. 2, p. 311-321, abr./jun. 2019
311 Psico-USF, Bragança Paulista, v. 24, n. 2, p. 311-321, abr./jun. 2019
311 Resumen
El El presente estudio buscó analizar de qué forma la percepción de “alcance de justicia”, a partir de la influencia de la posición
grupal, puede ser utilizada para legitimar la exclusión social. Participaron 103 estudiantes universitarios con edades entre 18
y 30 años (M=20.44, DP=1.98). El estudio fue compuesto por dos condiciones experimentales: los participantes justificaron
la situación planteada por una condición experimental (restricción de “alcance de justicia”) y por el control (observadores)
por medio de cuestionarios y fue realizado análisis de contenido de las justificaciones encontradas. Los resultados mostraron
que, en la condición de restricción sucedió la legitimación de posición hegemónica del grupo dominante a través de discursos
meritocráticos. En el grupo control, los participantes percibieron desigualdad social y sugirieron ampliar el alcance de justicia,
o sea la no restricción de derechos para determinados grupos. De esta forma, se identificó el uso estratégico de argumentos de
justicia como ampliación / restricción de “alcance de justicia” para justificar desigualdades sociales de acuerdo con determinada
posición grupal.l Palabras clave: alcance de justicia; legitimidad; conflicto inter-grupal O Uso Estratégico do Escopo de Justiça na Legitimação do Conflito Intergrupal O Uso Estratégico do Escopo de Justiça na Legitimação do Conflito Intergrupal O Uso Estratégico do Escopo de Justiça na Legitimação do Conflito Intergrupal O Uso Estratégico do Escopo de Justiça na Legitimação do Conflito Intergrupal Disponível em www.scielo.br Resumo O presente estudo buscou analisar como a percepção do escopo de justiça, a partir da influência da posição grupal, pode ser
utilizada para legitimar a exclusão social. Participaram 103 estudantes universitários com idade entre 18 e 30 anos (M = 20,44,
DP = 1,98). O estudo foi composto por duas condições experimentais: os participantes justificavam a situação colocada por
uma condição experimental (restrição do escopo de justiça) ou controle (observadores) por meio de questionários. Realizou-se
a análise de conteúdo das justificativas encontradas. Os resultados mostraram que, na condição de restrição, aconteceu a legi-
timação da posição hegemônica do grupo dominante através de discursos meritocráticos. No grupo controle, os participantes
percebiam a desigualdade social e sugeriam ampliar o escopo de justiça, ou seja, não-restrição de direitos para determinados
grupos. Dessa forma, identifica-se o uso estratégico de argumentos de justiça como a ampliação/restrição do escopo de justiça
para justificar as desigualdades sociais de acordo com determinada posição grupal. Palavras-chave: escopo de justiça, legitimação, conflito intergrupal Abstract This study sought to analyze how the scope of justice perception, based on the influence of group position, can be used to
legitimize social exclusion. One hundred and three college students aged between 18 and 30 years (M = 20.44; SD = 1.98)
participated. The study was composed of two experimental conditions: the participants justified the situation posed by an exper-
imental condition (restriction of the scope of justice) or control (observers) through questionnaires. The content analysis was
made from found justifications. The results introduced that, in restriction condition, the legitimization of the hegemonic posi-
tion from dominant group occurred through meritocratic discourses. In control group, participants perceived social inequality
and suggested widening the scope of justice, that is, non-restriction of rights for certain groups. Therefore, the strategic use of
arguments of justice is identified as the widening/restriction of the scope of justice to justify social inequalities according to a
certain group position.l g
p p
Keywords: scope of justice; legitimation; intergroup conflict Prejudice, Discrimination and Justice Prejudice is strictly related to discriminatory
practices and is conceptualized as forms of intergroup
relations contextualized by a power relationship, where
majority groups develop and express negative atti-
tudes and discriminatory behaviors because members
of social minorities participate in those groups (Lima-
Nunes, Lins, Camino, & Torres, 2010). Stereotypes
also have a close relationship with categorizations,
as one of the possible consequences of prejudice,
functioning as justificatory factors of discrimination
(Pereira & Souza, 2016). At the root of the social exclusion processes exist
the intergroup conflict concerning social contexts (eg.,
Sherif, 1967). Therefore, the intergroup difference itself
raises ways of dealing with interest conflicts, sometimes
excluding certain rights groups that theoretically should
be extended to all (Torres & Camino, 2013). In fact, discrimination and prejudice have an
interdependent relationship, because prejudice is
related to discriminatory practices (the manifesting
prejudice action). This relationship is mediated by
non-prejudiced basic justifications in order to enforce
discriminatory action, thus prejudice becomes more
veiled (Pereira, Vala & Leyens, 2009). Such mediation
takes the restricted vision form of the scope of justice
(Lima-Nunes, Pereira, & Correia, 2013a). According to Pereira, Torres and Almeida (2003),
power relations generate ideological representations,
which justify discrimination processes against minor-
ity groups. Thus, prejudice can be considered as the
result of power relations between majority and minor-
ity groups, either through the hierarchy belief or the
perception that groups should or should not have their
rights guaranteed. Recent research show how social
inequalities based on prejudiced attitudes can be justi-
fied by a legitimation complex process (Costa-Lopes,
Dovidio, Pereira, & Jost, 2013). The “scope of justice” (Deustch, 2006; Opotow,
1990; Staub, 1989) is defined in this paper as the per-
ception that justice principles should be applied only
to the ingroup (eg. Hafer & Olson 2003; Olson et al. 2011). There is a dichotomy between outgroup and
ingroup, and therefore, the justice principles application
to the outgroup must be different from that applied to
the ingroup, based on morality incongruities, belief sys-
tem and the justice principles that mark these groups
otherness. Thus, the scope of justice restriction only to
the ingroup excludes the justice principles application
to outgroup as equality, benefits, resources division, and
delimits access to rights to those who are not part of
the ingroup (Opotow 1990, 1995). Introduction the years, despite struggle movements and resistance
against these discriminatory processes. Discrimination against certain minorities in Bra-
zil is still a constant. According to Rodrigues (1998
apud Mello, 2005), a study on “cordial racism” in 1995
shows that although 89% of Brazilians agree that there
is prejudice against blacks in Brazil, only 10% admit
some prejudice degree in relation to this group. On Social justice is a present objective on the basis of
democratic societies and must be ensured as a funda-
mental right of citizens. Conversely, for certain minority
groups (eg., blacks, gypsies, indigenous people), there
is a restriction on their access to social justice. Thus,
social/racial/gender exclusion in Brazil continues over http://dx.doi.org/10.1590/1413-82712019240208 Lima-Nunes, A. & Farias, K. G. Scope of justice
312 the other hand, 87% of the participants agree, admit
or pronounce statements or behaviors with prejudiced
content. These data present the prejudice dimension in
Brazil, in which the vast majority can use veiled forms
of prejudice, such as expressions and jokes, and still
maintain a fair citizen self image. 2009). Justifications and rationalizations that subsidize
moral exclusion are difficult to detect (Opotow, 1995). These veiled perceptions are elements that can legiti-
mize support for discriminatory policies against social
minorities. Specifically, the legitimizing role of justice percep-
tions as the scope of justice restriction (Opotow, 1990;
Lima-Nunes, Pereira, & Correia, 2013a) can be applied
to several minority groups discrimination, as a justice
argument strategically legitimize the status quo mainte-
nance. Nonetheless, expanding the scope of justice can
help integrate these groups as citizens with all rights
ensured, so that justice arguments can be used as pro-
tective behaviors (Bilewicz, Imhoff & Drogosz, 2011). In this way, it is questioned why the issues of racial
discrimination are invisibilized and disregarded, even
seeming to be non-existent? Possibly the answers to
these questions correspond to aspects related to three
concepts raised in this work: the intergroup relationship,
the “scope of justice” and the Justified Discrimination
Model (JDM) (Pereira, 2011; Pereira & Vala, 2010). The differentiation between certain groups is
formed from a belonging sense, as well as through the
belief that there are crucial differences between groups,
that is, the own group (ingroup) is perceived as more
human and less animalistic in relation to another group
(outgroup). Introduction Through this ethnocentric perspective,
groups are characterized by an intrinsic essence and
not through circumstances and events, in which certain
conceptions are naturalized (Pereira, Vala & Leyens,
2009; Tajfel, 1970). Psico-USF, Bragança Paulista, v. 24, n. 2, p. 311-321, abr./jun. 2019 Instrument The questionnaire was the instrument used to col-
lect data, being composed of sociodemographic issues
(sex, age and course) and the problem situation resulting
from manipulation. Two questionnaires models were
collected, each corresponding to an experimental con-
dition: restriction of the scope of justice and neutral. Prejudice, Discrimination and Justice Certain theories and theoretical models (Pereira,
2012; Pereira & Souza, 2016) analyze how legitima-
tion can be a psycho-sociological strategy to explain
the psychological tension created by the contrasting
motivations of expressing prejudice and, at the same
time, being able to maintain the fair citizen self-image. In fact, justifying factors such as: the outgroup percep-
tion as threatening to society, with immutable cultural
differences and infra-humanization, in the sense of
dehumanizing outgroup people (Pereira, Vala & Leyens, Psico-USF, Bragança Paulista, v. 24, n. 2, p. 311-321, abr./jun. 2019 Psico-USF, Bragança Paulista, v. 24, n. 2, p. 311-321, abr./jun. 2019 Lima-Nunes, A. & Farias, K. G. Scope of justice
313 313 Therefore, the scope of justice restriction is the
psychological mechanism that lies between prejudice
and discrimination. Thus, the greater the prejudice,
the greater the need to reduce the scope of justice only
to ingroup individuals (Lima-Nunes, Pereira & Cor-
reia, 2013a). The consequence of this restriction may
be to favor the ingroup (eg. Tajfel & Turner, 1979),
but it can also be a type of discrimination in other
spheres, such as support for discriminatory policies
against outgroup (active) or non-support for policies
that favor outgroup (passive). context, a qualitative experimental study was designed
in order to identify its strategic use in two specific situ-
ations: when people are part of the dominant group
(situation of intergroup conflict) and when they are
merely observers of the conflict (assuming a neutral
position). Thus, it was expected that, in the intergroup con-
flict presence, people would make use of justificatory
discourses to restrict resources only to their belonging
group (eg., “racial quota is one way of discriminat-
ing whites who have everything on merit without the
Brazilian State help”). It was also hoped that, in the
intergroup conflict absence, people would broaden the
resources use to all possible groups (eg., “racial quota is
one way for all, truly, have the same rights to university
and work in Brazil”). Thus, an explanation for the perpetuation of vio-
lence or disregard for certain groups in society may be
within the scope of discourses justifying prejudice. The
Justified Discrimination Model (JDM) is presented as
an explanatory hypothesis in order to explore this issue. Specifically, the JDM examines how, between prejudice
and discrimination, there is mediation of a (non-biased)
discrimination justification to be understood as fair. Participants A total of 103 (one hundred and three) university
students enrolled in a university in the Paraíba country-
side participated in this study. They come from courses
in three knowledge fields: human (administration,
design and geography), exact (mathematics, materials
engineering, mining engineering, production engineer-
ing, petroleum engineering and chemical engineering)
and biological (nursing) courses. From these partici-
pants, 68 reported being female and 35 male. It was a
predominantly young sample, with ages ranging from
18 to 30 years (M = 20.44; SD = 1.98), with a people
predominance between 18 and 23 years of age, corre-
sponding to 90% of the respondents. These devices are necessary in societies with
egalitarian ideals, when through justifying factors, indi-
viduals seek to find a coherence between these ideals
and internalized attitudes (Modesto et al., 2017; Pereira,
Vala & Leyens, 2009). In this sense, the answer to the
question “Why the restriction perpetuation on the
scope of justice is insufficiently fought?” may be the
idea that social inequalities are perceived as legitimized
and thus the withdrawal of rights is seen as valid. In this sense, this study referred to the analysis of
justice perceptions (eg., the scope of justice) and its role
in the process of legitimizing intergroup conflict, that
is, how these perceptions can legitimize social inequali-
ties in a Brazilian students population. For that, it was
analyzed how the justice arguments are used in the
mechanisms implementation directed to social justice,
specifically, investigating the scope of justice role and
its application in the Brazilian context. Procedures Before conducting the research, the project was
submitted to the Ethics Committee by the Plataforma
Brasil (CAAE 47911615.1.0000.5182). By being in
agreement with the ethical procedures, it obtained its
approval. Participants signed the Free and Informed
Consent Term, where the research procedures and
doubts were elucidated, praising the respondents ano-
nymity in the questionnaires, with the initiative to make
them aware of the process. In order to analyze the legitimation forms of this
intergroup conflict type, this study aimed to analyze if
the scope of justice can be used as a base discourse to
interfere in the justice application to depend on social
position. That is, to identify whether, according to the
group position presented in the situation (dominant or
neutral), people use the scope of justice strategically to
justify their exclusion intentions or social inclusion. Psico-USF, Bragança Paulista, v. 24, n. 2, p. 311-321, abr./jun. 2019 Method In order to analyze the justifying justice role
(Lima-Nunes, Pereira & Correia, 2013b) in the Brazilian Psico-USF, Bragança Paulista, v. 24, n. 2, p. 311-321, abr./jun. 2019 Psico-USF, Bragança Paulista, v. 24, n. 2, p. 311-321, abr./jun. 2019 Lima-Nunes, A. & Farias, K. G. Scope of justice
314 Their distribution was random relative to the experi-
mental condition (restriction or neutral), in which only
one of the two conditions was presented to the par-
ticipants, guaranteeing a greater diversity of responses. The research scenarios were classrooms, in the classes
beginning or end, making it difficult to spread. following scenario: There is one country where justice
is not for everyone, because there are dominant groups
and dominated groups. You, the participant, are neu-
tral and do not make part of either group, being only
an observer, without power in the rights application to
neither of them. How do you, a neutral party in the
situation, justify the rights application in this country? Write at least three sentences. “ g
g
gi
p
After reading the problem situation, after any
doubts heeded, the participants were summoned to
justify their position depending on the experimental
condition, writing at least three sentences to do so. The instruction was to write what they thought about
their justice perception in that problem situation, and
to seek to position themselves from the proposed
statement, justifying the situation. In this sense, the
conditions were intended to verify the following
hypothesis: the participant’s group position influences
his/her justice perception, and consequently, the
scope of justice application. The answers analysis was based on Content Anal-
ysis, based on Bardin (2011), which is a methodological
set applied to a variety of discourses. It is a controlled
hermeneutics where deduction is applied, uniting
two extremes, both the subjectivity fecundity and the
objectivity rigor. The categorizations were elaborated
through three stages: pre-analysis, material exploration
and results treatment. Results Both experimental conditions had the same initial
information: “We are interested in understanding how
our justice perception depends on where we are. So we
want you to be part of a hypothetical situation, assum-
ing the role we present below.” After this statement, the
problem situation corresponding to the experimental
condition or control condition was presented. The participants’ responses categorization was
divided according to experimental conditions in the
“dominant group” (condition restriction of the scope
of justice) and “neutral group” (control condition). The dominant group condition obtained 50 respon-
dents, while the neutral group condition counted on
53 respondents. Some questionnaires were considered
as “unclassified” because they presented answers that
deviated from the study purpose or the theme.i Condition Restriction of the scope of justice:
the statement presented a scenario with the scope of
justice restriction, so that the participants justified
a position of rights restriction to outgroup without
threatening their fair citizen self-image, because it is in
the hypothetical situation statement. It is worth men-
tioning that the dominant group was not named, which
would be in charge of the respondent’s understanding. The complementary statement of the condition was:
“Imagine the following scenario: There is one coun-
try where justice is not for everyone because there
are dominant groups and dominated groups. You, the
participant, are part of the dominant group, with the
power to restrict the rights application to be applied
more to your group than to the dominated group. How do you, part of the dominant group, justify this
difference in the rights application in this country? Write at least three sentences.” In general, the same categories were identified in
the two experimental conditions. Differences between
conditions are presented from two components: 1)
prevalence of a given category in one condition and
absence in the other; 2) discursive positioning from the
same content (justified social inequality in the restriction
condition of the scope of justice is different from justi-
fied social inequality in the control/neutral condition). Subsequently, the categories will be presented
according to frequency (in percentage) and, therefore,
relevance in the groups derived from the experimen-
tal conditions. Psico-USF, Bragança Paulista, v. 24, n. 2, p. 311-321, abr./jun. 2019 Social inequality the main justification for the difference in the rights
application, just as economic inequality was associ-
ated with the dominant group existence. A relevant
component in the justifications in this experimental
condition refers to the meritocratic discourses use
to justify their dominant position towards the other
group, indicating the effectiveness of the restriction
manipulation. The social inequality conception is related to the
dominant group economic condition, and as one of
its consequences, to have impunity and the corrupting
justice possibility. Being 18% of the total restriction
condition, this category presents corruption as the
main cause of inequality. The favoring forms of the dominant group refer
here to financial status, formal education and high posi-
tions: D18: “a dominant group is often responsible for high
positions and high purchasing power, for having this set of char-
acteristics linked to higher education for finding loopholes in the
laws and being favored by others for passing on an idea that
they are the society basis.” According to respondents, such
privileges carry some advantages, such as supposedly
facilitated corruption, favoring inequality in the way the
law is enforced and rights enforced. Dominant group (the scope of justice condition restriction) In this experimental condition, the participants’
difficulty arose to position themselves as a dominant
group, as requested in the problem situation state-
ment. This effect may have arisen as a criticism from
the participants, although some respondents put
themselves as “I” and “we” rather than as an external
actor to justification. In the responses, the scope of
justice retriction based on meritocracy was considered Condition Control: the statement also presented
a scenario, but the participant should justify his/her
response from a neutral position. The idea in this con-
dition was to equate the tasks in the two conditions,
that is, to be placed only as an observer, without being
part of the context. Thus, the control condition com-
plement to the general statement was: “Imagine the Psico-USF, Bragança Paulista, v. 24, n. 2, p. 311-321, abr./jun. 2019 Lima-Nunes, A. & Farias, K. G. Scope of justice
315 Lima-Nunes, A. & Farias, K. G. Scope of justice
315 Scope of general justice The scope of general justice refers to a justice
broader argument, in agreeing with the problem-sit-
uation statement, approaching the scope of justice
concept itself. In this category, the participants confirm
the inconsistencies present in the justice, legitimating
the asymmetric power relations. In the scope of jus-
tice restriction condition, this category corresponded to
18% of participants’ responses. Here is an example of
an answer: The scope of justice restriction The scope of justice restriction was associated
with the privileges justification and leadership of the
dominant group, and the reasons that enabled this
group to gain power. It was also mentioned the favor-
ing of people belonging to the ingroup, in this sense,
meritocracy is a crucial justification. This categorization
refers to 38% of the questionnaires with statements
from the dominant group. Individualization The dominant group attention is directed towards
the ingroup participants and their counterparts, where
the outgroup rights are secondary to their own inter-
ests, as the following argument illustrates: “It is a right
matter, of merit, therefore, while dominant, I believe that my right
worths more than the others right”(D35).i In this condition, individualization is described
as a tendency to associate personality traits with the
dominant group superiority, especially characteristics
considered negative, such as egoism or “machiavel-
lian traits.” This category corresponded to 10% of the
responses found in the experimental condition. The differentiation justification in the rights appli-
cation is through meritocracy: “The justice application in a
differentiated manner can be justified on the merit basis (D47).”
This conception is associated with neoliberal ideals, rec-
ognizing the individual acquisitions quality, legitimizing
through exploitation ideology over a majority and a
minority dominating social production (Helal, 2007). D40: “Differentiated treatment may be a result of the
dominant group awareness lack, aiming only for self-improve-
ment. The overlapping of self-will before the will of others shows
the justice sense lack and selfishness. “ The justice conception on the part of the domi-
nant group is put as distorted by only favoring the own
group, but due to a psychological factor or sense lack. The reflection lack is placed before the effect of their
acts on the dominated group, giving the dominant
group a purely individual trait. Psico-USF, Bragança Paulista, v. 24, n. 2, p. 311-321, abr./jun. 2019 The scope of justice expansion This category, in the restriction condition, refers
to the participants difficulty participants to place them-
selves in the dominant group position. Respondents
proposed solutions to reduce inter-group inequal-
ity, both towards basic education and fundamentally
through justice, that can reach all people, that is, widen
the scope of justice for the outgroup people. Only 10%
of participants responded according to this category. D17: “As a human being grows in the environment in
which society causes you to grow economically, there will always
be dominant and dominated classes as the relation employer and
employee.” D26: “There is no way to do justice if a portion or group
of this imaginary country has fewer rights than others. There-
fore the real way to do justice is in the rights application for all
without distinction of color, race, financial condition or social
status (...)”. Thus, the legitimation of one subordinate group
is presented, where the privilege is perennial for the
dominant group, while the rights restriction faced by
the dominated group entails a series of losses. Psico-USF, Bragança Paulista, v. 24, n. 2, p. 311-321, abr./jun. 2019 Lima-Nunes, A. & Farias, K. G. Scope of justice
316 ‘sets them apart’ from others. We have, for example, these groups:
politicians, judges, police...” This type of discourse is agrees with what Helal
(2007) indicates about education and the merit perspec-
tive. When education is related to a leveling inequality
model, it puts the individual in charge of the society
transformation, which is part of a meritocratic vision. On the other hand, education can be seen from this
point of view of a factor that maintains social inequali-
ties and, thus, is fundamental for social stratification. In the aforementioned answer, the fact that,
through friendship or some family bond, to live with
people with high purchasing power, is already a way
of entering the dominant group, and thus, obtain
privileges. Other determining factors for the rights
application are placed beyond the financial question,
such as the ethnic question, considered as a factor of
intergroup inequalities. The racial difference is put in
some answers as the intergroup difference justification,
just as the gender difference and sexual orientation, in
this neutral category, it is named which groups are more
disadvantaged than others. Mobilization lack The tone of the mobilization lack category follows
the disinterest direction, conformation and even passiv-
ity of the dominated group, as a possible way to blame
this group. However, the mobilization is considered in
these justifications as preponderant to change the dom-
inated group condition, guaranteeing rights extension. This category represents 6% of the responses from the
dominant group condition. Neutral group (control condition) N38: “It is natural that in a country where the laws
are written by a certain group that has been in power for some
time, this group is always interested in the privileges maintenance
guaranteed by that power. On the other hand, it is becoming
increasingly difficult for the oppressed group to break these barri-
ers, since it is necessary to be in power to guarantee rights.” In this condition, the category “social inequality”
has been highlighted, related to nepotism, corruption
and power, in a similar way the dominant group, but
tending to name those who participate in the domi-
nant and dominated groups. There were also speeches
related to the scope of justice expansion, that is, the
rights application for all, equally distributed. Due to the
participant’s position as an observer, as requested by the
experimental condition, it was possible to give visibility
to the specific issues of certain struggles (racial, eth-
nic and sexual orientation issues), illustrated in excerpts
from the participants’ justifications. Thus, a dichotomy between practice and theory
in the justice application is evident, because the law is
not at the good service for all regardless of the group
position in which they are. However, in order to assert
power over the dominated group and as a mode of
control, the laws drafting are on the dominant group
hands, retaining the privileges to aim for its own good. Scope of general justice This category was relevant in the control con-
dition, once 28% of the responses of this condition
fit within it. Some discourses are based on the justice
questioning, which tends to provide the right for few
and even unworthy means, either through corruption
or with the oppressing purpose and securing the power
that its position reserves. Here’s an example of this
argument: D43: “I believe that the difference in the rights applica-
tion has been present for a long time in this country, and that the
population is conformed to this situation and does not mobilize to
modify this type of thing.” Psico-USF, Bragança Paulista, v. 24, n. 2, p. 311-321, abr./jun. 2019 Discussion The scope of justice extension in this section
is proposed through the political reform possibility
to achieve equal rights for each other and for justice. Therefore, the discourse in this fragment considers
the transformation power being exclusively in charge
of the political class, that is, of the dominant group. Politics also depends on popular participation, through
claims, as well as politicians are elected by a population,
which still has a certain choice power. The results point to the existence of the strate-
gic use of the justice arguments (scope of justice) to
justify the inequality posed in the problem situation. The group position, manipulated from the experimen-
tal conditions, influenced the emergence of certain
contents to the detriment of others, and consequently,
discursive categories. The categories variety that have arisen in response
to experimental conditions show the perceptions plu-
rality even when a specific situation exists. The way
the situation is justified illustrates a differentiated
perspective on what justice is for everyone. Thus, the
arguments that emerged represent relevant justice per-
ceptions from a place in society, posited in the present
study as a hypothetical situation, in an unequal country. The scope of justice expansion In this experimental condition, the financial con-
dition presents itself as an interference for the proper
rights application and/or compliance with the law. About half of the respondents (40%) in the neutral
group justified the problem situation through the dif-
ference in law enforcement. Respondents have evoked a trend towards the
ingroup rights application to the outgroup for all-
inclusive justice. Another positioning form refers to
the end of the differentiation between dominated and
dominant group, through the search of a repair to the
dominated group, with the purpose of guaranteeing
the equal justice for all, an example concerns the quo-
tas. This category corresponds to 22% of the neutral
group’s answers. N34: “(...) People with greater purchasing power, those
with influential relatives and/or friends became ‘participants in
dominant groups’. And the rest of society ends up being ‘the
dominated group’, being subject to any decision of the dominant
group. Law enforcement is for everyone, but ‘dominant groups’
are those who end up being left out, because their social condition N52: “The application of rights in this country occurs
in the wrong way, because, as neutral, I take position thinking Psico-USF, Bragança Paulista, v. 24, n. 2, p. 311-321, abr./jun. 2019 Lima-Nunes, A. & Farias, K. G. Scope of justice
317 group. The rights struggle must reach the field of equality: consid-
ering, each individual is entirely equal to others.” that there should be no dominant and dominated groups. Every-
one should have equal rights, both before men and before justice. Accordingly, improvements should be made in groups of politi-
cians to improve equality of justice between groups.” Lack of mobilization This category lists justifications that express a
conformation of the dominated groups in not claiming
their rights for the justice application, either because
they have a knowledge lack of their rights or because
there are no claims movements. This category is related
to about 6% of justifications. In general, different justifying arguments have
emerged to depend on position in intergroup con-
flict. For example, in the neutral group, there were no
respondents whose response was categorized as “the
scope of justice restriction”, while the category “social
inequality” emerges as the main justification for the dif-
ference in the rights application in this experimental
condition. That is, the problem situation, posed from a
group positioning, caused the justice perception to be
changed, changing the narrative justification focus. N36: “And on the other hand the dominated go to the
streets to claim what they want, but the fact of having no degree
of kinship with people of powers lose their voice, but they claim
and in some cases they are heard.” The dominated group invisibility and, because
of this, influence and voice lack are placed as fac-
tors that make it impossible to perceive this group
demands, that is, own belonging to the dominated
group would be an exclusion factor because they did
not have access to outgroup. However, their demands
can be met due to the mobilization, considered a
fundamental factor for the representativeness effec-
tiveness and the rights guarantee. However, in relation to the dominant group con-
dition, the category “the scope of justice restriction”
stood out among others, with a greater occurrence,
while the category “social inequality” was less promi-
nent. The “restriction of the scope of justice” category
emergence in this condition may be related to an
ingroup favoring, due to the dominant group social
prestige, since the outgroup rights should be restricted
(Tajfel & Turner, 1979). Psico-USF, Bragança Paulista, v. 24, n. 2, p. 311-321, abr./jun. 2019 Individualization This category has as main point to attribute per-
sonality characteristics and affections to dominant
group members. There is an exclusion of the social
question in these discourses, possibly in an attempt to
minimize the intergroup conflicts consequences exist-
ing in the scenario. It would be an attempt to hold each
subject responsible in the relationship, for it shifts the
action of a dominant abstract group to the individual
plane. This category corresponds to approximately 4%
of the questionnaires. As expected, the most relevant results refer to the
scope of justice restriction condition (dominant group),
where a greater occurrence was obtained regarding
justification of the justice application restricted to the
ingroup. Specifically, the justification for the domi-
nant and dominated group position argues, through
meritocratic discourses, the resources restriction to the
dominated group. N37: “I believe that the rights difference in whatever the
locality does not start from the dominant group action, but part
of the individual action of each person contained in the dominant The belief in meritocracy as a descriptive norm
makes it possible to support and sustain various dis-
crimination forms and inequality maintenance, but Lima-Nunes, A. & Farias, K. G. Scope of justice
318 318 it depends on whether the context has norms with
egalitarian values. Meritocracy can be motivated by dis-
crimination (eg., racism), by perceiving that individuals
(eg., blacks) are responsible for their own social posi-
tion, where this merit idea is a legitimating discourse
that mediates racism and opposition to affirmative
action, considered to be unfair for opposing the merit
idea (Pereira, 2012, Pereira & Souza, 2016). groups between dominant and dominated. Moreover,
in this experimental condition, the participants present
greater occurrence of content related to the scope of
justice expansion, that is, 22% of the arguments in the
neutral condition to the detriment of 10% of the argu-
ments in the restriction condition. That is, when the
group position was not directly influencing participants’
perception of the problem situation, the possibility of
allowing the rights application to be extended to all
groups seems more feasible. (
)
In the scope of the present study, even when
faced with a hypothetical situation, the discursive pat-
tern follows a legitimizing logic to the superiority of
one group over others. Final considerations The arguments existence based on justice per-
ceptions make possible to deepen the scope of justice
role in discriminating against certain minority groups,
supporting previous research on the subject (eg.,
Hadarics & Kende, 2018; Lima-Nunes, Pereira, &
Correia, 2013a). Field research is fundamental to broaden the hori-
zons of theoretical development, thus finding empirical
evidence of the hypotheses discussed in this paper. Although the scope of justice strategic use in legitimiz-
ing arguments of intergroup conflict has been identified,
some methodological limitations of the study should
be pointed out. On the one hand, the exploratory and
qualitative character of the present study made possible
to access the psychological mechanism of scope of
justice restricting in a broader and more complex way,
corresponding to the construct itself. Therefore, when the participant was summoned
to position himself/herself as part of a dominant
group in the hypothetical situation, the arguments used
to justify the restriction of resources were: the status
quo maintenance and the position it occupies in society. While in the observer position (neutral position), the
justifications were to give visibility to groups that suffer
social exclusion and the attempt to seek equitable jus-
tice. These results corroborate studies that point to the
scope of justice restriction as an effective social exclu-
sion justification, showing how justice arguments can
help in the derogation of certain groups (Coryn & Bor-
shuk, 2006, Kals, & Strubel, 2017; Lima-Nunes, Pereira,
& Correia, 2013b). On the other hand, the subjective response to the
manipulated problem situation made it difficult to dif-
ferentiate between experimental conditions, with similar
categories in the two conditions. The main difference
between the conditions was identified in the category
that presented only in the dominant group condition:
the scope of justice restriction by meritocracy. Likewise, we identify the limitation when respond-
ing to the dominant condition (the scope of justice
restriction). Participants hesitated to justify their posi-
tion, and there was a difficulty in naming who would
be responsible for restricting the rights application. This hesitation was understood as an attempt to avoid
a possible ingroup blame and the responsibility induced
by the manipulation. This inability may be related to
a mechanism concerning cognitive dissociation where
people tend to deny that they are prejudiced by impu-
tizing prejudice to an abstraction or even, in the present
case, avoiding being held accountable (Pereira, Torres
& Almeida, 2003). Individualization That is, even if “real groups”
are not identified, the group positioning has influenced
the psychological process that underlies the justification
based on a justice perception. Such a mechanism rein-
forces the asymmetrical relationship of power that was
presented, by the argument characteristic - justification
by justice. In this sense, the Justified Discrimination
Model premise (JDM, Pereira, 2011) can be applied to
the process identified in the discourse based on justice
arguments, where the group effect position on the dif-
ferentiation behavior is not direct, but mediated by the
scope of justice restriction. Psico-USF, Bragança Paulista, v. 24, n. 2, p. 311-321, abr./jun. 2019 References In relation to the control condition (neutral group),
it was easier to name the actors in each group and the
justice restriction due to the participant position as an
observer. In fact, the justification most present in this
second group is social inequality, followed by the scope
of general justice and the scope of justice expansion,
which in the dominant group did not appear as more
relevant discourses. Despite the neutrality requested
by the problem-situation in this experimental condi-
tion, such positioning in reality is impossible. Thus,
the attempt to place itself in this position may have
generated an artificiality in the arguments to justify the
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568–604. Retrieved from http://nsuworks.nova. edu/tqr/vol11/iss3/9 In general, in order to resolve such limitations,
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developed to measure the scope of justice are needed
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229-237. doi: 10.1002/ejsp.1966 In order to face discrimination, the struggle for the
rights achievement must be constant, since the perpetu-
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intricate (Pereira, Vala & Leyens, 2009). The reconfigu-
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co, CA: Jossey-Bass, 2nd Edition. p. 41-64. Final considerations In this sense, the scope of justice strategic use
concerns the restriction application to the problem sit-
uation, justifying the group positioning, starting from
the respondent situational perception. Thus, the restric-
tion application of the scope of justice to the ingroup
occurs because such an argument seems “reasonable
and appropriate (from the actor’s point of view) to
accept passively or even to contribute to the one indi-
vidual suffering and not of another.” (Hafer & Olson,
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no mercado de trabalho gaúcho. (Dissertação de Mes-
trado). Universidade Federal do Rio Grande do
Sul (UFRGS), Porto Alegre, RS. Retrieved from
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humanization to discrimination: The mediation of
symbolic threat needs egalitarian norms. Journal
of Experimental Social Psychology, 45, 336–344. doi:
10.1016/j.jesp.2008.10.010 Modesto, J. G. et al. (2017). Racismo e Políticas Afir-
mativas: Evidências do Modelo da Discriminação
Justificada. Psicologia: Teoria e Pesquisa., 33, 1-8. doi:
10.1590/0102.3772e3353. Sherif, M. (1967) Social interaction: processes and products. Chicago: Aldine. doi: 10.2307/144927 Olson, J. M et al. (2011) Distinguishing two meanings of
moral exclusion: exclusion from moral principles
or principled harm-doing? Social Justice Research, 24,
365-390. doi: 10.1007/s11211-011-0141-8 Staub, E. (1989) The roots of evil: Origins of genocide and
other group violence. New York: Cambridge Univer-
sity Press. Opotow, S. (1990) Moral exclusion and injustice: An
introduction. Journal of Social Issues, 46, 1-20. doi:
10.1111/j.1540-4560.1990.tb00268.x Tajfel, H. (1970). Experiments in intergroup discrimi-
nation. Scientific American, 223, 96-102. Opotow, S. (1995). Drawing the line: Social categori-
zation, moral exclusion, and the scope of justice. In B. B. Bunker & J. Z. Rubin (Eds.), Conflict, co-
operation, and justice (pp. 347–369). San Francisco:
Jossey-Bass. Tajfel, H., & Turner, J. C. (1979) An integrative the-
ory of intergroup conflict. In W. G. Austin & S. Worchel (Eds.), The social psychology of inter-
group relations, Monterey, CA: Brooks/Cole. p. 33-47 Pereira, C. R. (2011). References doi:
10.1007/978-1-4419-9994-8_5 In this perspective, the scope of justice strategic
use can act both to prevent the general development
of social justice, facilitating segregation (restricting
the scope of justice), as well as stimulating coopera-
tion between groups, allowing for greater openness of
justice to other groups (widening the scope of justice). Thus, the scope of justice strategic use depends on
what position the subject occupies in the intergroup
relationship. Hadarics, M., & Kende, A. (2018). Moral foundations
of positive and negative intergroup behaviour:
Moral exclusion fills the gap. International Journal
of Intercultural Relations, 64, 67-76. doi: 10.1016/j. ijintrel.2018.03.006 Hafer, C. L., & Olson, J. M. (2003). An Analysis of
Empirical Research on the Scope of Justice. Per-
sonality and Social Psychology Review, 7, 311–323. doi:
10.1207/s15327957pspr0704_04 Thus, this study constitutes as an advance in the
Brazilian literature in the social psychology of justice
scope, for addressing the scope of justice in relation
to the group context. It is hoped that this research can
serve as a basis for the development of a research line
that relates the justice perception to aspects related to
intergroup relations. The fact of perceiving the justice
application as (a) restricted to certain groups or (b)
extended to many or all groups can have great effects
on the derogation of historically minority groups, with
great difficulties in conquering rights. Future studies
should explore the relationship between the scope of
justice and specific forms of prejudice and discrimina-
tion, as well as seek the validation of the scale of the
scope of justice in Brazil. Helal, D. H. (2007) O papel da educação na sociedade e
organizações modernas: criticando a meritocracia. Revista Eletrônica de Administração, 13(2), 386-408. Retrieved from http://seer.ufrgs.br/index.php/
read/article/view/39926/25461 Kals, E., & Strubel, I. T. (2017). Volunteering to Support
Refugees: A Question of One’s Scope of Justice. Refuge, 33(2), 66-77. Retrieved from https://ref-
uge.journals.yorku.ca/index.php/refuge/article/
viewFile/40459/36462 Lima-Nunes, A., Pereira, C. R., & Correia, I. (2013a)
Restricting the scope of justice to justify discrimi-
nation: The role played by justice perceptions in Psico-USF, Bragança Paulista, v. 24, n. 2, p. 311-321, abr./jun. 2019 Lima-Nunes, A. & Farias, K. G. Scope of justice
320 discrimination against immigrants. European Jour-
nal of Social Psychology, 43, 627-636. doi: 10.1002/
ejsp.1981 (Eds.), Normas, atitudes e comportamentos sociais (pp. 171-207). Lisboa: Imprensa de Ciências Soci-
ais. Retrieved
from
http://repositorio.ul.pt/
handle/10451/20549 Lima-Nunes, A., Pereira, C. R., & Correia, I. (2013b). Sobre as autoras: Aline Lima-Nunes, Adjunct Professor at Universidade Federal de Campina Grande (UFCG). PhD in Psychology
from Instituto Universitário de Lisboa (ISCTE-IUL), Portugal. Master’s in Social Psychology from UFPB. Bachelor’s
and Licentiate degrees in Psychology from UFPB. Researcher in the area of Social Psychology of Justice and Social
Exclusion Processes. ORCID: https://orcid.org/0000-0002-3042-5451 Email: limanunes.aline@gmail.com Email: limanunes.aline@gmail.com Kamila Gadelha Farias, Graduated in Psychology at Universidade Federal de Campina Grande (UFCG). Holds a
Post-graduation in Mental Health and Psychosocial Care Network by IESM (Instituto Superior Mútiplo). Researches
include themes relevant to Social Psychology and Health, mainly in the scope of public policies related to political
minorities. ORCID: https://orcid.org/0000-0002-5199-838X
Email: kamila.gd@live.com ORCID: https://orcid.org/0000-0002-5199-838X
Email: kamila.gd@live.com ORCID: https://orcid.org/0000-0002-5199-838X
Email: kamila gd@live com References A legitimação da discriminação
em diferentes contextos normativos. In E. M. Te-
chio & M. E. O. Lima (Eds.), Cultura e produção
das diferenças: Estereótipos e preconceito no
Brasil, Espanha e Portugal (pp. 363-404). Brasília:
Technopolotik. Torres, A. R. R., & Camino, L. (2013). Grupos soci-
ais, relações intergrupais e identidade social. In: L. Camino et al. (Eds.), Psicologia Social: temas e teorias
(2ª ed.), revista e ampliada. p. 513-538. Brasília:
TechnoPolitik. Recebido em: 23/05/2018
Reformulado em: 30/07/2018
Aprovado em: 29/08/2018 Torres, A. R. R., & Camino, L. (2013). Grupos soci-
ais, relações intergrupais e identidade social. In: L. Camino et al. (Eds.), Psicologia Social: temas e teorias
(2ª ed.), revista e ampliada. p. 513-538. Brasília:
TechnoPolitik. Pereira, C. R. (2012). Normas sociais e legitimação da
discriminação. In C. R. Pereira & R. Costa-Lopes Psico-USF, Bragança Paulista, v. 24, n. 2, p. 311-321, abr./jun. 2019 Nota das autoras: This research was funded by CNPq within the framework of the PIBIC/UFCG Grant awarded to the second author. Lima-Nunes, A. & Farias, K. G. Scope of justice
321 Psico-USF, Bragança Paulista, v. 24, n. 2, p. 311-321, abr./jun. 2019
Aline Lima-Nunes
Universidade Federal de Campina Grande (UFCG), Academic Unit of Psychology
Rua Juvêncio Arruda, 795, Bodocongó
Campina Grande-PB, Brazil
CEP: 58429-600
Institutional email: aline.nunes@ufcg.edu.br Aline Lima-Nunes
Universidade Federal de Campina Grande (UFCG), Academic Unit of Psychology
Rua Juvêncio Arruda, 795, Bodocongó
Campina Grande-PB, Brazil
CEP: 58429-600
Institutional email: aline.nunes@ufcg.edu.br Aline Lima-Nunes
Universidade Federal de Campina Grande (UFCG), Academic Unit of Psychology
Rua Juvêncio Arruda, 795, Bodocongó
Campina Grande-PB, Brazil
CEP: 58429-600
Institutional email: aline.nunes@ufcg.edu.br Institutional email: aline.nunes@ufcg.edu.br Institutional email: aline.nunes@ufcg.edu.br Psico-USF, Bragança Paulista, v. 24, n. 2, p. 311-321, abr./jun. 2019 Psico-USF, Bragança Paulista, v. 24, n. 2, p. 311-321, abr./jun. 2019
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https://journals.plos.org/plospathogens/article/file?id=10.1371/journal.ppat.1009842&type=printable
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English
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Broadly directed SARS-CoV-2-specific CD4+ T cell response includes frequently detected peptide specificities within the membrane and nucleoprotein in patients with acute and resolved COVID-19
|
PLOS pathogens
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cc-by
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PLOS PATHOGENS PLOS PATHOGENS OPEN ACCESS Citation: Heide J, Schulte S, Kohsar M, Brehm TT,
Herrmann M, Karsten H, et al. (2021) Broadly
directed SARS-CoV-2-specific CD4+ T cell
response includes frequently detected peptide
specificities within the membrane and
nucleoprotein in patients with acute and resolved
COVID-19. PLoS Pathog 17(9): e1009842. https://
doi.org/10.1371/journal.ppat.1009842 ¤ Current address: Department of Obstetrics and Gynecology, University of Chicago, Chicago, Illinois, United
States of America
* julianszw@gmail.com ¤ Current address: Department of Obstetrics and Gynecology, University of Chicago, Chicago, Illinois, United
States of America
* j li
@
il Broadly directed SARS-CoV-2-specific CD4+ T
cell response includes frequently detected
peptide specificities within the membrane
and nucleoprotein in patients with acute and
resolved COVID-19 Janna Heide1,2¤, Sophia Schulte1, Matin Kohsar1, Thomas Theo BrehmID1,2,
Marissa HerrmannID1,2, Hendrik KarstenID1, Matthias Marget3, Sven Peine3, Alexandra
M. JohanssonID4, Alessandro SetteID5, Marc Lu¨tgehetmannID6, William W. KwokID4,
John Sidney7, Julian Schulze zur WieschID1,2* a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 1 Infectious Diseases Unit, I. Department of Medicine, University Medical Center, Hamburg-Eppendorf,
Germany, 2 German Center for Infection Research (DZIF), Partner Site, Hamburg-Lu¨beck-Borstel-Riems,
Germany, 3 Department of Transfusion Medicine, University Medical Center, Hamburg-Eppendorf,
Germany, 4 Benaroya Research Institute at Virginia Mason, Seattle, Washington, United States of America,
5 Center for Infectious Disease and Vaccine Research, La Jolla Institute for Immunology (LJI), La Jolla,
California, United States of America, 6 Institute of Medical Microbiology, Virology and Hygiene, University
Medical Center, Hamburg-Eppendorf, Germany, 7 Department of Medicine, Division of Infectious Diseases
and Global Public Health, University of California, San Diego (UCSD), La Jolla, California, United States of
America PLOS PATHOGENS SARS-CoV-2 N, M, and E-specific CD4+ T cells cell peptide responses demonstrates that SARS-CoV-2 infection universally primes a broad
T cell response directed against multiple specificities located within the N, M and E structural
protein. cell peptide responses demonstrates that SARS-CoV-2 infection universally primes a broad
T cell response directed against multiple specificities located within the N, M and E structural
protein. Information files. The amino acid sequence can be
accessed in GenBank under the accession number
MT318827. Information files. The amino acid sequence can be
accessed in GenBank under the accession number
MT318827. Funding: This project was funded by Deutsches
Zentrum fu¨r Infektionsforschung (DZIF) (JH,
JSzW). JSzW was additionally funded by the DFG
(SFB 841 and SFB1328). The project was partially
funded by the NIH NIAID under awards AI 142742
(Cooperative Centers for Human Immunology)
(AS), National Institutes of Health contract Nr. 75N9301900065 (AS), and U19 AI118626 (AS). WWK and AMJ are supported by NIH grant
3U19AI135817-04S1. The funders had no role in
study design, data collection and analysis, decision
to publish, or preparation of the manuscript. Author summary The SARS-CoV-2 genome encodes for 25 different viral proteins. However, many immu-
nological studies have focused on the immune response against the spike protein. This
current study was designed to get a detailed understanding of the breadth and specificity
of the CD4+ T cell response directed against the other structural proteins, namely the
envelope (E), membrane (M) and nucleoprotein (N) using a comprehensive overlapping
peptide set in a cohort of patients during early and resolved COVID-19. We detected a
universally broad T cell response with on average more than 20 peptide responses per
patient. Three peptides elicited CD4+ T cell responses in more than 55% of all patients,
two located within the M protein, and one located within the N protein. These peptides
were further defined in terms of length and HLA restriction, and we developed a novel
MHC class II tetramer based on this data, which enabled us to investigate the ex vivo phe-
notype of SARS-CoV-2-specific CD4+ T cells in one patient. Competing interests: The authors have declared
that no competing interests exist. This large immunological data set on individual immune responses will be useful for
further detailed studies on the immunopathogenesis of SARS-CoV-2 infection and vac-
cine design. Abstract Editor: Benhur Lee, Icahn School of Medicine at
Mount Sinai, UNITED STATES Editor: Benhur Lee, Icahn School of Medicine at
Mount Sinai, UNITED STATES
Received: May 19, 2021
Accepted: July 27, 2021
Published: September 16, 2021 The aim of this study was to define the breadth and specificity of dominant SARS-CoV-2-
specific T cell epitopes using a comprehensive set of 135 overlapping 15-mer peptides cov-
ering the SARS-CoV-2 envelope (E), membrane (M) and nucleoprotein (N) in a cohort of 34
individuals with acute (n = 10) and resolved (n = 24) COVID-19. Following short-term virus-
specific in vitro cultivation, the single peptide-specific CD4+ T cell response of each patient
was screened using enzyme linked immuno spot assay (ELISpot) and confirmed by single-
peptide intracellular cytokine staining (ICS) for interferon-γ (IFN-γ) production. 97% (n = 33)
of patients elicited one or more N, M or E-specific CD4+ T cell responses and each patient
targeted on average 21.7 (range 0–79) peptide specificities. Overall, we identified 10 N, M
or E-specific peptides that showed a response frequency of more than 36% and five of them
showed high binding affinity to multiple HLA class II binders in subsequent in vitro HLA bind-
ing assays. Three peptides elicited CD4+ T cell responses in more than 55% of all patients,
namely Mem_P30 (aa146-160), Mem_P36 (aa176-190), both located within the M protein,
and Ncl_P18 (aa86-100) located within the N protein. These peptides were further defined
in terms of length and HLA restriction. Based on this epitope and restriction data we devel-
oped a novel DRB*11 tetramer (Mem_aa145-164) and examined the ex vivo phenotype of
SARS-CoV-2-specific CD4+ T cells in one patient. This detailed characterization of single T Peer Review History: PLOS recognizes the
benefits of transparency in the peer review
process; therefore, we enable the publication of
all of the content of peer review and author
responses alongside final, published articles. The
editorial history of this article is available here:
https://doi.org/10.1371/journal.ppat.1009842 Copyright: © 2021 Heide 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. Data Availability Statement: All relevant data are
within the manuscript and its Supporting 1 / 24 PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1009842
September 16, 2021 PLOS PATHOGENS PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1009842
September 16, 2021 Introduction Coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coro-
navirus type 2 (SARS-CoV-2) is a severe flu-like illness which is associated with hyperinflam-
mation and immune dysfunction. SARS-CoV-2 has led to a pandemic with more than 200
million confirmed cases and more than 4 million deaths (https://covid19.who.int/). Only a
small percentage of patients with COVID-19 develop a severe disease course, with the main
established risk factors being old age and comorbidities like hypertonus, adiposity or diabetes
[1]. SARS-CoV-2 has a single-stranded RNA genome of approximately 30 kb which includes
four structural proteins and open reading frames (ORFs) encoding for the nonstructural poly-
proteins [2]. The structural proteins are the spike (S), envelope (E), membrane (M) and nucle-
oprotein (N) [3]. The S protein is 1273 amino acids, the M protein 222 amino acids, and the E
protein 75 amino acids long. Together, these three antigens are part of the viral coat. The M
protein is the most abundant structural protein and it defines the shape of the viral envelope
[4]. It has a small N-terminal glycosylated ectodomain, three transmembrane domains, and a
much larger C-terminal endodomain that extends 6–8 nm into the viral particle [4]. The 419
amino acids long N protein is involved in the packaging of the RNA genome [5]. The E protein
is the smallest of the four structural proteins. This transmembrane protein has an N-terminal
ectodomain and a C-terminal endodomain with ion channel activity, which is associated with
pathogenesis [6]. Previous studies suggest that SARS-CoV-2-specific T cells play a key role in COVID-19 dis-
ease resolution and modulation of disease severity [7–11]. The relationship between T cell
immunity against the different SARS-CoV-2 antigens (or pre-existing cross-reactive immune PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1009842
September 16, 2021 2 / 24 PLOS PATHOGENS SARS-CoV-2 N, M, and E-specific CD4+ T cells responses against other coronaviruses) and the clinical course of a SARS-CoV-2 infection are
currently being unraveled [7, 9, 12–16]. In case of the original SARS-CoV, most T cell
responses were directed against the structural proteins as compared to the non-structural pro-
teins [17]. Furthermore, the T cell responses directed against the M and N protein were among
the most dominant and long-lasting [17]. How the adaptive immune response, and in particular T cell response pattern, influence the
kinetics of viral loads and the duration of a SARS-CoV-2 infection remains unclear [3, 11]. Introduction Additionally, the longevity of the naturally acquired immune memory to SARS-CoV-2 in com-
parison to the SARS-CoV-2 vaccine-induced response will be a substantial question to address
in future studies [11]. The definition of SARS-CoV-2-specific T cell epitopes is important to
evaluate potential influences of mutations on acquired immunity and vaccine efficacy. The
role of potential cross-reactivity between SARS-CoV-2 and other coronaviruses like SARS-
CoV and common cold coronaviruses can also be investigated with the knowledge of estab-
lished immune epitopes [15, 16]. In this study, we screened a cohort of patients with resolved
or acute COVID-19 using a comprehensive, overlapping panel of synthetic 15-mer peptides
derived from the SARS-CoV-2 N, M and E protein sequences, rather than relying on prese-
lected and in silico predicted epitope specificities [7, 9]. We found a universally broad N, M
and E-specific CD4+ T cell response regardless of the clinical course of the disease. The large
SARS-CoV-2 epitope data set presented here will be a useful tool for further investigations of
the ex vivo phenotype of SARS-CoV-2-specific T cells e.g., by tetramer technology, and could
also be helpful for efficient peptide-based vaccine design. Clinical features of the study cohort The clinical data of the patient cohort are summarized in Tables 1 and S1. The cohort con-
sisted of 34 patients infected with SARS-CoV-2. Infection was verified by a positive RT-PCR of
nasopharyngeal swabs as previously described [18]. For further analysis, the patient cohort was
stratified into patients with acute and resolved COVID-19. PBMC samples of ten patients dur-
ing acute infection [average days since diagnosis 6.3 (range: 2–16)] and of 24 patients after
convalescence [average days since diagnosis 96.36 (range: 40–184)] were collected. 12 patients
were female (35%) and 22 were male (65%), the average age was 47.2 years (range: 25–78
years). All patients were treated as inpatients for SARS-CoV-2 infection or attended an outpa-
tient clinic at University Medical Center Hamburg-Eppendorf: 26 patients (76%) were from
Germany, eight patients (24%) from the Philippines, Afghanistan, Croatia, Greece, or Syria. 22
patients (65%) had a mild or moderate course of disease as defined by the WHO, while 12
patients (35%) suffered from a severe or critical infection [19]. Seven patients needed to be
treated in the intensive care unit during their hospitalization (21%). Five patients were on
long-term treatment with immunosuppressive medication due to comorbidities: patients
aCov-04, aCov-07 and rCov-03 were treated with mycophenolic acid, doxorubicin/ifosfamide
and imatinib, respectively (S1 Table) and rCov-12 and rCov-16 received B-cell depleting med-
ication (rituximab and obinutuzumab, respectively). PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1009842
September 16, 2021 https://doi.org/10.1371/journal.ppat.1009842.t001 High response rate and broadly directed N- and M-specific T cell responses Using ELISpot as previously described [20–22], we first looked at the ex vivo N, M and E-spe-
cific IFN-γ production of PBMC from COVID-19 patients upon stimulation with single pep-
tides. A set of 43 overlapping 15-mer peptides covering the M protein, 82 peptides covering
the N protein and ten peptides covering the E protein of SARS-CoV-2, were utilized (S2
Table). As described earlier [23] and depicted in S1 Fig, the ex vivo ELISpot after stimulation 3 / 24 PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1009842
September 16, 2021 PLOS PATHOGENS SARS-CoV-2 N, M, and E-specific CD4+ T cells Table 1. Clinical and immunological patient characteristics of acute and resolved COVID-19 patients. Data are expressed as absolute numbers n (n/N) or n (range)
or mean with standard deviation. [normal range]
Acute COVID-19 (n = 10)
Resolved COVID-19 (n = 24)
Age in years
50.8 (25–76)
46.58 (26–78)
Sex
Female
1 (10%)
11 (46%)
Male
9 (90%)
13 (54%)
Disease severity
Mild
5 (50%)
10 (42%)
Moderate
2 (20%)
5 (21%)
Severe
2 (20%)
5 (21%)
Critical
1 (10%)
4 (17%)
Days since diagnosis
6.3 (2–16)
96.36 (40–184)
Days since start of symptoms
9.88 (2–24)
Comorbidities
None
3 (30%)
12 (50%)
Hypertension
1 (10%)
7 (29%)
Heart disease
2 (20%)
2 (8%)
Diabetes
3 (30%)
4 (17%)
Lung disease
1 (10%)
2 (8%)
Cancer
2 (20%)
3 (13%)
Other
3 (30%)
9 (38%)
Blood cell count at time of analysis
White blood cell count
3.8–11.0 Mrd/l
6.98 (± 2.78)
Lymphocyte count
1.0–3.6 Mrd/l
1.15 (± 0.53)
Hemoglobin
14.0–17.5 g/dl
14.05 (± 4.18)
Platelet count
150–400 Mrd/l
226.5 (± 87.84)
Immunology at time of analysis
T lymphocyte count
900–2,900/µl
962.9 (± 550.7)
T lymphocyte %
55–84 %
68.3 (± 8.9)
CD4 count
500–1,350/µl
587.9 (± 306.7)
CD4 %
31–60 %
42.8 (± 8.4)
CD8 count
290–930/µl
327.4 (± 250.3)
CD8 %
13–41 %
22 (± 6)
CD4/CD8 ratio
0.6–3.6
2.1 (± 0.8)
Tregs %
5.7–10.1 %
7.5 (± 2.0)
Clinical parameters at time of analysis
CRP
-5 mg/l
89.63 (± 81.54)
IL-6
<7 ng/l
61.58 (± 86.51)
Ferritin
22–322 µg/l
1066.03 (±1013.16)
Procalcitonin
-0.5 µg/l
0.39 (± 0.68)
D-dimer
0.21–0.52 mg/l
0.69 (±0.49) with single peptides showed a low overall IFN-γ response with a magnitude barely above the
limit of detection of this assay (number of spots range: 0–9 spots per 100,000 cells). High response rate and broadly directed N- and M-specific T cell responses We
detected IFN-γ responses in all three patients with an average number of nine M peptide PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1009842
September 16, 2021 PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1009842
September 16, 2021 4 / 24 PLOS PATHOGENS SARS-CoV-2 N, M, and E-specific CD4+ T cells Fig 1. The envelope, membrane and nucleoprotein-specific CD4+ T cell response. (A) Comparison of the IFN-y response after stimulation with M-Pool 4 ex vivo
and after ten-day T cell culture with M-Pool 4 (patient rCov-01). Gated on CD4+ T cells. R10 and DMSO were added to the negative control. (B) Comparison of the
total number of envelope, membrane and nucleoprotein-specific CD4+ T cell responses of each patient after 10-day in vitro culture. Mean with standard deviation. NS = not significant. Fig 1. The envelope, membrane and nucleoprotein-specific CD4+ T cell response. (A) Comparison of the IFN-y response after stimulation with M-Pool 4 ex vivo
and after ten-day T cell culture with M-Pool 4 (patient rCov-01). Gated on CD4+ T cells. R10 and DMSO were added to the negative control. (B) Comparison of the
total number of envelope, membrane and nucleoprotein-specific CD4+ T cell responses of each patient after 10-day in vitro culture. Mean with standard deviation. NS = not significant. https://doi.org/10.1371/journal.ppat.1009842.g001 https://doi.org/10.1371/journal.ppat.1009842.g001 responses per patient (range 5–15 peptide responses), and an average of 15.6 peptide responses
(range 9–24 peptide responses) against the N protein. In order to increase the overall detection rate of individual peptide responses, we employed
a well-established highly sensitive in vitro approach to detect virus-specific T cell responses
[20, 22]. In short, our peptide set was divided into 13 different peptide pools (S2 Table), and
13 individual cell cultures were started in parallel per patient with each peptide pool for ten
days, followed by a single peptide ELISpot for IFN-γ-production. Each positive T cell response
in the ELISpot assay was confirmed in a subsequent ICS assay for IFN-γ production after re-
stimulation with the respective single peptide. Fig 1A shows an exemplary ICS result of an M
peptide pool specific CD4+ T cell response ex vivo and after expansion of antigen-specific T
cells with M-Pool 4 for ten days. With this approach, we were able to detect a broad range of IFN-γ CD4+ T cell responses
against the M, N and E protein with the majority of responses directed against the N protein
(Fig 1B). PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1009842
September 16, 2021 High response rate and broadly directed N- and M-specific T cell responses 97% of all patients showed a peptide response against the N protein (29/30) (Fig 2). In the resolved patient group, all patients showed at least three or more peptide responses with
a mean of 16.67 (range 3–51; median 6) peptide specific CD4+ T cell responses per patient. In
the group of acutely infected COVID-19 patients, all but one patient recognized at least two
peptide specificities of the N protein with an average number of 12.44 (range 0–19; median 16)
CD4+ T cell responses (Fig 1B). One resolved patient (rCov-09) and one acute patient (aCov-07) did not show a M protein
peptide-specific CD4+ T cell response. Of the other patients, the resolved patient group had an
average of 6.96 (range 1–22; median 6) M-specific CD4+ T cell responses (Fig 3). The acute
patient group showed an average of 6.2 (range 0–12, median 5.5) individual M peptide specific
CD4+ T cell responses. The pattern of the CD4+ T cell response against the overlapping peptide set of the smaller E
protein is shown in S3 Table. From the resolved patient group, 13 patients (54.2%; n = 24) The pattern of the CD4+ T cell response against the overlapping peptide set of the smaller E
protein is shown in S3 Table. From the resolved patient group, 13 patients (54.2%; n = 24)
showed a detectable response against E, whereas four of the acutely infected patients showed a
peptide specific CD4+ T cell response (40%; n = 10). An average of 1.67 responses per resolved showed a detectable response against E, whereas four of the acutely infected patients showed a
peptide specific CD4+ T cell response (40%; n = 10). An average of 1.67 responses per resolved PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1009842
September 16, 2021 5 / 24 PLOS PATHOGENS SARS-CoV-2 N, M, and E-specific CD4+ T cells Fig 2. Overview of the SARS-CoV-2 nucleoprotein-specific CD4+ T cell responses in patients with acute (n = 9) and resolved (n = 21) COVID-19. For
each patient, the HLA class II molecules DRB1 and DQB1 are listed. aCov = acute COVID-19 patient, rCov = resolved COVID-19 patient. = patient was
treated with immunosuppressant medication or received chemotherapy. https://doi.org/10.1371/journal.ppat.1009842.g002 Fig 2. Overview of the SARS-CoV-2 nucleoprotein-specific CD4+ T cell responses in patients with acute (n = 9) and resolved (n = 21) COVID-19. For
each patient, the HLA class II molecules DRB1 and DQB1 are listed. High response rate and broadly directed N- and M-specific T cell responses aCov = acute COVID-19 patient, rCov = resolved COVID-19 patient. = patient was
treated with immunosuppressant medication or received chemotherapy. https://doi.org/10.1371/journal.ppat.1009842.g002 https://doi.org/10.1371/journal.ppat.1009842.g002 patient were detected (range 0–6) and in the acute patients, we saw an average of 0.8
CD4+ responses (range 0–3). Overall, 84% of the 135 overlapping SARS-CoV-2 peptides (116/135) elicited at least one
virus-specific CD4+ T cell response with 23.2 CD4+ T cell responses/ per resolved patient
(total number of 557 CD4+ T cell responses) and 18.2 CD4+ T cell responses /per acute patient
(total number of 182 CD4+ T cell responses) (Fig 4). Overall, 84% of the 135 overlapping SARS-CoV-2 peptides (116/135) elicited at least one
virus-specific CD4+ T cell response with 23.2 CD4+ T cell responses/ per resolved patient
(total number of 557 CD4+ T cell responses) and 18.2 CD4+ T cell responses /per acute patient
(total number of 182 CD4+ T cell responses) (Fig 4). Fig 3. Overview of the SARS-CoV-2 membrane protein-specific CD4+ T cell responses in patients with acute (n = 10) and resolved (n = 24) COVID-19. For each patient, the HLA class II molecules DRB1 and DQB1 are listed. aCov = acute COVID-19, rCov = resolved COVID-19. = patient was treated with
immunosuppressant medication or received chemotherapy. Fig 3. Overview of the SARS-CoV-2 membrane protein-specific CD4+ T cell responses in patients with acute (n = 10) and resolved (n = 24) COVID-19. For each patient, the HLA class II molecules DRB1 and DQB1 are listed. aCov = acute COVID-19, rCov = resolved COVID-19. = patient was treated with
immunosuppressant medication or received chemotherapy. Fig 3. Overview of the SARS-CoV-2 membrane protein-specific CD4+ T cell responses in patients with acute (n = 10) and resolved (n = 24) COVID-19. For each patient, the HLA class II molecules DRB1 and DQB1 are listed. aCov = acute COVID-19, rCov = resolved COVID-19. = patient was treated with
immunosuppressant medication or received chemotherapy. https://doi.org/10.1371/journal.ppat.1009842.g003 https://doi.org/10.1371/journal.ppat.1009842.g003 6 / 24 PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1009842
September 16, 2021 PLOS PATHOGENS SARS-CoV-2 N, M, and E-specific CD4+ T cells Fig 4. Distribution of CD4+ T cell responses against the nucleo, membrane and envelope-protein. (A) Overview of the nucleoprotein-specific CD4+ T cell
response against 82 single nucleoprotein peptides. A total number of 30 patients were tested. (B) Overview of the SARS-CoV-2 membrane protein-specific CD4+ T
cell response against 43 single membrane peptides. PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1009842
September 16, 2021 High response rate and broadly directed N- and M-specific T cell responses A total number of 34 patients were tested. (C) Overview of the SARS-CoV-2 envelope protein-specific CD4+ T
cell response against 13 single envelope peptides. A total number of 34 patients were tested. Response frequency (RF) was calculated by dividing the number of
patients that had a specific T cell response against a single peptide by the total number of patients who were tested. PLOS PATHOGENS
SARS CoV 2 N, M, and E specific CD4+ T cells Fig 4. Distribution of CD4+ T cell responses against the nucleo, membrane and envelope-protein. (A) Overview of the nucleoprotein-specific CD4+ T cell
response against 82 single nucleoprotein peptides. A total number of 30 patients were tested. (B) Overview of the SARS-CoV-2 membrane protein-specific CD4+ T
cell response against 43 single membrane peptides. A total number of 34 patients were tested. (C) Overview of the SARS-CoV-2 envelope protein-specific CD4+ T
cell response against 13 single envelope peptides. A total number of 34 patients were tested. Response frequency (RF) was calculated by dividing the number of
patients that had a specific T cell response against a single peptide by the total number of patients who were tested. https://doi.org/10.1371/journal.ppat.1009842.g004 https://doi.org/10.1371/journal.ppat.1009842.g004 In S2 Fig, all of the detected peptide-specific CD4+ T cell responses of patient rCov-17 are
shown. This patient with resolved COVID-19 infection (129 days after first positive PCR)
showed 26 CD4+ T cell responses against the N protein, 18 against the M protein and five
against the E protein. All but one patient responded to at least one peptide located within the N, M or E antigen
(97%, 33/34). Patient aCov-07 who did not show a T cell response had an acute asymptomatic
infection, was treated for a high-grade sarcoma with Doxorubicin/Ifosphamid, and was tested
early into the infection (two days after the first positive PCR test). In our healthy control group (n = 12), five subjects showed a N, M or E-specific CD4+ T
cell response (42%) (S4 Table) with a mean of 1.4 CD4+ T cell responses per healthy control
(range 0–9). Of note, on average these responses had a lower magnitude than peptide-specific
T cell responses measured in the patient cohort. Correlation of the virus-specific CD4+ T cell response pattern with patient
characteristics, clinical course and previous exposure to commonly
circulating coronaviruses (CCC) It should be noted that in contrast to previous studies, no significant correlation was found
between age or gender and the N, M and E-specific CD4+ T cell response using our assays
(S3E Fig). Similarly, no correlation was found between acute and convalescence status of the
infection (Fig 1B) or the time after infection and the breadth or pattern of the virus-specific
CD4+ T cell response (S3A Fig). The disease severity (mild/moderate against severe/critical) Table 2. Overview of the most frequently detected peptides of the SARS-CoV-2 nucleoprotein by CD4+ T cells in the current study. Peptides detected in the current
study with a response frequency (RF) 20%. Red and bold font indicates the optimal epitope length validated by truncation experiments for the peptides Mem_P30,
Mem_P36 and Ncl_P18. High response rate and broadly directed N- and M-specific T cell responses 7 / 24 PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1009842
September 16, 2021 PLOS PATHOGENS SARS-CoV-2 N, M, and E-specific CD4+ T cells The response frequency (RF), meaning the number of CD4+ T cell responses divided by
the number of patients tested, was assessed for every peptide of the N, M and E protein (Tables
2–4). Tables 2–4 show all peptides with a RF above 20% in this study. The two most frequently
detected peptides are Mem_P30 and Mem_P36, both located in the endodomain of the M pro-
tein [4]. 26 patients (RF: 72.2%; 26/34) showed a CD4+ T cell response against Mem_P30
(aa146-160), and 24 patients responded to Mem_P36 (aa176-190) (RF: 66.7%, 14/34). Within
the N protein, Ncl_P17 (aa81-95) and Ncl_P18 (aa86-100) showed the highest RF of 56%. These two peptides are located within the N-terminal domain of the protein [24]. A number of
epitopes described in the current study, have previously been reported (Table 5) [15, 25–28]. The RF from the respective previous reports as well as the HLA restriction are shown. From
the peptides listed in Tables 2–4, eleven peptides have not been published before according to
the IEDB database (www.iedb.org), including peptide Mem_P02, Ncl_P24, Ncl_P34, Ncl_P10,
Ncl_P09, Ncl_P23, Ncl_P35, Ncl_P08, Ncl_P40, Ncl_P39, Env_P11. While the in vitro cultivation of PBMC with 15-mer peptides is potentially biased towards
the enrichment of antigen-specific CD4+ T cells, we also analyzed the peptide specific CD8+ T
cell response (S5 Table). 70% of the acute patients (7/10) and 79% of the resolved patients (19/
24) showed a N, M or E-specific CD8+ T cell response in this study. The highest number of
CD8+ T cell responses were directed against peptide Mem_P30 with a total number of nine
responses (RF: 26.5%). Most CD8+ T cell responses coincided with a parallel SARS-CoV-
2-specific CD4+ T cell response and the average magnitude of these CD8+ T cell responses
was smaller compared to the average peptide specific CD4+ T cell response. Correlation of the virus-specific CD4+ T cell response pattern with patient
characteristics, clinical course and previous exposure to commonly
circulating coronaviruses (CCC) Most frequently detected peptides of the Nucleoprotein
Peptide
aa position
Sequence
RF
Peptide
aa position
Sequence
RF
Ncl_P17
81–95
D
D
Q
I
G
Y
Y
R
R
A
T
R
R
I
R
56%
Ncl_P58
286–300
F
G
D
Q
E
L
I
R
Q
G
T
D
Y
K
H
34%
Ncl_P18
86–100
Y
Y
R
R
A
T
R
R
I
R
G
G
D
G
K
56%
Ncl_P10
46–60
P
N
N
T
A
S
W
F
T
A
L
T
Q
H
G
31%
Ncl_P70
346–360
F
K
D
Q
V
I
L
L
N
K
H
I
D
A
Y
53%
Ncl_P03
11–25
N
A
P
R
I
T
F
G
G
P
S
D
S
T
G
28%
Ncl_P26
126–140
N
K
D
G
I
I
W
V
A
T
E
G
A
L
N
50%
Ncl_P09
41–55
R
P
Q
G
L
P
N
N
T
A
S
W
F
T
A
28%
Ncl_P11
51–65
S
W
F
T
A
L
T
Q
H
G
K
E
D
L
K
47%
Ncl_P23
111–125
Y
Y
L
G
T
G
P
E
A
G
L
P
Y
G
A
28%
Ncl_P71
351–365
I
L
L
N
K
H
I
D
A
Y
K
T
F
P
P
47%
Ncl_P35
171–185
F
Y
A
E
G
S
R
G
G
S
Q
A
S
S
R
28%
Ncl_P44
216–230
D
A
A
L
A
L
L
L
L
D
R
L
N
Q
L
44%
Ncl_P59
291–305
L
I
R
Q
G
T
D
Y
K
H
W
P
Q
I
A
28%
Ncl_P45
221–235
L
L
L
L
D
R
L
N
Q
L
E
S
K
M
S
44%
Ncl_P65
321–335
G
M
E
V
T
P
S
G
T
W
L
T
Y
T
G
28%
Ncl_P54
266–280
K
A
Y
N
V
T
Q
A
F
G
R
R
G
P
E
44%
Ncl_P08
36–50
R
S
K
Q
R
R
P
Q
G
L
P
N
N
T
A
25%
Ncl_P25
121–135
L
P
Y
G
A
N
K
D
G
I
I
W
V
A
T
38%
Ncl_P27
131–145
I
W
V
A
T
E
G
A
L
N
T
P
K
D
H
25%
Ncl_P22
106–120
P
R
W
Y
F
Y
Y
L
G
T
G
P
E
A
G
34%
Ncl_P40
196–210
N
S
T
P
G
S
S
K
R
T
S
P
A
R
M
25%
Ncl_P24
116–130
G
P
E
A
G
L
P
Y
G
A
N
K
D
G
I
34%
Ncl_P66
326–340
P
S
G
T
W
L
T
Y
T
G
A
I
K
L
D
25%
Ncl_P34
166–180
T
L
P
K
G
F
Y
A
E
G
S
R
G
G
S
34%
Ncl_P78
386–400
Q
K
K
Q
Q
T
V
T
L
L
P
A
A
D
L
25%
Ncl_P53
261–275
K
R
T
A
T
K
A
Y
N
V
T
Q
A
F
G
34%
Ncl_P20
96–110
G
G
D
G
K
M
K
D
L
S
P
R
W
Y
F
22%
https://doi.org/10.1371/journal.ppat.1009842.t002 ly detected peptides of the SARS-CoV-2 nucleoprotein by CD4+ T cells in the current study. Correlation of the virus-specific CD4+ T cell response pattern with patient
characteristics, clinical course and previous exposure to commonly
circulating coronaviruses (CCC) Peptides detected in the curre
0%. Red and bold font indicates the optimal epitope length validated by truncation experiments for the peptides Mem_P30, erview of the most frequently detected peptides of the SARS-CoV-2 nucleoprotein by CD4+ T cells in the current study. Peptides detected in the current
response frequency (RF) 20%. Red and bold font indicates the optimal epitope length validated by truncation experiments for the peptides Mem_P30, Table 2. Overview of the most frequently detected peptides of the SARS-CoV-2 nucleoprotein by CD4+ T cells in the current study. Peptides detected in the current
study with a response frequency (RF) 20%. Red and bold font indicates the optimal epitope length validated by truncation experiments for the peptides Mem_P30,
Mem_P36 and Ncl_P18. Table 2. Overview of the most frequently detected peptides of the SARS-CoV-2 nucleoprotein by CD4+ T cells in the current study. Peptides detected in the current
study with a response frequency (RF) 20%. Red and bold font indicates the optimal epitope length validated by truncation experiments for the peptides Mem_P30,
Mem_P36 and Ncl_P18. 8 / 24 PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1009842
September 16, 2021 PLOS PATHOGENS SARS-CoV-2 N, M, and E-specific CD4+ T cells Table 3. Overview of the most frequently detected peptides of the SARS-CoV-2 membrane protein by CD4+ T
cells in the current study. Peptides detected in the current study with a response frequency (RF) 20%. Red and bold
font indicates the optimal epitope length validated by truncation experiments for the peptides Mem_P30, Mem_P36
and Ncl_P18. Table 3. Overview of the most frequently detected peptides of the SARS-CoV-2 membrane protein by CD4+ T
cells in the current study. Peptides detected in the current study with a response frequency (RF) 20%. Red and bold
font indicates the optimal epitope length validated by truncation experiments for the peptides Mem_P30, Mem_P36
and Ncl_P18. Correlation of the virus-specific CD4+ T cell response pattern with patient
characteristics, clinical course and previous exposure to commonly
circulating coronaviruses (CCC) Most frequently detected peptides of the Membrane protein
Peptide
aa position
Sequence
RF
Mem_P30
146–160
R
G
H
L
R
I
A
G
H
H
L
G
R
C
D
72%
Mem_P36
176–190
L
S
Y
Y
K
L
G
A
S
Q
R
V
A
G
D
67%
Mem_P34
166–180
K
E
I
T
V
A
T
S
R
T
L
S
Y
Y
K
42%
Mem_P29
141–155
G
A
V
I
L
R
G
H
L
R
I
A
G
H
H
33%
Mem_P33
161–175
I
K
D
L
P
K
E
I
T
V
A
T
S
R
T
28%
Mem_P35
171–185
A
T
S
R
T
L
S
Y
Y
K
L
G
A
S
Q
28%
Mem_P41
201–215
I
G
N
Y
K
L
N
T
D
H
S
S
S
S
D
28%
Mem_P03
11–25
E
E
L
K
K
L
L
E
Q
W
N
L
V
I
G
25%
Mem_P14
66–80
V
L
A
A
V
Y
R
I
N
W
I
T
G
G
I
25%
Mem_P02
6–20
G
T
I
T
V
E
E
L
K
K
L
L
E
Q
W
22%
Mem_P28
136–150
S
E
L
V
I
G
A
V
I
L
R
G
H
L
R
22%
Mem_P40
196–210
Y
S
R
Y
R
I
G
N
Y
K
L
N
T
D
H
22%
https://doi.org/10.1371/journal.ppat.1009842.t003 Most frequently detected peptides of the Membrane protein https://doi.org/10.1371/journal.ppat.1009842.t003 in this small cohort also did not correlate with the breadth of the CD4+ T cell response in this
study (S3B Fig). With the exception of patients aCov-07 (see above), the four other immuno-
suppressed patients including patient rCov-12 and rCov-16, who were both B cell-depleted,
showed a comparable number of T cell responses (Fig 1B) [30]. in this small cohort also did not correlate with the breadth of the CD4+ T cell response in this
study (S3B Fig). With the exception of patients aCov-07 (see above), the four other immuno-
suppressed patients including patient rCov-12 and rCov-16, who were both B cell-depleted,
showed a comparable number of T cell responses (Fig 1B) [30]. The aim of this study was to define dominant SARS-CoV-2-specific CD4+ T cell epitopes
located within the N, M and E protein. Larger studies are necessary to investigate correlations
between the clinical status and the SARS-CoV-2-specific T cell response. Fine mapping and restriction experiments The optimal length of the epitopes Mem_P30 (aa146-160), Mem_P36 (aa176-190) and
Ncl_P18 (aa86-100) was assessed by additional experiments using peptide truncations. PBMC
of patients aCov-03 and rCov-17, who had shown a strong CD4+ T cell response against
Mem_P30, Mem_P36 and Ncl_P18 were cultivated with the respective peptides and their cor-
responding set of truncations. For the truncations, peptides were synthesized either shortened
at the N- or the C-terminus by two or four amino acids, respectively (Fig 5A). Both patients
showed a similar distribution of IFN-y responses across all truncations and both patients
expressed HLA-DRB111:01, which is likely one of the restricting HLA molecule for all three
peptides. This was supported by the in vitro data (Tables 6 and 7) that showed binding to
HLA-DRB111:01 with high affinity of all three peptides Mem_P30, Mem_P36 and Ncl_P18. Interestingly, healthy control HC-06 responded to Ncl_P17 and Ncl_P18 and also expressed
HLA-DRB111:01. To confirm restriction of Mem_P30, Mem_P36 and Ncl_P18, we used
PBMC as antigen presenting cells (APC) from healthy donors who expressed the respective
HLA-molecule (Fig 5B). Original FACS-plots are shown in S4 Fig. Mem_P30, and Ncl_P18
were most pronouncedly presented by matched and peptide-pulsed PBMC expressing
HLA-DRB111:01, and Mem_P36 by PBMC expressing DRB101:01. The in vitro HLA class II binding data (Tables 6 and 7) indicated that the peptides
Env_P11, Mem_P30, Mem_P34, Mem_P36 and Ncl_P70 peptides that were most frequently
recognized in this study (RF range: 36.1–72.2%) had the capacity to bind 12 or more of the 22
HLA-molecules tested with an affinity of 1000 nM or better. y
We then compared the pattern of in vitro HLA binding with the HLA-molecules expressed
by the patients from this study. The most likely binding HLA-DRB1 molecules are shown in
S8 Table. The Mem_P30 peptide was consistently recognized by all five patients expressing
HLA molecule DRB107:01 and all six expressing DRB111:01 (S8 Table). Correspondingly,
the in vitro binding data revealed that the peptide bound both molecules with high affinity
(132 nM and 34 nM, respectively). Furthermore, Mem_P30 is also likely restricted by
DRB104:01, given that it binds the molecule with an affinity of 998nM, and all three
DRB104:01 positive patients responded to this peptide. The Mem_P36 peptide was found to bind DRB101:01 with very high affinity (0.83 nM)
and elicited a CD4+ T cell response in three out of four DRB101:01 patients. Correlation of the virus-specific CD4+ T cell response pattern with patient
characteristics, clinical course and previous exposure to commonly
circulating coronaviruses (CCC) To investigate the possibility of cross-reactivity with CCC, we analyzed sequence homology
of the most frequently detected SARS-CoV-2 epitopes in this study with the CCC 229E,
HKU1, OC43, and NL63 (S6 Table) [27]. Overall, sequence homology was low to intermediate
(range 27–87%) with the highest sequence homology located in Ncl_P22 (range: 60–87%). Interestingly, this peptide was detected by two of our healthy controls (HC-06, HC-12). To investigate past exposure to CCC, we measured IgG antibody responses against 229E,
HKU1, OC43, and NL63 and SARS-CoV-2 in the frozen plasma from 30 patients in our cohort
as well as four healthy controls using a commercial recomLine SARS-CoV-2 IgG Immunoblot
assay (S7 Table). All healthy controls were seronegative for SARS-CoV-2 antibodies. In our
patient cohort five out of ten (50%) acutely infected patients had detectable IgG antibodies,
whereas in the resolved patient group all but one patient showed antibody responses (95%). The one patient who did not show an antibody response was patient rCov-12 who was B-cell
depleted due to Rituximab treatment. The prevalence of antibodies to CCC or SARS-CoV-2
did not influence the breadth of antigen-specific CD4+ T cell responses detected in this study
(S3C and S3D Fig). Table 4. Overview of the most frequently detected peptides of the SARS-CoV-2 envelope protein by CD4+ T cells
in the current study. Peptides detected in the current study with a response frequency (RF) 20%. Most frequently detected peptides of the Envelope antigen
Peptide
aa position
Sequence
RF
Env_P12
56–70
F
Y
V
Y
S
R
V
K
N
L
N
S
S
R
V
36%
Env_P11
51–65
L
V
K
P
S
F
Y
V
Y
S
R
V
K
N
L
33%
Env_P06
26–40
F
L
L
V
T
L
A
I
L
T
A
L
R
L
C
22%
https://doi.org/10.1371/journal.ppat.1009842.t004 Table 4. Overview of the most frequently detected peptides of the SARS-CoV-2 envelope protein by CD4+ T cells
in the current study. Peptides detected in the current study with a response frequency (RF) 20%. PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1009842
September 16, 2021 9 / 24 PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1009842
September 16, 2021 PLOS PATHOGENS SARS-CoV-2 N, M, and E-specific CD4+ T cells Fine mapping and restriction experiments Similarly,
Mem_P36 is likely restricted by DRB104:01, 07:01, 11:01 and 15:01 (S8 Table). The Ncl_17 and Ncl_18 peptides, which where both recognized by all six patients with
DRB111:01, bound DRB111:01 with very high affinities of 20 and 71 nM, respectively
(Tables 7 and S8). These in vitro data are in concordance with the restriction data that was
previously published (Table 5). A number of directly neighboring peptides elicited a similar CD4+ T cell response in indi-
vidual patients, for example in Ncl_P17 and P18 (Fig 4). Truncation 3 of Ncl_P18 is shortened
towards Ncl_P17 and did not elicit a CD4+ T cell response (Fig 5A). This indicates that the
epitope is most likely located between the overlapping peptides Ncl_P17 and P18. PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1009842
September 16, 2021 Ex vivo CD4+ T cell phenotype We stimulated PBMC from six acute COVID-19 patients with the ten most frequently detected
peptides (listed in Table 5), in order to investigate the frequency and functionality of the ex
vivo SARS-CoV-2-specific CD4+ T cell response. The flow cytometry panel is shown in S9
Table. The ex vivo cytokine response was measured by ICS and the results are shown in S5A
and S5B Fig. Due to their relatively low frequency, SARS-CoV-2-specific CD4+ T cells were
difficult to assess ex vivo. However, three of six acute patients showed an IFN-y response above 10 / 24 PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1009842
September 16, 2021 PLOS PATHOGENS SARS-CoV-2 N, M, and E-specific CD4+ T cells Table 5. Overview of the most frequently detected peptides of the SARS-CoV-2 nucleo, membrane and envelope-protein by CD4+ T cells in previously published studies. Ex vivo CD4+ T cell phenotype Protein
aa
position
Sequence
RF in
previous
study
Peptide in
this study
HLA restriction
Reference
Membrane
protein
145–160
R
G
H
L
R
I
A
G
H
H
L
G
R
C
D
46%
Mem_P30
DRB115:01
DRB114:01
DRB113:01
DRB1 07:01
[25, 29]
175–190
L
S
Y
Y
K
L
G
A
S
Q
R
V
A
G
D
56%
Mem_P36
DQB103:01
DRB116:02
DRB116:01
DQB106:03
DQB106:02
DRB115:01
DRB101:01
DRB114:06
DRB107:01
[25, 26,
28]
Nucleo-
protein
50–65
S
W
F
T
A
L
T
Q
H
G
K
E
D
L
K
20%
Ncl_P11
not defined
[25, 29]
80–95
D
D
Q
I
G
Y
Y
R
R
A
T
R
R
I
R
6%
Ncl_P17
DRB113:01
DRB115:01
DRB114:06
DRB114:01
DRB116:02
[25, 27,
29]
85–100
Y
Y
R
R
A
T
R
R
I
R
G
G
D
G
K
23%
Ncl_P18
DRB113:01
DRB114:06
[25]
125–140
N
K
D
G
I
I
W
V
A
T
E
G
A
L
N
29%
Ncl_P26
DQB102:01
DRB107:01
DRB104:04
DQB105:03
DQB102:02
DQB103:02
[25]
215–230
D
A
A
L
A
L
L
L
L
D
R
L
N
Q
L
14%
Ncl_P44
DQB105:03
DRB114:01
DQB106:02
DRB103:01
DRB115:01
DRB112:01
[25, 27]
345–360
F
K
D
Q
V
I
L
L
N
K
H
I
D
A
Y
15%
Ncl_P70
DRB116:02
DRB115:01
DRB114:06
DRB114:01
DRB103:01
[25]
350–365
I
L
L
N
K
H
I
D
A
Y
K
T
F
P
P
24%
Ncl_P71
DRB114:06
DRB115:01
[25]
Envelope
protein
55–70
F
Y
V
Y
S
R
V
K
N
L
N
S
S
R
V
31%
Env_P12
not defined
[26]
1371/journal.ppat.1009842
September 16, 2021 Table 5. Overview of the most frequently detected peptides of the SARS-CoV-2 nucleo, membrane and envelope-protein by CD4+ T cells in previously published studies. PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1009842
September 16, 2021 Ex vivo CD4+ T cell phenotype In vitro binding capacity of 14 SARS-CoV-2-specific peptides to 22 frequent HLA class II MHC molecules. Binding capacities are expressed as IC50 nM val-
ues measured in classical in vitro binding assays based on inhibition of binding of a high affinity radiolabeled ligand to purified HLA molecules. High affinity binding is
defined as IC50 < 1,000 nM and highlighted by bold font. For reasons of comprehensibility, values larger than 40,000 nM are indicated by a dash. The total number of
alleles bound as well as the response frequency (RF) of the responding peptide is shown. Mem_P30 and Mem_P36, with the highest RFs, bound to 12 and 13 HLA mole-
cules respectively Table 6. In vitro binding capacity of 14 SARS-CoV-2-specific peptides to 22 frequent HLA class II MHC molecules. Binding capacities are expressed as IC50 nM val-
ues measured in classical in vitro binding assays based on inhibition of binding of a high affinity radiolabeled ligand to purified HLA molecules. High affinity binding is
defined as IC50 < 1,000 nM and highlighted by bold font. For reasons of comprehensibility, values larger than 40,000 nM are indicated by a dash. The total number of
alleles bound as well as the response frequency (RF) of the responding peptide is shown. Ex vivo CD4+ T cell phenotype Protein
aa
position
Sequence
RF in
previous
study
Peptide in
this study
HLA restriction
Reference
Membrane
protein
145–160
R
G
H
L
R
I
A
G
H
H
L
G
R
C
D
46%
Mem_P30
DRB115:01
DRB114:01
DRB113:01
DRB1 07:01
[25, 29]
175–190
L
S
Y
Y
K
L
G
A
S
Q
R
V
A
G
D
56%
Mem_P36
DQB103:01
DRB116:02
DRB116:01
DQB106:03
DQB106:02
DRB115:01
DRB101:01
DRB114:06
DRB107:01
[25, 26,
28]
Nucleo-
protein
50–65
S
W
F
T
A
L
T
Q
H
G
K
E
D
L
K
20%
Ncl_P11
not defined
[25, 29]
80–95
D
D
Q
I
G
Y
Y
R
R
A
T
R
R
I
R
6%
Ncl_P17
DRB113:01
DRB115:01
DRB114:06
DRB114:01
DRB116:02
[25, 27,
29]
85–100
Y
Y
R
R
A
T
R
R
I
R
G
G
D
G
K
23%
Ncl_P18
DRB113:01
DRB114:06
[25]
125–140
N
K
D
G
I
I
W
V
A
T
E
G
A
L
N
29%
Ncl_P26
DQB102:01
DRB107:01
DRB104:04
DQB105:03
DQB102:02
DQB103:02
[25]
215–230
D
A
A
L
A
L
L
L
L
D
R
L
N
Q
L
14%
Ncl_P44
DQB105:03
DRB114:01
DQB106:02
DRB103:01
DRB115:01
DRB112:01
[25, 27]
345–360
F
K
D
Q
V
I
L
L
N
K
H
I
D
A
Y
15%
Ncl_P70
DRB116:02
DRB115:01
DRB114:06
DRB114:01
DRB103:01
[25]
350–365
I
L
L
N
K
H
I
D
A
Y
K
T
F
P
P
24%
Ncl_P71
DRB114:06
DRB115:01
[25]
Envelope
protein
55–70
F
Y
V
Y
S
R
V
K
N
L
N
S
S
R
V
31%
Env_P12
not defined
[26]
https://doi.org/10.1371/journal.ppat.1009842.t005 11 / 24 PLOS PATHOGENS SARS-CoV-2 N, M, and E-specific CD4+ T cells Fig 5. Fine mapping of the most frequently detected peptides Mem_P30 (aa146-160), Mem_P36 (aa176-190) and Ncl_P18 (aa86-100). (A) Truncation
experiments of Mem_P30, Mem_P36 and Ncl_P18 in patients aCov-03 and rCov-17. For Mem_P30 one variant that contained the amino acid H instead of R was
also tested. (B) Restriction experiments of Mem_P30, Mem_P36 and Ncl_P18 in patients aCov-03 and rCov-17. PBMC from matched donors were used as antigen-
presenting cells. Autologous cells served as a positive control. Most likely HLA-restriction is indicated by bold font. https://doi.org/10.1371/journal.ppat.1009842.g005 Fig 5. Fine mapping of the most frequently detected peptides Mem_P30 (aa146-160), Mem_P36 (aa176-190) and Ncl_P18 (aa86-100). (A) Truncation
experiments of Mem_P30, Mem_P36 and Ncl_P18 in patients aCov-03 and rCov-17. Ex vivo CD4+ T cell phenotype For Mem_P30 one variant that contained the amino acid H instead of R was
also tested. (B) Restriction experiments of Mem_P30, Mem_P36 and Ncl_P18 in patients aCov-03 and rCov-17. PBMC from matched donors were used as antigen-
presenting cells. Autologous cells served as a positive control. Most likely HLA-restriction is indicated by bold font. https://doi.org/10.1371/journal.ppat.1009842.g005 Fig 5. Fine mapping of the most frequently detected peptides Mem_P30 (aa146-160), Mem_P36 (aa176-190) and Ncl_P18 (aa86-100). (A) Truncation
experiments of Mem_P30, Mem_P36 and Ncl_P18 in patients aCov-03 and rCov-17. For Mem_P30 one variant that contained the amino acid H instead of R was
also tested. (B) Restriction experiments of Mem_P30, Mem_P36 and Ncl_P18 in patients aCov-03 and rCov-17. PBMC from matched donors were used as antigen-
presenting cells. Autologous cells served as a positive control. Most likely HLA-restriction is indicated by bold font. https://doi.org/10.1371/journal.ppat.1009842.g005 https://doi.org/10.1371/journal.ppat.1009842.g005 https://doi.org/10.1371/journal.ppat.1009842.g005 the detection level (detection level: 0.02% of all CD4+ T cells for IFN-y, Il-2, and TNF-a). The
responses ranged from 0% - 0.1% (IFN-y), 0.01% - 0.21% (TNF-α) and 0% - 0.08% (IL-2) of
CD4+ T cells. CD4+ T cells predominantly produced TNF-α and the frequencies of multifunc-
tional IFN-y+TNFα+IL-2+ CD4+ T cells was low. No Th2 cytokines such as IL-4 were
assessed. PBMC of two resolved COVID-19 patients were also stimulated with the SARS-CoV-
2 peptide pool but no ex vivo CD4+ T cell response could be detected. Based on our experimental epitope truncation and restriction data (Fig 5), we were able to
develop an M-specific DRB11-restricted MHC class II tetramer containing the 20-mer pep-
tide sequence Mem_aa145-164 (LRGHLRIAGHHLGRCDIKDL) containing Mem_P30, the
most frequently detected peptide in this study. We performed an ex vivo staining of PBMC Table 6. In vitro binding capacity of 14 SARS-CoV-2-specific peptides to 22 frequent HLA class II MHC molecules. Binding capacities are expressed as IC50 nM val-
ues measured in classical in vitro binding assays based on inhibition of binding of a high affinity radiolabeled ligand to purified HLA molecules. High affinity binding is
defined as IC50 < 1,000 nM and highlighted by bold font. For reasons of comprehensibility, values larger than 40,000 nM are indicated by a dash. The total number of
alleles bound as well as the response frequency (RF) of the responding peptide is shown. Mem_P30 and Mem_P36, with the highest RFs, bound to 12 and 13 HLA mole-
cules, respectively Table 6. Ex vivo CD4+ T cell phenotype Mem_P30 and Mem_P36, with the highest RFs, bound to 12 and 13 HLA mole-
l
i
l p
y
Peptide
aa position
Sequence
Alleles
bound
RF
DPB1
02:01
DPA101:03
DPB104:01
DQA105:01
DQB102:01
DQA105:01
DQB103:0
DQA103:01
DQB103:02
DQA101:01
DQB105:01
DQA101:02
DQB106:02
Env_11
46–60
L
V
K
P
S
F
Y
V
Y
S
R
V
K
N
L
13
36.1
42
63
-
5766
-
-
24387
Mem_30
146–160
R
G
H
L
R
I
A
G
H
H
L
G
R
C
D
12
72.2
569
445
-
15141
-
37659
-
Mem_34
166–180
K
E
I
T
V
A
T
S
R
T
L
S
Y
Y
K
13
41.7
1909
3337
13645
649
17391
-
1827
Mem_36
176–190
L
S
Y
Y
K
L
G
A
S
Q
R
V
A
G
D
13
66.7
13822
-
22448
48
-
15193
428
Ncl_11
51–65
S
W
F
T
A
L
T
Q
H
G
K
E
D
L
K
9
46.9
-
35760
30440
1304
19748
-
952
Ncl_17
81–95
D
D
Q
I
G
Y
Y
R
R
A
T
R
R
I
R
7
56.3
34655
-
-
5067
-
-
-
Ncl_18
86–100
Y
Y
R
R
A
T
R
R
I
R
G
G
D
G
K
6
56.3
-
-
-
6720
-
-
-
Ncl_22
106–120
P
R
W
Y
F
Y
Y
L
G
T
G
P
E
A
G
5
34.4
10053
-
79
1278
414
2239
7882
Ncl_25
121–135
L
P
Y
G
A
N
K
D
G
I
I
W
V
A
T
2
37.5
13013
20045
11706
14585
3795
-
203
Ncl_26
126–140
N
K
D
G
I
I
W
V
A
T
E
G
A
L
N
9
50
4140
8348
422
4027
103
-
2643
Ncl_44
216–230
D
A
A
L
A
L
L
L
L
D
R
L
N
Q
L
7
43.8
1162
2779
2487
22499
860
8458
2499
Ncl_45
221–235
L
L
L
L
D
R
L
N
Q
L
E
S
K
M
S
9
43.8
285
701
3315
-
11904
23089
8081
Ncl_54
266–280
K
A
Y
N
V
T
Q
A
F
G
R
R
G
P
E
9
43.8
8438
6056
-
794
33174
-
2936
Ncl_70
346–360
F
K
D
Q
V
I
L
L
N
K
H
I
D
A
Y
13
53.1
287
794
30656
33658
9176
6010
8602
https://doi.org/10.1371/journal.ppat.1009842.t006 12 / 24 PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1009842
September 16, 2021 PLOS PATHOGENS SARS-CoV-2 N, M, and E-specific CD4+ T cells Table 7. Ex vivo CD4+ T cell phenotype In vitro binding capacity of 14 SARS-CoV-2-specific peptides to 22 frequent HLA class II MHC molecules. Binding capacities are expressed as IC50 nM values measured in classical in
vitro binding assays based on inhibition of binding of a high affinity radiolabeled ligand to purified HLA molecules. High affinity binding is defined as IC50 < 1,000 nM and highlighted by bold font. For reasons of comprehensibility, values larger than 40,000 nM are indicated by a dash. The total number of alleles bound as well as the response frequency (RF) of the responding peptide is shown. Mem_P30 and Mem_P36, with the highest RFs, bound to 12 and 13 HLA molecules, respectively. PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1009842
September 16, 2021 Ex vivo CD4+ T cell phenotype Peptide
aa
position
Sequence
Alleles
bound
RF
DRB1
01:01
DRB1
03:01
DRB1
04:01
DRB1
04:05
DRB1
07:01
DRB1
08:02
DRB1
09:01
DRB1
11:01
DRB1
12:01
DRB1
13:02
DRB1
15:01
DRB3
01:01
DRB3
02:02
DRB4
01:01
DRB5
01:01
Env_11
46–60
L
V
K
P
S
F
Y
V
Y
S
R
V
K
N
L
13
36.1
13
-
869
470
5.3
27
52
14
1340
7253
78
104
160
38933
7.5
Mem_30
146–160
R
G
H
L
R
I
A
G
H
H
L
G
R
C
D
12
72.2
59
14579
998
7618
132
65
92
34
1232
900
55
1277
1458
159
24
Mem_34
166–180
K
E
I
T
V
A
T
S
R
T
L
S
Y
Y
K
13
41.7
9.3
416
341
3904
38
228
17
14
174
205
6.2
5959
18244
770
41
Mem_36
176–190
L
S
Y
Y
K
L
G
A
S
Q
R
V
A
G
D
13
66.7
0.83
28371
21
63
5.3
74
9
187
16415
2734
91
169
28
26279
1.2
Ncl_11
51–65
S
W
F
T
A
L
T
Q
H
G
K
E
D
L
K
9
46.9
40
-
192
875
715
795
445
493
7871
-
4938
-
5940
13917
13
Ncl_17
81–95
D
D
Q
I
G
Y
Y
R
R
A
T
R
R
I
R
7
56.3
43
-
8423
9123
111
17
565
20
-
-
340
1029
1023
-
2.7
Ncl_18
86–100
Y
Y
R
R
A
T
R
R
I
R
G
G
D
G
K
6
56.3
62
-
9044
13721
111
78
732
71
16525
-
1388
5089
8484
9570
16
Ncl_22
106–120
P
R
W
Y
F
Y
Y
L
G
T
G
P
E
A
G
5
34.4
8.5
-
3013
18
3633
1226
1131
3391
23302
-
775
2770
3172
-
1097
Ncl_25
121–135
L
P
Y
G
A
N
K
D
G
I
I
W
V
A
T
2
37.5
1310
39342
20706
7532
1475
10551
3830
13942
19091
-
937
13782
6365
10562
1667
Ncl_26
126–140
N
K
D
G
I
I
W
V
A
T
E
G
A
L
N
9
50
42
4705
184
3688
219
6498
141
9109
-
-
596
14419
2483
843
735
Ncl_44
216–230
D
A
A
L
A
L
L
L
L
D
R
L
N
Q
L
7
43.8
9037
2446
6091
840
-
1141
12530
317
824
486
7139
85
-
43
26925
Ncl_45
221–235
L
L
L
L
D
R
L
N
Q
L
E
S
K
M
S
9
43.8
95
1195
9776
1852
12332
591
8386
94
481
673
134
135
-
1702
6347
Ncl_54
266–280
K
A
Y
N
V
T
Q
A
F
G
R
R
G
P
E
9
43.8
12
-
647
11692
29
389
105
196
-
6728
988
7837
3663
2488
0.75
Ncl_70
346–360
F
K
D
Q
V
I
L
L
N
K
H
I
D
A
Y
13
53.1
42
6458
14275
1318
620
152
1559
96
89
187
68
309
205
719
168 13 / 24 PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1009842
September 16, 2021 PLOS PATHOGENS SARS-CoV-2 N, M, and E-specific CD4+ T cells Fig 6. Ex vivo CD4+ T cell phenotype The ex vivo peptide-specific CD4+ T cell response in patient rCov21. (A) Frequency of tetramer+CD4+ T cells ex vivo, after magnetic bead enrichment,
and after 16-day expansion with the corresponding peptide and IL-2. (B) Tetramer+CD4+ T cells differentiated to an effector and central memory type. (C)
Antigen-specific CD4+ T cells show an activated and exhausted phenotype. CM = central memory, EM = effector memory, EMRA = terminally differentiated
effector memory cells. Fig 6. The ex vivo peptide-specific CD4+ T cell response in patient rCov21. (A) Frequency of tetramer+CD4+ T cells ex vivo, after magnetic bead enrichment,
and after 16-day expansion with the corresponding peptide and IL-2. (B) Tetramer+CD4+ T cells differentiated to an effector and central memory type. (C)
Antigen-specific CD4+ T cells show an activated and exhausted phenotype. CM = central memory, EM = effector memory, EMRA = terminally differentiated
effector memory cells. https://doi.org/10.1371/journal.ppat.1009842.g006 https://doi.org/10.1371/journal.ppat.1009842.g006 from patient rCov21 (DRB107:01P, 11:01P) who had the corresponding HLA-type and who
had shown a CD4+ T cell response against Mem_P30 (Fig 6A). The patient had a severe course
of COVID-19, and the blood sample used for this MHC class II tetramer staining was collected
20 days after the first positive PCR. The antigen-specific tetramer+ CD4+ T cells were mainly
effector memory cells (EM) and showed upregulation of the activation molecules CD38 and
HLA-DR and low CD127 expression, as well as ubiquitous expression of PD-1 and slight upre-
gulation of LAG-3 and TIM-3 compared to bulk CD4+ cells (Fig 6B and 6C). PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1009842
September 16, 2021 Discussion We did not find any correlation between the virus-specific T cell response directed against
the different structural proteins and clinical parameters such as gender, age, or disease severity
(S3 Fig). Of note, our study was rather designed for epitope characterization than to detect dif-
ferences between immunological and clinical status. At the same rate, the T cell pattern of this
acute resolving viral infection is not as dichotomous as in a chronically evolving infection like
HCV and the universally broad T cell response seems to be a characteristic for a SARS-CoV-2
infection [20]. We detected few CD4+ T cell responses with low magnitude in five out of twelve healthy
controls. These responses that were measured after stimulation with SARS-Cov-2-specific pep-
tide pools might possibly stem from cross-reactive memory responses originally primed
against one or more of the CCC [37, 38]. The serological assessment of preexisting antibodies
against CCC revealed that the majority of the samples tested, regardless of the infection status,
had serologically been exposed to CCC (S7 Table). This did not seem to have influenced the
breadth of the antigen-specific CD4+ T cell response as we did not find differences in the
prevalence of preexisting antibodies and the number or distribution of SARS-CoV-2-specific
CD4+ T cell responses using our methodology. Further studies with higher numbers of unex-
posed healthy controls, testing individual SARS-CoV-2-specific peptides are necessary to
define cross-reactive immune epitopes [39]. Multiple SARS-CoV-2 variants are circulating globally [40], and the three variants Alpha
(first emerged in the UK), Beta (South Africa), Gamma (Brazil), Delta (India) were labeled by
the CDC as variants of concern (https://www.cdc.gov/coronavirus/2019-ncov/more/science-
and-research/scientific-brief-emerging-variants.html). Most mutations of these three variants
are located in the spike protein: The Gamma lineage for example contains three mutations in
the spike protein receptor binding domain: K417T, E484K, and N501Y. Furthermore, there is
evidence that the E484K mutation may affect neutralizing antibodies [41]. Interestingly, only
few mutations are located in the N, M or E antigen (S6 Fig). The Beta lineage for example only
shows one mutation in the E protein (P71L), and one in N (T205I), and Alpha and Gamma
variant only show one mutation in the N protein, respectively (S235F, P80R), whereas the
Delta variant contains three mutations in the N protein (D63G, R203M, D377Y) (https://cov-
lineages.org/). Discussion The main result of our study was the detection of a universally broad CD4+ T cell response
specific for the N, M and to a lesser degree E-protein, regardless of the clinical course. With
our sensitive in vitro testing approach, we were able to detect T cell responses in 33 of 34
patients (97%) and found 44 N, M and E-specific peptides with a RF of 20% or higher. One
must keep in mind that the three peptide sets only covered a small number of the structural
and non-structural proteins encoded by the SARS-CoV-2 genome [31]. To date, most studies have utilized pools of predicted peptides or pools of overlapping pep-
tides spanning entire antigens, to probe responses to different SARS-CoV-2 antigens [3, 7–9,
12–14]. However, the exact T cell epitopes and immunodominant antigen regions have only
been determined in few studies [15, 25–27]. Notably, these studies have overall reported a sim-
ilar response pattern (Table 5), suggesting a reproducibility despite differences in the patient
cohorts and methodologies. The peptides described in Tables 2–4 elicited a CD4+ T cell
response in 22–72% of our study group that had an HLA-background representative of the PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1009842
September 16, 2021 14 / 24 PLOS PATHOGENS SARS-CoV-2 N, M, and E-specific CD4+ T cells European population. On the other hand, several of the most prevalent alleles in the general
worldwide population including DRB103:01, DRB104:01, DRB107:01, DRB111:01 and
DRB115:01 [32], were also well represented in our cohort. However, similar studies should be
conducted in varying populations. The broad SARS-CoV-2-specific T cell response detected here, is seemingly primed at an
early timepoint of infection, since we detected no significant difference between the number
and magnitude of T cell responses in early acute vs. resolved infection (Fig 1B). In further longitudinal studies, it will be important to expand our understanding of the ex
vivo kinetics of T cell priming, the role of antigen-specific T cells for viral control, and the
potential role of the antiviral T cell response in the pathogenesis of the cytokine storm [33–35]. Furthermore, the tissue-resident virus-specific T cell response will be of high interest [36]. MHC class I and II multimer technology using epitopes located within the different SARS-
CoV-2 structural proteins will be another tool for these investigations [37]. PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1009842
September 16, 2021 Discussion It has already been reported that overlapping peptide pools of the M, N and E protein have
been shown to induce SARS-CoV-2-reactive T cell responses with a relative dominance of
CD4+ over CD8+ T cells [9, 13]. Most virus-specific CD8+ T cell responses detected in this
study were weak and coincided with a CD4+ T cell response directed against the same peptide
in the same patient. Accordingly, we did not follow-up on fine-mapping these subdominant
CD8+ T cell responses. Notably, the patients rCov-12 and rCov-16 were B cell-depleted but still exhibited a broad
range of T cell responses—the results of the T cell assays of one B cell depleted patient were
previously reported [30]. Despite a broad T cell response, patient rCov-16 showed a chronical
course of SARS-CoV-2 infection while other patients were able to clear the virus [44]. It will be
important to study the complex and complimentary roles of coordinated B and T cell
responses in establishing viral clearance [7, 9]. We also know from previous studies that anti-
body responses against coronaviruses can be short-lived [45, 46]. Coronavirus-induced cellular
immunity is predicted to be more sustained, and it would be highly interesting to further
investigate in how far the SARS-CoV-2 specific T cell response alone can confer immunity. Additionally, it will be important to understand if a broad natural T cell memory response will
render better protection than the vaccine-induced T cell response since most vaccines e.g. the
Moderna (mRNA-1273) or Pfizer-BioNTech (BNT162b2) vaccine are designed to prime neu-
tralizing antibodies against the binding region of the S protein[44]. In summary, we present a detailed immunological study of the SARS-CoV-2-specific T cell
response against the E, M and N protein on a single epitope level. PBMC of a well-character-
ized patient cohort with verified SARS-CoV-2 infection status were examined and stratified
into acute and resolved infection. A broadly directed SARS-CoV-2-specific CD4+ T cell
response was detectable in 97% of patients regardless of the clinical course. The results of this
study add to the body of literature that demonstrates a broad and functional T cell response in
most patients with COVID-19. While many peptides elicited a T cell response in one or more
patients, there were ten highly recognized peptides that were each recognized by more than a
third of patients. Discussion With the extensive breadth of the CD4+ T cell response in each individual
patient and low mutation rates of the N, M and E antigen, viral escape from the N, M or E-spe-
cific T cell response does not appear very likely [37]. However, the relative role of neutralizing
antibodies and T cell responses for protection against symptomatic reinfection has not been
defined for SARS-CoV-2. Of note, immunodominant regions priming virus-specific CD4+ T
cells were reported to have minimal overlap with antibody epitopes [25]. PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1009842
September 16, 2021 15 / 24 PLOS PATHOGENS SARS-CoV-2 N, M, and E-specific CD4+ T cells Previously, a direct role of CD4+ T cells for anti-viral immunity has been suggested in ani-
mal models [42, 43] and in humans [7, 11, 13]. With the generated data, we were able to estab-
lish a M-specific MHC II tetramer which allowed us to perform an ex vivo analysis from one
patient 20 days after diagnosis. We detected an ex vivo frequency of 0.8% of
LRGHLRIAGHHLGRCDIKDL-specific CD4+ T cells that showed upregulation of several
activation, and co-inhibitory molecules (Fig 6). With this MHC class II tetramer and further
SARS-CoV-2-specific MHC II tetramers that will be established in due time, it will be easier to
perform longitudinal and phenotypical analysis of antigen-specific CD4+ T cells during
SARS-CoV-2 infection and will allow live-sort of antigen-specific CD4+ T cells for further
functional assays. Previously, a direct role of CD4+ T cells for anti-viral immunity has been suggested in ani-
mal models [42, 43] and in humans [7, 11, 13]. With the generated data, we were able to estab-
lish a M-specific MHC II tetramer which allowed us to perform an ex vivo analysis from one
patient 20 days after diagnosis. We detected an ex vivo frequency of 0.8% of Using a peptide pool with the most commonly detected peptides from this study, we were
additionally able to detect virus-specific ex vivo T cell responses by IFN-y, TNF-α, and IL-2
cytokine staining (S5 Fig). Intriguingly, it has been reported that T cells with different SARS-
CoV-2 epitope specificities have different phenotypes and functionalities: whereas S-specific
CD4+ T cells were skewed towards a circulating T follicular helper profile, N and M-specific
CD4+ T cells showed a Th1 or a Th1/Th17 profile [13]. PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1009842
September 16, 2021 HLA typing High definition molecular HLA class I and II typing was available for 33 of 34 patients and was
performed at the Institute of Transfusion Medicine at the University Medical Center Ham-
burg-Eppendorf, by PCR-sequence specific oligonucleotide (PCR-SSO) using the commercial
kit SSO LabType as previously described (One Lambda, Canoga Park, CA, USA)[48]. E, M, and N protein peptides 15-mer peptides overlapping by ten amino acids corresponding to the complete E, M and N
protein amino sequences present in the first patient diagnosed in Hamburg [47] were synthe-
sized (peptides & elephants, Hennigsdorf, Germany). 43 peptides cover the M protein, 82 pep-
tides the N protein and 10 peptides the smaller E protein. The synthesis for the peptides
Env_P2, Env_P4, and Env_P5 failed, we therefore only tested 10 overlapping peptides span-
ning the E protein. All peptides were formulated into 13 pools of either 10 or 11 peptides (S2
Table). For in vitro culture peptide pools were used at a concentration of 1 μg/ml per single
peptide. For the enzyme linked immunospot assays (ELISpot) the final concentration of each
single peptide was 10 μg/ml. Further peptides for truncation experiments and variants were
also synthesized. Ethics statement The study was approved by the local ethics board of the A¨rztekammer Hamburg (PV4780,
PV7298) and written consent was obtained by all study participants. Patient cohort Peripheral blood mononuclear cells (PBMC) from SARS-CoV-2 infected patients (n = 34) and
uninfected healthy controls (n = 12) were collected at the University Medical Center Ham-
burg-Eppendorf. For investigation of ex vivo responses an additional eight patients with acute
and resolved COVID-19 were recruited (S1 Table). Antibody tests confirmed the absence of
SARS-CoV-2 antibodies in the uninfected healthy controls. Virus-specific in vitro cell cultures
were started with fresh, unfrozen PBMC. SARS-CoV-2 infection was verified by at least one
positive reverse transcription polymerase chain reaction (RT-PCR) of nasopharyngeal swab in
all patients as previously described [18]. Patient disease status was defined as recovered if the
first positive PCR was more than 40 days ago, the most recent PCR was negative, and symp-
toms were resolved. Acute infection was defined as acute symptoms compatible with COVID-
19 and/or first positive PCR less than 16 days ago. For patients with acute infections and those
with resolved infections, the disease severity, time since onset of symptoms, comorbidities,
and clinical outcome were assessed. For patients with acute infection additional clinical
parameters including white blood cell and lymphocyte count, c-reactive protein (CRP), hemo-
globin, platelet count, further T cell sub-classification as well as comorbidities were assessed at
the day of blood sampling for this study (+/- two days) (Tables 1 and S1). PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1009842
September 16, 2021 Discussion These detailed data on SARS-CoV-2-specific T cell epitopes will be helpful
for the development of tools like additional SARS-CoV-2-specific MHC class II multimers or
to monitor the immune response on an epitope level during future vaccine trials. 16 / 24 PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1009842
September 16, 2021 PLOS PATHOGENS SARS-CoV-2 N, M, and E-specific CD4+ T cells Intracellular cytokine staining and flow cytometry ICS was performed as previously described [20, 49]. 5 x 10^5 PBMC were stimulated with the
corresponding SARS-CoV-2 peptide at a final concentration of 10 μg/ml before blocking the
secretion with 5 μg/ml Brefeldin A (Sigma Aldrich) one hour after stimulation. Cells were then
incubated at 37˚C overnight and stained with the Zombie NIR Fixable Viability kit for live
cells as well as surface antibodies against anti-CD3 (clone: Okt3; AlexaFluor 700), anti-CD4
(clone: SK3; PerCP-Cy5.5) and anti-CD8 (clone: RPA-T8; Brilliant Violet 786) (all antibodies
by BioLegend). After fixation and permeabilization (eBioscience, Foxp3/Transcription Factor
Staining Buffer Set), cells were stained with anti-IFN-γ-antibodies (clone: 4S.B3; PE-Texas red;
BioLegend). Cells were then analyzed on a BD LSRFortessa (BD Biosciences). We defined a T
cell response as positive when the percentage of CD4+ T cells within the gate for IFN-γ was
three times higher than the negative control, above 0.02%, and if the population could be
clearly separated from the negative control [20, 49, 51]. R10 and DMSO were added to the neg-
ative control. Fig 1A shows an exemplary ICS result of an M-specific CD4+ T cell response. For ex vivo ICS, PBMC from COVID-19 patients were stimulated overnight with a peptide
pool of the ten most frequently detected N, M and E-specific peptides (Mem_P30, Mem_P36,
Ncl_P17, Ncl_P18, Ncl_P70, Ncl_P26, Ncl_P11, Ncl_P71, Ncl_P44, and Env_P12) at a final
concentration of 10 μg/ml before blocking the secretion with 5 μg/ml Brefeldin A (Sigma
Aldrich) one hour after stimulation. Cells were then stained for IFN-y (clone: 4S.B3; PE-Texas
red; BioLegend), TNFα (clone: Mab11; BV605; BioLegend) and IL-2 (clone: MQ1-17H12;
BUV737; BD Biosciences) as described above. The threshold for positivity for the cytokines
IFN-y, TNF-a and IL-2 was set at 0.02% of all CD4+ T cells. The antibody panel used for this
assay is shown in S9 Table. ELISpot assay ELISpot assays were performed as previously described [20, 22, 49]. Cultivated PBMC were
stimulated with the respective 10 or 11 peptides from the peptide pool. We used 30.000 cells
per well and responses were considered positive if the number of spots was at least three times
the number of spots in the negative control and at least a total number of 30 spots. Single pep-
tides were used at a concentration of 10 μg/ml. Anti-CD3-antibodies served as a positive con-
trol, R10 + DMSO as a negative control [50]. All positive responses were confirmed by ICS
assays following stimulation with the respective peptide. Bulk stimulation of peripheral blood mononuclear cells (PBMC) 30–50 x 10^6 fresh PBMC were divided in 13 wells with 1500 μl of R10 medium (RPMI 1640
medium with 10% FCS (Sigma Aldrich), 1% HEPES buffer and 1% Penicillin-Streptomycin). PBMC of each well were stimulated with one of the 13 peptide pools (S2 Table) at a final con-
centration of 10 μg/ml, together with 1 μg/ml of anti-CD28 and anti-CD49d antibodies (BD
FastImmune™, clone: L293 (CD28), clone: L25 (CD49d)) for 10 days. Medium with PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1009842
September 16, 2021 17 / 24 17 / 24 PLOS PATHOGENS SARS-CoV-2 N, M, and E-specific CD4+ T cells recombinant IL-2 (50 U/ml) was added when necessary. After 10 days, cells were re-stimulated
with single SARS-CoV-2-peptides (final concentration of 10 μg/ml) and then assayed for inter-
feron-γ (IFN-γ) production by ELISpot and intracellular cytokine staining (ICS) on day 11 as
previously described [20]. PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1009842
September 16, 2021 Statistical analysis All flow cytometric data were analyzed using FlowJo 10.5.0 software (Treestar, Ashland, OR,
USA). Statistical analyses were carried out using the Prism 7.0 software (GraphPad software,
San Diego, CA). Mann Whitney test or the Kruskal-Wallis test with Dunn´s post-test was per-
formed throughout all samples for inter-group comparisons. Spearman´s correlation was per-
formed for bivariate correlation analyses. Data are expressed as means with standard
deviations (SD) or with standard error of mean (SEM). P-values less than or equal to 0.05 were
considered significant. Tetramer staining The MHC class II tetramer used in this study is specific for a 20-mer peptide sequence (aa145-
164 LRGHLRIAGHHLGRCDIKDL) from the SARS-CoV-2 membrane protein restricted by
the MHC class II molecule DRB111:01. MHC class II tetramer enrichment was performed as
previously described [53]. In short, cryopreserved PBMC were thawed and stained with the
PE-labelled MHC class II tetramer. Tetramer enrichment was performed using MACS tech-
nology with anti-PE microbeads (Miltenyi Biotec, Germany) according to the manufacturer’s
protocol. Pre-, enriched, and depleted tetramer fractions were further analyzed by flow cytom-
etry using the BD LSRFortessa. The antibody panel can be found in S9 Table. HLA restriction and epitope fine mapping For epitope fine mapping experiments, PBMC were stimulated with SARS-CoV-2 peptides
and corresponding truncated peptides (final concentration of 10 μg/ml) in the presence of IL-
2. After 12 days, the cells were re-stimulated with the single peptides and truncations (final
concentration of 10 μg/ml) and stained for IFN-γ (ICS). For restriction experiments, the cells
were restimulated after T cell culture with PBMC (APC) from healthy, seronegative donors
loaded with the respective peptide. The healthy donors matched one HLA-DRB1 molecule of
the COVID-19 patients. PBMC from the healthy donors were incubated with the respective
peptide (concentration of 10 μg/ml) for 20 minutes and then washed six times. After re-stimu-
lation in the presence of 1:10 donor PBMC, ICS was performed to assess the IFN-y production PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1009842
September 16, 2021 18 / 24 PLOS PATHOGENS SARS-CoV-2 N, M, and E-specific CD4+ T cells as described above. PBMC from the matched donors served as a negative control, separately
peptide-loaded autologous PBMC from the respective patients served as a positive control. as described above. PBMC from the matched donors served as a negative control, separately
peptide-loaded autologous PBMC from the respective patients served as a positive control. In vitro binding assays with 14 of the peptides that elicited a N, M or E-specific CD4+ T cell
response were performed using purified HLA-DR molecules, as previously described [52]. as described above. PBMC from the matched donors served as a negative control, separately
peptide-loaded autologous PBMC from the respective patients served as a positive control. In vitro binding assays with 14 of the peptides that elicited a N M or E specific CD4+ T cell In vitro binding assays with 14 of the peptides that elicited a N, M or E-specific CD4+ T cell
response were performed using purified HLA-DR molecules, as previously described [52]. Antibody screening IgG antibodies specific to the RBD, the N protein as well as S1 of SARS-CoV-2 and the N pro-
tein of the endemic coronaviruses 229E, NL63, OC43 and HKU1 were measured utilizing the
commercially available recomLine SARS-CoV-2 IgG (Mikrogen Diagnostik, Neuried, Ger-
many) according to the manufacturer’s instructions. In short, 30 samples of acute and resolved
COVID-19 patients and four healthy donors were incubated with recombinant antigens of
SARS-CoV-2 and endemic coronaviruses. After a washing step, anti-human IgG conjugate
antibodies coupled to horseradish peroxidase were added and subsequently unbound conju-
gate antibodies were washed away. The color reaction catalyzed by the peroxidase was then
evaluated in a semiquantitative manner (ranging from “-“, indicating no reaction, to “+++”,
indicating a “very strong intensity”). The whole process from dilution of patient samples to
scanning of results was carried out on a CarL “complete automation of recomLine strip assays”
device (Mikrogen Diagnostik, Neuried, Germany). Supporting information S1 Table. Clinical and immunological patient characteristics. (XLSX) S1 Table. Clinical and immunological patient characteristics. (XLSX) S2 Table. SARS-CoV-2 peptide sequences of the envelope-, membrane-, and nucleoprotein. (XLSX) S2 Table. SARS-CoV-2 peptide sequences of the envelope-, membrane-, and nucleoprotein. (XLSX) S3 Table. Overview of SARS-CoV-2 envelope protein-specific CD4+ T cell responses in
patients with acute and resolved COVID-19. For each patient the HLA class I and II mole-
cules are listed. aCov = acute COVID-19 patient, rCov = resolved COVID-19 patient. = 19 / 24 PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1009842
September 16, 2021 PLOS PATHOGENS SARS-CoV-2 N, M, and E-specific CD4+ T cells patient was treated with immunosuppressant medication or received chemotherapy. (XLSX)
S4 Table. SARS-CoV-2 envelope, membrane and nucleoprotein-specific CD4+ and CD8
+ T cell responses in 12 healthy donors. (XLSX)
S5 Table. Overview of SARS-CoV-2 membrane and nucleoprotein-specific CD8+ T cell
responses in acute and resolved COVID-19 patients. For each patient, the HLA class I mole-
cules are listed. aCov = acute COVID-19 patient, rCov = resolved COVID-19 patient. =
patient was treated with immunosuppressant medication or received chemotherapy. (XLSX)
S6 Table. Sequence homology of CCC and the most frequently detected SARS-CoV-2 epi-
topes in this study. (XLSX)
S7 Table. Antibody prevalence to CCC as well as SARS-CoV-2. (XLSX)
S8 Table. (A) In vitro binding value and number of responding patients in this cohort. (B)
Most likely binding HLA-DRB1 molecules for SARS-CoV-2 peptides that elicited a CD4+ T
cell response. (XLSX)
S9 Table. Flow cytometry panels used for ex vivo ICS (A) and tetramer analysis (B). (XLSX)
S1 Fig. Ex vivo ELISpot of two recovered and one acute COVID-19 patient. (A) Ex vivo ELI-
Spot with PBMCs from two resolved and one acute COVID-19 patient stimulated with all 43
membrane peptides. The spots in the negative control ranged from 0 to 1 spot per 100.000
cells. (B) Ex vivo ELISpot with PBMCs from two resolved and one acute COVID-19 patients
stimulated with all 82 nucleoprotein peptides. The spots in the negative control ranged from 0
to 1 spot per 100.000 cells. (TIF)
S2 Fig. Breadth of the CD4+ T cell response against the SARS-CoV-2 envelope, membrane,
and nucleoprotein of patient rCov-17. All IFN-y+ CD4+ T cell responses of patient rCov-17
against the envelope, membrane, and nucleoprotein. Gated on CD4+ T cells. All cytokine
gates are set based on the respective negative control (R10 and DMSO). Supporting information (TIF)
S3 Fig. Breadth of the CD4+ T cell response in correlation with the time after infection (A),
disease severity (B), seroprevalence of IgG antibodies against SARS-CoV-2 (C) or CCC (D)
and gender (E). (TIF)
S4 Fig. Original FACS-plots of restriction assay. (TIF)
S5 Fig. Ex vivo ICS with PBMCs of six acute COVID-19 patients after stimulation with the
ten most frequently detected peptides of the envelope, membrane, and nucleoprotein
from this study. (A) Exemplary IFN-y, TNF-α and IL-2 CD4+ T cell response, pre-gated on
CD4+ T cells. (B) Frequencies of IFN-y, TNF-α and IL-2 of CD4+ T cells and the distribution S5 Table. Overview of SARS-CoV-2 membrane and nucleoprotein-specific CD8+ T cell
responses in acute and resolved COVID-19 patients. For each patient, the HLA class I mole-
cules are listed. aCov = acute COVID-19 patient, rCov = resolved COVID-19 patient. =
patient was treated with immunosuppressant medication or received chemotherapy. (XLSX)
S6 Table. Sequence homology of CCC and the most frequently detected SARS-CoV-2 epi-
topes in this study. (XLSX)
S7 Table. Antibody prevalence to CCC as well as SARS-CoV-2. (XLSX)
S8 Table. (A) In vitro binding value and number of responding patients in this cohort. (B)
Most likely binding HLA-DRB1 molecules for SARS-CoV-2 peptides that elicited a CD4+ T
cell response. (XLSX)
S9 Table. Flow cytometry panels used for ex vivo ICS (A) and tetramer analysis (B). (XLSX)
S1 Fig. Ex vivo ELISpot of two recovered and one acute COVID-19 patient. (A) Ex vivo ELI-
Spot with PBMCs from two resolved and one acute COVID-19 patient stimulated with all 43
membrane peptides. The spots in the negative control ranged from 0 to 1 spot per 100.000
cells. (B) Ex vivo ELISpot with PBMCs from two resolved and one acute COVID-19 patients
stimulated with all 82 nucleoprotein peptides. The spots in the negative control ranged from 0
to 1 spot per 100.000 cells. (TIF)
S2 Fig. Breadth of the CD4+ T cell response against the SARS-CoV-2 envelope, membrane,
and nucleoprotein of patient rCov-17. All IFN-y+ CD4+ T cell responses of patient rCov-17
against the envelope, membrane, and nucleoprotein. Gated on CD4+ T cells. All cytokine
gates are set based on the respective negative control (R10 and DMSO). (TIF)
S3 Fig. Breadth of the CD4+ T cell response in correlation with the time after infection (A),
disease severity (B), seroprevalence of IgG antibodies against SARS-CoV-2 (C) or CCC (D) S7 Table. Antibody prevalence to CCC as well as SARS-CoV-2.
(XLSX) (B) Frequencies of IFN-y, TNF-α and IL-2 of CD4+ T cells and the distribution 20 / 24 PLOS Pathogens | https://doi.org/10.1371/journal.ppat.1009842
September 16, 2021 PLOS PATHOGENS SARS-CoV-2 N, M, and E-specific CD4+ T cells of the frequencies of single, double, and triple positive CD4+ T cells. The threshold for positiv-
ity for the cytokines IFN-y, TNF-a and IL-2 was set at 0.02% of all CD4+ T cells. The back-
ground detected in the negative controls (R10 + DMSO) was subtracted from positive values. (TIF) S6 Fig. Overview of most frequently detected epitopes in this study and mutations in variants
of concern of the (A) nucleoprotein, (B) membrane protein and (C) envelope protein. Hori-
zontal, black parentheses indicate epitopes identified in this study. Red shade indicates asymp-
tomatic CD4+ T cell epitopes identified by Prakash et al. [28] with high conservancy among
human and animal coronaviruses. Highlighted amino acids indicate mutations found in vari-
ants of concern (according to PANGO lineages: https://cov-lineages.org/). Colour code: yel-
low, B.1.1.7; light blue, B.1.351; grey, P1 (no VOC-defining mutations are located in the M
protein). (TIF) Acknowledgments We thank all patients who participated in this study. We also thank all of the members of the
UKE ID COVID-19 study group for helping with the recruitment of the patients, Silke Kum-
mer, Robin Woost and Melanie Wittner for technical assistance, and Christin Ackermann for
the administrative support. S7 Table. Antibody prevalence to CCC as well as SARS-CoV-2.
(XLSX) S8 Table. (A) In vitro binding value and number of responding patients in this cohort. (B)
Most likely binding HLA-DRB1 molecules for SARS-CoV-2 peptides that elicited a CD4+ T
cell response. (XLSX) S9 Table. Flow cytometry panels used for ex vivo ICS (A) and tetramer analysis (B). (XLSX) S1 Fig. Ex vivo ELISpot of two recovered and one acute COVID-19 patient. (A) Ex vivo ELI-
Spot with PBMCs from two resolved and one acute COVID-19 patient stimulated with all 43
membrane peptides. The spots in the negative control ranged from 0 to 1 spot per 100.000
cells. (B) Ex vivo ELISpot with PBMCs from two resolved and one acute COVID-19 patients
stimulated with all 82 nucleoprotein peptides. The spots in the negative control ranged from 0
to 1 spot per 100.000 cells. (TIF) S1 Fig. Ex vivo ELISpot of two recovered and one acute COVID-19 patient. (A) Ex vivo ELI-
Spot with PBMCs from two resolved and one acute COVID-19 patient stimulated with all 43
membrane peptides. The spots in the negative control ranged from 0 to 1 spot per 100.000
cells. (B) Ex vivo ELISpot with PBMCs from two resolved and one acute COVID-19 patients
stimulated with all 82 nucleoprotein peptides. The spots in the negative control ranged from 0
to 1 spot per 100.000 cells. (TIF) S2 Fig. Breadth of the CD4+ T cell response against the SARS-CoV-2 envelope, membrane,
and nucleoprotein of patient rCov-17. All IFN-y+ CD4+ T cell responses of patient rCov-17
against the envelope, membrane, and nucleoprotein. Gated on CD4+ T cells. All cytokine
gates are set based on the respective negative control (R10 and DMSO). (TIF)
S3 Fig. Breadth of the CD4+ T cell response in correlation with the time after infection (A),
disease severity (B), seroprevalence of IgG antibodies against SARS-CoV-2 (C) or CCC (D)
and gender (E). (TIF)
S4 Fig. Original FACS-plots of restriction assay. (TIF)
S5 Fig. Ex vivo ICS with PBMCs of six acute COVID-19 patients after stimulation with the
ten most frequently detected peptides of the envelope, membrane, and nucleoprotein
from this study. (A) Exemplary IFN-y, TNF-α and IL-2 CD4+ T cell response, pre-gated on
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Karsten, Julian Schulze zur Wiesch. Formal analysis: Janna Heide, Julian Schulze zur Wiesch. Funding acquisition: Julian Schulze zur Wiesch. Funding acquisition: Julian Schulze zur Wiesch. Investigation: Janna Heide, Sophia Schulte, Julian Schulze zur Wiesch. Investigation: Janna Heide, Sophia Schulte, Julian Schulze zur Wiesch. Methodology: Janna Heide, Sophia Schulte, Marissa Herrmann. Project administration: Julian Schulze zur Wiesch. Resources: Matthias Marget, Sven Peine, Alexandra M. Johansson, Marc Lu¨tgehetmann, Wil-
liam W. Kwok, Julian Schulze zur Wiesch. Supervision: Julian Schulze zur Wiesch. Supervision: Julian Schulze zur Wiesch. Validation: Alessandro Sette, John Sidney. Visualization: Janna Heide, Sophia Schulte. Writing – original draft: Janna Heide, Alessandro Sette, John Sidney, Julian Schulze zur
Wiesch. Writing – review & editing: Janna Heide, Alessandro Sette, John Sidney, Julian Schulze zur
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OneShotDA: Online Multi-Object Tracker With One-Shot-Learning-Based Data Association
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Received January 15, 2020, accepted February 15, 2020, date of publication February 24, 2020, date of current version March 3, 2020. Digital Object Identifier 10.1109/ACCESS.2020.2975912 KWANGJIN YOON
1, JEONGHWAN GWAK
2, YOUNG-MIN SONG
3,
YOUNG-CHUL YOON
4, AND MOONGU JEON
3 ,
1SI-Analytics Company, Ltd., Daejeon 34051, South Korea
2Department of Software, Korea National University of Transportation, Chungju 27469, South Korea
3School of Electrical Engineering and Computer Science, Gwangju Institute of Science Technology, Gwangju 61005, South Korea
4Robot Center, LG Electronics, Seoul 07336, South Korea p
,
y
p
,
gj
,
3School of Electrical Engineering and Computer Science, Gwangju Institute of Science Technology, Gwangju 61005, South Korea
4Robot Center, LG Electronics, Seoul 07336, South Korea Corresponding author: Moongu Jeon (mgjeon@gist.ac.kr) This work was supported in part by the Institute of Information and Communications Technology Planning and Evaluation (IITP) grant
funded by the Korean Government (MSIT) under Grant 2014-3-00077, in part by the AI National Strategy Project, and in part by the
National Research Foundation of Korea (NRF) grant funded by the Korean Government (MSIT) under Grant 2019R1A2C2087489 and
Grant 2017R1D1A1B03036423. ABSTRACT
Tracking multiple objects in a video sequence can be accomplished by identifying the
objects appearing in the sequence and distinguishing between them. Therefore, many recent multi-object
tracking (MOT) methods have utilized re-identification and distance metric learning to distinguish between
objects by computing the similarity/dissimilarity scores. However, it is difficult to generalize such
approaches for arbitrary video sequences, because some important information, such as the number of
objects (classes) in a video, is not known in advance. Therefore, in this study, we applied a one-shot learning
framework to the MOT problem. Our algorithm tracks objects by classifying newly observed objects into
existing tracks, irrespective of the number of objects appearing in a video frame. The proposed method, called
OneShotDA, exploits the one-shot learning framework based on an attention mechanism. Our neural network
learns to classify unseen data samples using labels from a support set. Once the network has been trained,
it predicts correct labels for newly received detection results based on the set of existing tracks. To analyze
the effectiveness of our method, it was tested on the MOTchallenge benchmark datasets (MOT16 and
MOT17 datasets). The results reveal that the performance of the proposed method was comparable with
those of current state-of-the-art methods. In particular, it is noteworthy that the proposed method ranked first
among the online trackers on the MOT17 benchmark. INDEX TERMS
Data association, deep learning, multi-object tracking, object recognition, one-shot
learning. his work is licensed under a Creative Commons Attribution 4.0 License. For more information, see http://creativecommons.org/licenses/by/4.0/ I. INTRODUCTION detection result, and ends if there is no detection result. A subtle difference between such approaches is the num-
ber of repeated detections (misdetections) used for track
initiation (termination). In [1]–[4], a new track hypothe-
sis was generated at every frame for each detection result
that was not associated with an existing track. A track
was terminated if the number of consecutive misdetections
exceeded a predefined threshold. To eliminate false tra-
jectories, tracks shorter than a predefined threshold were
deleted from the track set after the tracking process was
completed. Multi-object tracking (MOT) is considered one of the most
challenging problems in computer vision research. Recently,
tracking-by-detection methods have attracted significant
interest, because they can isolate the problem of object detec-
tion from object tracking, which helps them focus on the
tracking tasks, such as track management, initiation, and
termination, as well as data association. There are several methods available for track initia-
tion/termination. Several studies [1]–[4] have adopted a
straightforward rule wherein tracking starts if there is a Another strategy involves optimizing an objective func-
tion over the space of trajectories [11]–[13]; it is nec-
essary
to
perform
both
track
management
and
data The associate editor coordinating the review of this manuscript and
approving it for publication was Victor Sanchez
. 38060 VOLUME 8, 2020 VOLUME 8, 2020 VOLUME 8, 2020 K. Yoon et al.: OneShotDA: Online Multi-Object Tracker With One-Shot-Learning-Based Data Association Let Q be the query set, and x(i)
q be the i-th query sample. G is the gallery set. G = {(x(j)
g , y(j)
g )}|G|
j=1, where x(j)
g and y(j)
g
are the j-th sample and label in the gallery set, respectively. In Figure 1(a), Tk−1, which is the track set at frame k −1,
represents the query set, and Zk, which is the detection set
at frame k, represents the gallery set. The feature embedding
network (FEN) in OneShotDA takes a sample from both Q
and G as an input, and generates a feature vector f (·). Note
that the FEN processes samples in Q and G using the same
weights. Next, conditional embedding networks (CENs) are
used to embed feature vectors to generate more robust fea-
tures. CEN_Q and CEN_G are the CENs for the query set and
gallery set, respectively. I. INTRODUCTION Additionally, we include a network
called TD_clf that estimates the probability of accurate detec-
tion for a given input (detection response). We shall detail
each component of the OneShotDA in the following sections. association simultaneously, based on the optimization results. Zhang et al. [11] proposed a network-flow-based global
optimization method for MOT. They constructed a network
using a set of detection results from a video, and computed the
global best trajectories by identifying the min-cost flow of the
network. Initialization and termination of trajectories were
handled intrinsically after the solution had been computed. Pirsiavash et al. [12] used an approach similar to that of
Zhang et al. , except that they adopted a greedy algorithm
(shortest path) for a flow network. In [13], the authors used
the multiple-hypothesis tracking (MHT) for track manage-
ment. The MHT saves track proposals in a tree structure that
grows with new detections for each frame, that is, the tree
describes all the possible data association results originating
from a single detection result. Track initiation and termina-
tion and data association in the MHT are treated as solving
an optimization problem, that is, the maximum weighted
independent set (MWIS), within a certain time window. As shown in Figure 1(b), the OneShotDA maps the class
of a query sample to labels in the gallery set, which is defined
by p(yg|xq, G). The left table in Figure 1(b) contains the
probability distribution of data associations for the scenario
depicted in Figure 1(a). For example, q3, an embedded vector
of T (3)
k−1, has a low probability of data association, because
the object is completely occluded in frame k. The right table
in Figure 1(b) contains the probabilities of accurate detection
for the gallery set, which are estimated by TD_clf. We also
demonstrate that the proposed data association mechanism
can be easily integrated with any online MOT system. In the
experiments section, we track objects using a combination
of the MHT framework [7] and the proposed OneShotDA. Tables 1 and 2 summarize the notations and abbreviations
used in this article. In the tracking-by-detection paradigm, data association
entails connecting detection outputs across video frames and
screening misdetections. This problem can be considered a
form of statistical estimation such as the likelihood estimation
of p(Z|T ), where Z is the set of detections and T is the set
of trajectories. 1The use of detection results as a query set or the use of existing tracks as
a query set is possible. 2The MHT tracker used in this research is now available on the web:
https://github.com/yoon28/pymht I. INTRODUCTION (a): Architecture of the proposed OneShotDA method. Our network consists of FEN, CEN_Q, CEN_G, and TD_clf. (b): Outputs of OneShotDA. FIGURE 1. (a): Architecture of the proposed OneShotDA method. Our network consists of FEN, CEN_Q, CEN_G, and TD_ the proposed OneShotDA method. Our network consists of FEN, CEN_Q, CEN_G, and TD_clf. (b): Outputs of OneShotDA. TABLE 2. Abbreviation summary: These abbreviations are used in our
model. TABLE 2. Abbreviation summary: These abbreviations are used in our
model. generated using MOT datasets such as the MOTChal-
lenge datasets [8]. • We demonstrate that OneShotDA can be easily inte-
grated with any online MOT system (MHT in this
study).2 • We demonstrate that the effectiveness of the proposed
MOT system can match those of the state-of-the-art
methods. It is noteworthy that the proposed method
ranks first among online trackers when evaluated on the
MOT17 benchmark. I. INTRODUCTION The distribution of likelihood determines the
probability of associated detections belonging to the same
object when the track proposal T has been satisfied. Online
tracking methods recursively estimate the likelihood based
on the detection set up to the current frame [1]–[4], [13]. In contrast, offline/batch methods [11], [12] use the detection
results for an entire video sequence. Recently, MOT has been accomplished by identifying and
distinguishing between objects appearing in the sequence. Therefore, many recent MOT methods have focused on
re-identification and distance metric learning to distinguish
between objects by computing the similarity/dissimilarity
scores between them. However, it is difficult to generalize
such approaches for arbitrary video sequences, because some
important information, e.g., the number of objects (classes)
in a video, is not known in advance. In this study, we pro-
pose a novel data association strategy called OneShotDA
that exploits one-shot learning frameworks such as those
in [14]–[16]. In such frameworks, the class of a query sample
is determined by the samples in a gallery set. For exam-
ple, in [16], predictions for the samples in a query set are
obtained based on a relation module that computes the dis-
tance between a query feature and the features in a gallery
set. By following the protocol of the one-shot framework,
our method classifies a newly received detection result (query
sample) into an existing track (gallery set), or vice versa.1
Specifically, our model can predict the label of a query
sample based on the labels of the gallery set by using an
attention mechanism that indicates a corresponding sample
in the gallery set. TABLE 1. Notation summary: We define the mathematical notations used
for formalizing problems in this article. TABLE 1. Notation summary: We define the mathematical notations used
for formalizing problems in this article. TABLE 1. Notation summary: We define the mathematical notations used
for formalizing problems in this article. 1The use of detection results as a query set or the use of existing tracks as
a query set is possible. VOLUME 8, 2020 38061 K. Yoon et al.: OneShotDA: Online Multi-Object Tracker With One-Shot-Learning-Based Data Association FIGURE 1. (a): Architecture of the proposed OneShotDA method. Our network consists of FEN, CEN_Q, CEN_G, and TD_clf. (b): Outputs of OneShotDA. FIGURE 1. (a): Architecture of the proposed OneShotDA method. Our network consists of FEN, CEN_Q, CEN_G, and TD_clf. (b): Outputs of OneShotDA. FIGURE 1. II. RELATED WORKS Modern MOT methods based on the tracking-by-detection
paradigm can be categorized into offline and online meth-
ods. Offline methods utilize the detection results from all
the video frames to construct robust trajectories, whereas,
online approaches process videos sequentially in a frame-
by-frame manner by recursively updating existing tracks with
new detection results. The contributions of this study can be summarized as
follows: • We propose a novel data association mechanism called
OneShotDA that can classify newly generated detection
outputs into existing tracks using one-shot classification. The offline methods are commonly set up by represent-
ing the problem as a graph wherein each detection result
represents a node, and the edges represent possible links. • We adopt a training strategy that is customized for
one-shot learning and suitable for data association tasks. We also demonstrate the way training samples are 38062 38062 VOLUME 8, 2020 VOLUME 8, 2020 K. Yoon et al.: OneShotDA: Online Multi-Object Tracker With One-Shot-Learning-Based Data Association and tracklets. Voigtlaender et al. [29] extended the MOT
to MOT and segmentation. They also presented a track-
ing method that jointly addresses detection, tracking,
and segmentation using a single convolutional network. Long et al. [30] tackled unreliable detection by selecting
candidates from the outputs of both detection and track-
ing. They also demonstrated that the identification ability
of their tracker could be improved by using appearance
representations trained on a person re-identification dataset. In [31], the authors tracked the objects using recurrent neural
networks (RNNs), and demonstrated that RNNs can effec-
tively address the problems of trajectory estimation and data
association. Ma et al. [17] formulated the problem as a hierarchical corre-
lation clustering (HCC), a modified form of the correlation-
clustering-based tracking method [18]. Tang et al. [19] used
a combination of the lifted multicut problem (LMP) formu-
lation and body pose information. Henschel et al. [20] also
utilized body pose information; however, they formulated the
problem as a min-cost graph labeling problem [21]. p
g p
g p
It is known that online methods are often impeded by
long-term occlusion issues, because only the current frame
and previous frames are available [22]. The MHT [7], a robust
online tracking method, attempts to resolve this issue by
constructing a track tree that describes all the possible data
association results within a particular time window. II. RELATED WORKS Even
if this time window causes the MHT to produce delayed
tracking results, it is still considered an online tracker because
only the current detection set is used to update the scores
of previous tracks [7]. In other words, the MHT recur-
sively estimates the likelihood of the previous tracks reoccur-
ring based on current observations. Recently, various MHT
algorithms [13], [22], [23] have been proposed for MOT
tasks. In [13], long-term appearance modeling was incorpo-
rated into the MHT, where the tracker estimated the online
appearance features for each track. In [23], an LSTM network
was adopted to score track proposals in the MHT. The authors
also proposed a bilinear LSTM model, a modified version of
the original LSTM model [6] for a gating network. In [22],
the authors proposed an iterative MWIS algorithm for the
MHT, making it possible to solve the MWIS problem based
on the solutions of previous frames. One(few)-shot learning aims to train a classifier to recog-
nize unseen classes during training using only a single (few)
labeled example(s). Because many deep learning systems
require hundreds or thousands of samples, one(few)-shot
learning has attracted significant interest [14]–[16], [32]. In such frameworks, the class of a query sample is determined
by the samples in a gallery set. In [32], the authors proposed a
novel strategy for one-shot classification using Siamese neu-
ral networks for verification. In [14], the MatchingNet model
was proposed to map a small labelled gallery set and an unla-
beled query sample to the correct label. MatchingNet com-
pares the cosine distances between a query feature and each
gallery feature. Snell et al. [16] proposed ProtoNet, which
also predicts the class of a query sample based on distance;
however, ProtoNet uses the Euclidean distance between a
query and the gallery. Sung et al. [15] presented RelationNet,
which, apart from replacing distance with a learnable relation
module, is based on a similar concept. There have also been many recent studies based on MOT. Sun et al. [24] tracked objects using a novel deep network
that can infer object affinities across different frames by
analyzing exhaustive permutations of the extracted features. Their network also accounts for the appearance and disap-
pearance of objects between video frames. Yang et al. [25]
proposed an online MOT algorithm that uses two-step
data association combined with an improved sparse-based
appearance affinity model and rank-based motion affinity
model. II. RELATED WORKS They tracked objects by fusing trajectory dynamics
information, and proposed a novel two-step data association
framework. He et al. [26] proposed a tracking-by-animation
framework to achieve both label-free and end-to-end learn-
ing for MOT, unlike tracking-by-detection frameworks, that
isolate the detection task from the tracking task. Their
differentiable neural network first tracks objects in input
frames, and then animates the tracked objects in reconstructed
frames. Learning is driven by reconstruction error based
on backpropagation. Zhang et al. [27] tracked objects in
multi-modal scenarios by adopting a deep architecture that
can be trained in an end-to-end manner, thereby enabling
the joint optimization of the base feature extractors of each
modality and an adjacency estimator for cross-modality. Wen et al. [28] proposed an MOT algorithm based on a
non-uniform hypergraph that can model different degrees of
dependency among tracklets for a unified objective. Their
method can model higher-order dependencies among objects In OneShotDA, we adopt the one-shot learning frame-
work to classify new samples based on known examples,
which solves the data association problem in MOT. There
are multiple available options for this task, including existing
frameworks such as MatchingNet, RelationNet, or ProtoNet. Therefore, we consider the one-shot framework as a data
association solver for MOT. IV. DATA ASSOCIATION WITH ONE-SHOT LEARNING LR(T (i)
k ) =
Qt
l=1 pK(kin(z[i]
k−l+1)|kin(T (i)
k−l), H1)
Qt
l=1 pK(kin(z[i]
k−l+1)|H0)
×
Qt
l=1 pA(app(z[i]
k−l+1)|app(T (i)
k−l), H1)
Qt
l=1 pA(app(z[i]
k−l+1)|H0)
P0(H1)
P0(H0),
(3) p(ˆy(i)
q |x(i)
q , G) = softmax(
|G|
X
j=1
⟨qi, gj⟩y(j)
g ),
(5) (5) (3) where ⟨·, ·⟩is the inner product between two vectors. qi and gj are the corresponding embedding vectors of x(i)
q
and x(j)
g , respectively. In Eq. 5, the probability distribution
of the query sample’s label ˆy(i)
q
is computed by applying
the softmax function over the gallery set’s labels. There-
fore, the class of the query sample is computed from the
gallery set with the maximum probability (i.e., arg maxˆy(i)
q ∈G
p(ˆy(i)
q |x(i)
q , G)). This process can be viewed as an attention
mechanism pointing to a corresponding sample in the gallery
set. It is important to note that the estimated label of the query
sample is not the same as the label in the query set (i.e.,
ˆy(i)
q
̸= y(i)
q ). This is because the label of query sample only
represents its class [14]. In addition, as mentioned previously,
the labels in the query set are used for network training,
meaning we can train the network twice per query-gallery pair
by swapping the two sets. where, app(·) is a function that returns the appearance feature
of a given input and kin(·) returns the kinematic component
of an input (e.g., the coordinate of the bounding box). The
kinematic term of LR(T (i)
k ) at frame (k −l + 1) under the
true-track hypothesis is assumed to be a Gaussian and is
estimated via Kalman filtering. Constants are set for the
false alarm hypotheses of both the appearance and kinematic
terms (pA(app(z[i]
k−l+1)|H0) and pK(kin(z[i]
k−l+1)|H0) are set
to Capp and Ckin, respectively). We use the log likelihood ratio for track scoring by tak-
ing the logarithm of Eq 3. Additionally, the track initiation
score is defined as ln[ P0(H1)
P0(H0)] and a constant Cβ is set. Track
termination is performed after a track is updated with dummy
detection results for τmiss consecutive frames. To maintain the
size of the feasible track proposals, the track tree is pruned
such that it does not exceed a tree depth of τD. Tree pruning
can be performed after finding the best set of proposals. IV. DATA ASSOCIATION WITH ONE-SHOT LEARNING For the data association task of MOT, we decided to adopt
the one-shot architecture of MatchingNet [14] because its
contextual embedding provides robust input features, partic-
ularly when two difficult examples are very close to each
other in the feature space [14]. However, simply applying
MatchingNet directly to our domain is not possible because
the data association problem does not match a detection to a
track and the proposed system must identify false alarms. LR(T (i)
k ) = p(T (i)
k |H1)
p(T (i)
k |H0)
P0(H1)
P0(H0)
(1) (1) In Eq. 1, P0(H1) and P0(H0) are the prior probabilities of the
true target and false alarm hypotheses, respectively. Based on
the chain rule, the likelihood is factorized as LR(T (i)
k ) =
Qt
l=1 p(z[i]
k−l+1|T (i)
k−l, H1)
Qt
l=1 p(z[i]
k−l+1|H0)
P0(H1)
P0(H0),
(2) Let Q and G be a set of query samples and a set of gallery
samples, respectively. Each sample consists of image-label
pairs (i.e., Q = {(x(i)
q , y(i)
q )}|Q|
i=1, and G = {(x(j)
g , y(j)
g )}|G|
j=1). |Q| and |G| represent the sizes of sets Q and G, respectively. The labels y(j)
g in the gallery set are |G|-sized vectors, each
of which is one-shot encoded such that the j-th component is
set to 1. Let Q and G be a set of query samples and a set of gallery
samples, respectively. Each sample consists of image-label
pairs (i.e., Q = {(x(i)
q , y(i)
q )}|Q|
i=1, and G = {(x(j)
g , y(j)
g )}|G|
j=1). |Q| and |G| represent the sizes of sets Q and G, respectively. The labels y(j)
g in the gallery set are |G|-sized vectors, each
of which is one-shot encoded such that the j-th component is
set to 1. (2) where we assume that detection results are conditionally
independent given the false alarm hypothesis and t is the
track length of T (i)
k . This equation can be further factorized
based on the independence assumption between kinematic
and appearance terms as follows: OneShotDA estimates the probability distribution of the
label of the i-th query sample for the labels in the gallery set
(Eq. 5). III. MULTIPLE HYPOTHESIS TRACKING In this section, we briefly review the MHT model presented
in [7] and discuss how we combine MHT with the proposed
OneShotDA. MHT maintains a track proposal by constructing a track
tree that describes all possible data association results orig-
inating from a single detection result (i.e., root node) and
its branches. Each node in the track tree can either cor-
respond to a real detection result obtained from an object
detector or be a dummy detection result representing a
misdetection. Let T (i)
k
be the i-th track proposal at frame
k maintained by the MHT and let t be its length. Then,
T (i)
k
= {z[i]
k−t+1, z[i]
k−t+2, . . . , z[i]
k−1, z[i]
k }, where z[i]
l
is a detec-
tion result chosen by the i-th track proposal at frame l. As mentioned previously, z[i]
l
can be a real detection result
from Z or a dummy detection result. Note that z(j)
l represents VOLUME 8, 2020 38063 38063 K. Yoon et al.: OneShotDA: Online Multi-Object Tracker With One-Shot-Learning-Based Data Association of Eq. 4 estimates the probability that a track T (i)
l−1 is classified
as cls(z[i]
l , Zl). the j-th detection at frame l, meaning z(j)
l
∈Zl in which Zl is
a set of detections at frame l (i.e., Zl ⊂Z). Following the original formulation in [7], the score for each
track proposal is defined as the likelihood ratio (LR) between
the true-track (H1) and false alarm (H0) hypotheses (Eq. 1). IV. DATA ASSOCIATION WITH ONE-SHOT LEARNING IV. DATA ASSOCIATION WITH ONE-SHOT LEARNING Once
the best set is identified, we select an ancestor of the best
proposal with a distance τD and prune the subtrees diverging
from that node. In Eq. 5, qi and gj are embedding vectors mapped from
the image space into a latent space. One potential method for
performing mapping for each sample is to train an embedding
network and apply the network to each sample independently
(e.g., qi = f (x(i)
q ), gj = f (x(j)
g ) for i = 1, ..., |Q|, j =
1, ..., |G|, where f is a CNN). We made f an FEN and
searched the ResNet family [33] to find the optimal FEN
structure. However, embedding each sample independently
means we cannot encode information regarding the entire
set, so the classification function in Eq. 5 is simply nearest
neighbor classification based on an inner product. To resolve
this issue, we train a CEN that embeds the feature vector
further by incorporating all other samples. This can improve Finally, the likelihood for the appearance term at frame l
under the true-track hypothesis is estimated by OneShotDA. The true-track likelihood at frame l is written as pA(app(z[i]
l )|app(T (i)
l−1), H1) = p(cls(z[i]
l , Zl)|T (i)
l−1, Zl),
(4) (4) where cls(z, Z) is a function that retrieves the label of a
detection result z determined by Z. Therefore, the right side 38064 VOLUME 8, 2020 VOLUME 8, 2020 K. Yoon et al.: OneShotDA: Online Multi-Object Tracker With One-Shot-Learning-Based Data Association the accuracy of classification, particularly in cases where
some samples are very close to each other (i.e., hard samples). the samples as true detection results and false alarms. True
detection results are detection results whose intersection over
union (IoU) is greater than the threshold τIoU, where each
ground truth bounding box has at most one true detection
result. This is an assignment problem based on the maximum
total IoU score. We solved this problem using the Hungarian
algorithm [34]. The remaining detection results that were
not chosen by the Hungarian method were classified as false
alarms. Note that because objects in the dataset are frequently
occluded by each other, we filter out small ground truth
bounding boxes using non-maximum suppression with an
IoU threshold τ GT
IoU prior to identifying true detection results. IV. DATA ASSOCIATION WITH ONE-SHOT LEARNING Specifically, CEN_Q is the CEN for set Q, which reads
samples in G through the softmax function over the cosine
similarity measures.3 Therefore, the conditional embedding
vectors qi are defined as qi = fq([f (x(i)
q ), ri]),
(6)
ri =
|G|
X
j=1
aijgj,
(7)
ai = softmax(
|G|
X
j=1
⟨f (x(i)
q ), gj⟩y(j)
g ). (8) qi = fq([f (x(i)
q ), ri]),
(6)
ri =
|G|
X
j=1
aijgj,
(7) (6) (7) ai = softmax(
|G|
X
j=1
⟨f (x(i)
q ), gj⟩y(j)
g ). (8) (8) IoU
Next, a training sample is constructed using two consecu-
tive video frames from the video sequence. Let l be a particu-
lar frame, then Q and G are the detection sets from l and l+1,
respectively. Additionally, Q and G can be detection results
from l+1 and l, respectively. In this manner, we compute loss
twice using one query-gallery pair by swapping the two sets. Let LCE be the cross entropy loss, where Q consists of the
detection set at l, and LCE′ be the loss, where Q consists of the
detection set at l + 1. These losses measure the classification
error for the query set. If a query sample is either a false
alarm or missing in the gallery set, that sample is not used
to compute loss. Note that the size of the query set |Q| will
be the batch size and the size of the gallery set |G| will be the
number of classes. Therefore, the class size and batch size
are not fixed. This can be achieved based on the attention
mechanism we adopted. f is the FEN (ResNet [33]). The last layer in f is activated
by the tanh function. In Eq. 6, fq is a fully connected
network whose output has the same size as f (x(i)
q ). [·, ·] is
a concatenation operator between two vectors. The output
of fq is also activated by the tanh function. In Eq. 7,
aij represents the j-th component of ai. Therefore, the con-
ditional embedding vector qi incorporates all elements in the
set G based on the weighted average gj. f is the FEN (ResNet [33]). The last layer in f is activated
by the tanh function. In Eq. 6, fq is a fully connected
network whose output has the same size as f (x(i)
q ). IV. DATA ASSOCIATION WITH ONE-SHOT LEARNING TABLE 3. Division of training and validation sets in which D, F, and S in
the MOT17 dataset represent the DPM, Faster R-CNN, and SDP,
respectively. Finally, the final loss L is defined as L = LCE + LCE′ + λTDLTD,
(12) (12) where λTD is the weight for LTD. Therefore, training
OneShotDA, including the FEN, CEN, and TD_clf, is accom-
plished using a single training sample by minimizing L in one
step. IV. DATA ASSOCIATION WITH ONE-SHOT LEARNING [·, ·] is
a concatenation operator between two vectors. The output
of fq is also activated by the tanh function. In Eq. 7,
aij represents the j-th component of ai. Therefore, the con-
ditional embedding vector qi incorporates all elements in the
set G based on the weighted average gj. Next, we present the CEN for the set G, which is denoted
as CEN_G. gj is generated by embedding an additional sam-
ple with the samples in set G using bidirectional LSTM
(Bi-LSTM) [6]. gj = −→
h j + ←−
h j + f (x(j)
g ),
(9)
−→
h j, −→c j = LSTM(f (x(j)
g ), −→
h j−1, −→c j−1),
(10)
←−
h j, ←−c j = LSTM(f (x(j)
g ), ←−
h j+1, ←−c j+1),
(11) (9) Additionally, identifying false alarms is crucial because
many false alarms are present in detector outputs. To that
end, we attach a fully connected layer (TD_clf) with a size
of one following the FEN. Therefore, the network takes a
feature vector f (·) from the FEN and outputs a prediction
pTD that indicates whether or not the input is a true detection
result. We then define LTD as the binary cross entropy loss,
which measures the classification error between the label of a
true detection result and the predicted probability of the true
detection result (i.e., pTD). where −→
h and ←−
h are the outputs of forward and backward
LSTM, respectively. −→c and ←−c are the cells of the corre-
sponding LSTM networks. CEN_G is similar to the network
in [14] as we also add a skip connection between the input
and output. where −→
h and ←−
h are the outputs of forward and backward
LSTM, respectively. −→c and ←−c are the cells of the corre-
sponding LSTM networks. CEN_G is similar to the network
in [14] as we also add a skip connection between the input
and output. TABLE 3. Division of training and validation sets in which D, F, and S in
the MOT17 dataset represent the DPM, Faster R-CNN, and SDP,
respectively. TABLE 3. Division of training and validation sets in which D, F, and S in
the MOT17 dataset represent the DPM, Faster R-CNN, and SDP,
respectively. TABLE 3. Division of training and validation sets in which D, F, and S in
the MOT17 dataset represent the DPM, Faster R-CNN, and SDP,
respectively. A. TRAINING OneShotDA As discussed above, we use a one-shot learning framework
for MOT because it has the ability to unravel the associa-
tion problem between an unseen object and existing tracks. In this work, our one-shot framework was derived from
MatchingNet [14] with many modifications. In this section,
we summarize the role of each component in OneShotDA,
namely the FEN, CEN (CEN_Q and CEN_G), and TD_clf. To train the network, we generated training samples from
the training sets in the MOTchallenge datasets [8]. Specifi-
cally, we used subsets of the MOT16 and MOT17 datasets
(Table 3). We used public detection methods and classified 3We use the similarity measure of an inner product space. Because similar-
ity can be influenced by not only the direction of the inner product, but also
the norm of the feature vector, we activate all feature vectors with a tanh
function. • FEN: This network generates a feature vector for a given
input (i.e., x(i)
q and x(j)
g ), where x(i)
q
∈Q and x(j)
g
∈G. • FEN: This network generates a feature vector for a given
input (i.e., x(i)
q and x(j)
g ), where x(i)
q
∈Q and x(j)
g
∈G. • FEN: This network generates a feature vector for a given
input (i.e., x(i)
q and x(j)
g ), where x(i)
q
∈Q and x(j)
g
∈G. 38065 VOLUME 8, 2020 K. Yoon et al.: OneShotDA: Online Multi-Object Tracker With One-Shot-Learning-Based Data Association We use the ResNet family for this network and investi-
gate the performance of each residual network in terms
of tracking accuracy. (section V-C) of the OneShotDA tracker. The training and validation
dataset separation is detailed in Table 3. The test set of
MOT17 includes a total of seven sequences, each of which
comes with three sets of public detection results. These
three public sets come from different detectors, namely the
deformable part model (DPM) [5], faster-RCNN [9], and
scale-dependent pooling (SDP) [10]. The MOT16 test set
consists of the same sequences as those in MOT17, but it
only contains the DPM detection set. It must be noted that
the ground truth labels are not shared for the same sequences
across MOT16 and MOT17. • CEN: This network further embeds a feature vector that
is outputted by the FEN to generate more robust features. A. TRAINING OneShotDA The features outputted by this network are sufficient for
distinguishing samples from each other, even when they
are close to each other. (section V-C) – CEN_Q: CEN for the set Q. This network helps a
sample in Q read entire elements of G and outputs
a conditional embedding vector for a given input. – CEN_G: CEN for the set G. A conditional embed-
ding vector for each element in G is generated using
bidirectional LSTM with the set G itself. The metrics used for measuring tracker performance are
the same as those used in [8]. MOT accuracy (MOTA)
measures performance by aggregating three error sources,
namely false positives, missed targets, and identity switches. IDF1 [35] computes the ratio of correctly identified detection
results over the average number of ground truth and com-
puted detection results. MOTA and IDF1 are considered the
main criteria for tracker performance. We also report mostly
tracked (MT) objects, mostly lost (ML) objects, the total
number of false positives (FP), false negatives (FN), and
identity switches (IDsw), and the total number of times a
trajectory is fragmented (Frag). • TD_clf: This network takes a feature vector f (·) and
outputs a prediction pTD, indicating whether or not the
input is a true detection result. V. EXPERIMENTS AND ANALYSIS In this section, we analyze the tracking performance of
the OneShotDA tracker on the MOTChallenge datasets
(MOT16 and MOT17). Additionally, ablation analysis is per-
formed to identify the best hyperparameter settings. A. IMPLEMENTATION DETAILS FIGURE 2. Ablation study on different FEN architectures. The x-axis is the
feature size and the y-axis is the MOTA score. In all the experiments, the values of the parameters τD, τIOU,
and τ GT
IOU were 30, 0.334, and 0.5, respectively. τmiss was
set to 2.3fps, where fps is the frames per second for
each sequence. Additionally, we have exhaustively searched
Capp, Ckin, and Cβ to find good parameters, and decided
to set to 0.1, 0.1, and 2.0, respectively. Input images were
resized to 288 × 96 pixels and normalized to the range
of 0 to 1. We also augmented the training set with uniform
random rotation in the angle range of [−8.5, 8.5], random
horizontal flipping, and random brightness changes. As men-
tioned previously, the ResNet family was used for the FEN. In the ablation analysis section, we investigate the perfor-
mances of the ResNet family, ResNet34, ResNet50, and
ResNet101, as well as their output sizes (feature vectors). Note that the size of a feature vector determines the size of the
Bi-LSTM in CEN_Q because the output cell of Bi-LSTM and
the corresponding feature vector are added inside CEN_Q. The ResNets were initialized with weights pre-trained on the
ImageNet datasets, except for the final fully connected layers. The final layer was replaced with our feature embedding layer
for various output sizes (see the ablation analysis section). The stochastic gradient descent optimizer with a momentum
0.9 was used for training all the networks, and the initial
learning rate was set to 10−4. The learning rate decreased
after every 3000 iterations based on exponential decay with
a decay rate of 0.95 until the minimum learning rate of 10−7
was achieved. λTD was set to 1. FIGURE 2. Ablation study on different FEN architectures. The x-axis is the
feature size and the y-axis is the MOTA score. C. ABLATION ANALYSIS We conducted ablation studies with different hyperparameter
settings to achieve optimal performance on the validation
set and its subsets. In Figure 2, we consider the ResNet
family and corresponding output size for the subsets of our
validation set (i.e., MOT17-05-{F,S} and MOT17-09-{F,S}). It is important to note that not only does the architecture of
the FEN affect the tracking performance, but the size of the
feature vectors is also a crucial aspect for performance. In this
study, all networks were trained for 5 epochs. The CENs
were consistently initialized for each setting and retrained
from the beginning. The sizes of the feature vectors were
sampled from a logarithmic scale ranging from 64 to 1024
(i.e., {64, 128, 256, 512, 1024}). The analysis results for var-
ious FEN settings are presented in Figure 2. According to the D. MOT PERFORMANCE ANALYSIS results in Figure 2, we selected the ResNet50 with 512 outputs
for our FEN. Our model achieves the maximum MOTA (59.3)
using ResNet50-512. Furthermore, it is important to note
that the model seems to suffer from overfitting when the
parameter size is greater than that in ResNet50 (25.6 M) or the
feature size is greater than 512. To determine if this assump-
tion was correct, we trained a ResNet101-1024 network with
additional epochs because the low performance of such a
large model could potentially result from a low convergence
rate based on its large parameter size. However, we found that
the MOTA of the large model continued to decrease or remain
constant while its training loss consistently decreased during
continued training. In this experiment, we used a ResNet50-512 network as the
FEN and trained the network with additional epochs. Our
network was trained for a total of 8 epochs. We first present the performance analysis of our network
as a binary classifier. Each prediction is considered the output
of a classification representing how likely it is for two objects
to be assigned the same identity. As shown in Figure 5,
the average precision of the precision-recall curve is 0.8957. The classification results for the validation set indicate that
OneShotDA is trained properly and makes precise association
predictions. FIGURE 5. Precision-recall curve. Finally, we present performance comparisons between the
OneShotDA tracker and existing state-of-the-art methods
such as HCC [17], LMP [19], GCRA [36], KCF16 [37],
MOTDT [30], JBNOT [20], eHAF17 [39], TLMHT [22],
EAGS16 [38], MHT_DAM [13], MHT_bLSTM [23], and
EDMT17 [40]. These methods were evaluated on the
MOTChallenge server.4 To provide a reasonable comparison,
only officially published and peer-reviewed entries in the
MOT16 and MOT17 benchmarks were considered. Addition-
ally, we collected MHT-based trackers, categorized as online FIGURE 5. Precision-recall curve. FIGURE 5. Precision-recall curve. FIGURE 4. w/ CEN vs w/o CEN, evaluated on the validation set. The tested
model is the ResNet50 with 512 output neurons. FIGURE 5. Precision-recall curve. Finally, we present performance comparisons between the
OneShotDA tracker and existing state-of-the-art methods
such as HCC [17], LMP [19], GCRA [36], KCF16 [37],
MOTDT [30], JBNOT [20], eHAF17 [39], TLMHT [22],
EAGS16 [38], MHT_DAM [13], MHT_bLSTM [23], and
EDMT17 [40]. These methods were evaluated on the
MOTChallenge server.4 To provide a reasonable comparison,
only officially published and peer-reviewed entries in the
MOT16 and MOT17 benchmarks were considered. B. MOTCHALLENGE DATASETS AND METRICS In this study, we used the MOTChallenge datasets [8] to train
our OneShotDA network and test the tracking performance 38066 38066 VOLUME 8, 2020 VOLUME 8, 2020 K. Yoon et al.: OneShotDA: Online Multi-Object Tracker With One-Shot-Learning-Based Data Association FIGURE 3. Analysis of pTD for the validation set. struggles to associate objects identified by the DPM detector
whose outputs are much noisier in comparison with those
of Faster R-CNN and SDP. Additionally, performance is
consistently improved by the CEN for the MOT17 dataset
(Figure 4(b)). The performance in terms of predicting true detection
results (pTD) was also investigated. This analysis helped us
in selecting a good threshold value for identifying true detec-
tion results in the detection outputs. Figure 3 presents the
average-precision (AP) score for each threshold value. The
values are evenly distributed at intervals of 0.15. We achieve
an AP of 0.981 at a threshold value 0.45. Therefore,
we chose 0.45 as the threshold value for pTD when testing
our OneShotDA tracker on the test set. FIGURE 3. Analysis of pTD for the validation set. 4https://motchallenge.net/ D. MOT PERFORMANCE ANALYSIS Addition-
ally, we collected MHT-based trackers, categorized as online FIGURE 4. w/ CEN vs w/o CEN, evaluated on the validation set. The tested
model is the ResNet50 with 512 output neurons. Next, we investigated the contribution of the CEN by
measuring its performance in terms of MOTA and IDF1 on
the validation set. Figure 4(a) presents comparative results
in terms of MOTA, and Figure4(b) presents comparative
results in terms of IDF1. It can be inferred from Fig-
ure 4(a) that our CEN efficiently improves the perfor-
mance on the MOT16 dataset. OneShotDA with the CEN
improves the tracking performance by 2.8% (MOTA) and
1.1% (IDF1). This is because OneShotDA, without the CEN, 38067 38067 VOLUME 8, 2020 K. Yoon et al.: OneShotDA: Online Multi-Object Tracker With One-Shot-Learning-Based Data Association FIGURE 6. Tracking results on the MOT17-09-FRCNN dataset. In the top row, (a)−(c), present the results for JBNOT [20] and in the bottom row,
(d)−(f) present our results for the same sequence. The man wearing a black jacket in the 100th frame is consistently tracked by our tracker. However,
JBNOT fails to track the man because he is occluded by other objects. FIGURE 6. Tracking results on the MOT17-09-FRCNN dataset. In the top row, (a)−(c), present the results for JBNOT [20] and in the bottom row,
(d)−(f) present our results for the same sequence. The man wearing a black jacket in the 100th frame is consistently tracked by our tracker. However,
JBNOT fails to track the man because he is occluded by other objects. FIGURE 7. Tracking results for the MOT17-11-SDP dataset. In the top row, (a)−(c) present the results for JBNOT [20]. In the bottom row, (d)−(f) present
our results for the same sequence. After the occlusion occurs, many ID-switches take place in the JBMOT tracker, but our tracker consistently tracks
objects, even during occlusions (e.g., ID-64, ID-65, ID-69, and ID-72). FIGURE 7. Tracking results for the MOT17-11-SDP dataset. In the top row, (a)−(c) present the results for JBNOT [20]. In the bottom row, (d)−(f) present
our results for the same sequence. After the occlusion occurs, many ID-switches take place in the JBMOT tracker, but our tracker consistently tracks
objects, even during occlusions (e.g., ID-64, ID-65, ID-69, and ID-72). D. MOT PERFORMANCE ANALYSIS The red numbers for each metric
represent the best performance (offline/online) and the blue numbers represent the second best performance (online). The methods marked with * are
MHT-based trackers. (Accessed on August 1, 2019.) TABLE 4. Results on the MOT16 dataset. We grouped methods according to their tracking mode (offline and online). The red numbers for each metric
represent the best performance (offline/online) and the blue numbers represent the second best performance (online). The methods marked with * are
MHT-based trackers. (Accessed on August 1, 2019.)
TABLE 5. Results on the MOT17 dataset. We grouped methods according to their tracking mode (offline and online). The red numbers for each metric
represent the best performance (offline/online) and the blue numbers represent the second best performance (online). The methods marked with * are
MHT-based trackers. (Accessed on August 2, 2019.) t. We grouped methods according to their tracking mode (offline and online). The red numbers for each metric
e/online) and the blue numbers represent the second best performance (online). The methods marked with * are
ust 2, 2019.) TABLE 5. Results on the MOT17 dataset. We grouped methods according to their tracking mode (offline and online). The red numbers for each metric
represent the best performance (offline/online) and the blue numbers represent the second best performance (online). The methods marked with * are
MHT-based trackers. (Accessed on August 2, 2019.) TABLE 5. Results on the MOT17 dataset. We grouped methods according to their tracking mode (offline and online). The red numbers for each metric
represent the best performance (offline/online) and the blue numbers represent the second best performance (online). The methods marked with * are
MHT-based trackers. (Accessed on August 2, 2019.)
typifying a track. Because the function app(·) can be of any
perform online MOT. The proposed network classifies exist- typifying a track. Because the function app(·) can be of any
type, incremental updates of appearance features can resolve
this issue. typifying a track. Because the function app(·) can be of any
type, incremental updates of appearance features can resolve
this issue. perform online MOT. The proposed network classifies exist-
ing tracks by pointing to corresponding detection results
using an attention mechanism. OneShotDA can solve the data
association problem of MOT and identify false positives in
detector outputs. To train the proposed network, we employed
a novel training strategy tailored for one-shot learning that
is suitable for data association tasks. REFERENCES [1] L. Chen, H. Ai, C. Shang, Z. Zhuang, and B. Bai, ‘‘Online multi-object
tracking with convolutional neural networks,’’ in Proc. IEEE Int. Conf. Image Process. (ICIP), Sep. 2017, pp. 645–649. [2] A. Sadeghian, A. Alahi, and S. Savarese, ‘‘Tracking the untrackable:
Learning to track multiple cues with long-term dependencies,’’ in Proc. IEEE Int. Conf. Comput. Vis. (ICCV), Oct. 2017, pp. 300–311. [3] K. Yoon, D. Kim, Y.-C. Yoon, and M. Jeon, ‘‘Data association for multi-
object tracking via deep neural networks,’’ Sensors, vol. 19, no. 3, p. 559,
Jan. 2019. [1] L. Chen, H. Ai, C. Shang, Z. Zhuang, and B. Bai, ‘‘Online multi-object
tracking with convolutional neural networks,’’ in Proc. IEEE Int. Conf.
Image Process. (ICIP), Sep. 2017, pp. 645–649. D. MOT PERFORMANCE ANALYSIS Our tracker seems to prefer DNN-based detectors to tra-
ditional detectors based on the fact that it ranks first among
online trackers on the MOT17 dataset but ranks lower on the
MOT16 dataset. We determined that our simple implemen-
tation of the function app(·) for tracks could degrade model
performance on the MOT16 dataset. The function app(T (i)
l )
simply retrieves an image patch of T (i)
l , which is an image of
the latest update with a detection result. Because the detec-
tor outputs in MOT16 are comparatively noisy, we believe
this function is insufficient for returning an image feature trackers in this study, for the purpose of simple comparisons. Trackers were grouped according to their tracking mode
(offline and online). In Tables 4 and 5, our method exhibits
performance comparable to those of existing state-of-the-art
methods. It is noteworthy that OneShotDA ranks first among
online trackers on the MOT17 benchmark. Our tracker out-
performs all other online trackers in the MOT17 group by
0.5% if we compare it with MOTDT. However, our methods
did not outperform the JBNOT, the state-of-the-art offline
method in MOT17. trackers in this study, for the purpose of simple comparisons. Trackers were grouped according to their tracking mode
(offline and online). In Tables 4 and 5, our method exhibits
performance comparable to those of existing state-of-the-art
methods. It is noteworthy that OneShotDA ranks first among
online trackers on the MOT17 benchmark. Our tracker out-
performs all other online trackers in the MOT17 group by
0.5% if we compare it with MOTDT. However, our methods
did not outperform the JBNOT, the state-of-the-art offline
method in MOT17. VOLUME 8, 2020 38068 VOLUME 8, 2020 38068 K. Yoon et al.: OneShotDA: Online Multi-Object Tracker With One-Shot-Learning-Based Data Association FIGURE 8. Qualitative results. (a)-(c): MOT16-05, (d)-(f): MOT16-06, (g)-(i): MOT17-01-FRCNN, (j)-(l): MOT17-03-SDP, (m)-(o): MOT17-07-SDP. FIGURE 8. Qualitative results. (a)-(c): MOT16-05, (d)-(f): MOT16-06, (g)-(i): MOT17-01-FRCNN, (j)-(l): MOT17-03-SDP, (m)-(o): MOT17-07-SDP. FIGURE 8. Qualitative results. (a)-(c): MOT16-05, (d)-(f): MOT16-06, (g)-(i): MOT17-01-FRCNN, (j)-(l): MOT17-03-SDP, (m)-(o): MOT17-07-SDP. FIGURE 8. Qualitative results. (a)-(c): MOT16-05, (d)-(f): MOT16-06, (g)-(i): MOT17-01-FRCNN, (j)-(l): MOT17- 38069 VOLUME 8, 2020 K. Yoon et al.: OneShotDA: Online Multi-Object Tracker With One-Shot-Learning-Based Data Association TABLE 4. Results on the MOT16 dataset. We grouped methods according to their tracking mode (offline and online). D. MOT PERFORMANCE ANALYSIS We also demonstrated
how training samples can be generated from MOTChallenge
datasets. In a series of experiments, our OneShotDA tracker
delivered performance comparable to the performances of
existing state-of-the-art methods. Additionally, our tracker
ranked first among online trackers on the MOT17 dataset. For future work, we plan to devise an incremental learn-
ing method to learn the appearance function of a track
(i.e., app(T (i)
l ) in this work). In online tracking mode, detec-
tion results come in a sequential order, meaning incremental
learning can help our tracker in updating learned appearance
features based on newly associated detection results. We further examined the performance of our method
by incorporating qualitative analysis. In Figures 6 and 7,
the robustness of our tracker against ID-switches is ana-
lyzed via frame-by-frame investigation. We compare the
results to JBNOT [20], which achieved the top rank on
the MOT17 dataset in terms of MOTA but with inferior
ID-switch performance compared to our tracker (Table 5). In Figure 6, the top row presents partial results for JBNOT on
the MOT17-09-FRCNN dataset, and the bottom row presents
the results for our method on the same sequence. This fig-
ure suggests that our tracker consistently tracks the man
with the black jacket (ID-6 in our results). However, this
object is lost by JBNOT, which initiates a new track with
ID-13 after the object is occluded by other objects. Figure 7
presents the results on the MOT17-11-SDP dataset, where
the top row represents JBNOT and the bottom row represents
our model. These results demonstrate the robustness of our
tracker against ID-switches. Our tracker consistently tracks
objects, even during heavy occlusions (e.g., ID-64, ID-65,
ID-69, and ID-72 in our results), while many switches occur
for the JBNOT tracker. Finally, we present the qualitative
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received the B.S. degree
in computer science from Sahmyook University,
Seoul, South Korea, in 2009, the M.S. degree in
image engineering from Chung-Ang University,
Seoul, in 2011, and the Ph.D. degree in electri-
cal engineering and computer science from the
Gwangju Institute of Science and Technology,
Gwangju, South Korea, in 2019. He is currently a
Research Scientist with SI-Analytics. His research
interests include multiobject tracking, computer
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tive multiple hypothesis tracking with tracklet-level association,’’ IEEE
Trans. Circuits Syst. Video Technol., vol. 29, no. 12, pp. 3660–3672,
Dec. 2019. JEONGHWAN GWAK received the Ph.D. degree
in machine learning and artificial intelligence from
the Gwangju Institute of Science and Technology
(GIST), Gwangju, South Korea, in 2014. From
2002 to 2007, he worked for several companies
and research institutes as a Researcher and a Chief
Technician. From 2014 to 2016, he worked as a
Postdoctoral Researcher with GIST. From 2016 to
2017, he worked as a Research Professor. From
2017 to 2019, he was a Research Professor with
the Department of Radiology, Biomedical Research Institute, Seoul National
University Hospital, Seoul, South Korea. In 2019, he joined the Korea
National University of Transportation, where he is currently the Director of
the Applied Machine Intelligence Laboratory. His current research interests
include deep learning, computer vision, image and video processing, evo-
lutionary computation, optimization, and relevant applications of medical
and visual surveillance systems. He has served as a PC/TPC member or
Chairman for many artificial intelligence, machine learning, and computer
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tion for multi-target tracking,’’ in Proc. IEEE Conf. Comput. Vis. Pattern
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Jul. 2019, pp. 8981–8988. 38071 VOLUME 8, 2020 VOLUME 8, 2020 K. Yoon et al.: OneShotDA: Online Multi-Object Tracker With One-Shot-Learning-Based Data Association MOONGU JEON received the B.S. degree in
architectural engineering from Korea University,
Seoul, South Korea, in 1988, and the M.S. and
Ph.D. degrees in computer science and scientific
computation from the University of Minnesota,
Minneapolis, MN, USA, in 1999 and 2001, respec-
tively. As a Postgraduate Researcher, he worked
on optimal control problems at the University of
California at Santa Barbara, Santa Barbara, CA,
USA, from 2001 to 2003. Then, he moved to the
National Research Council of Canada, where he worked on the sparse repre-
sentation of high-dimensional data and image processing until 2005. In 2005,
he joined the Gwangju Institute of Science and Technology, Gwangju, South
Korea, where he is currently a Full Professor with the School of Electrical
Engineering and Computer Science. His current research interests include
machine learning, computer vision, and artificial intelligence. YOUNG-MIN SONG received the B.S. degree
in
computer
science
and
engineering
from
Chungnam University, Daejeon, South Korea,
in 2013, and the M.S. degree in information and
communications from the Gwangju Institute of
Science and Technology (GIST), Gwangju, South
Korea, in 2015, where he is currently pursuing
the Ph.D. degree in electrical engineering and
computer science. His research interests include
multiobject tracking and data fusion. YOUNG-CHUL YOON received the B.S. KWANGJIN YOON degree
in electronics and communications engineering
from Kwangwoon University, Seoul, South Korea,
and the M.S. degree in electrical engineering and
computer science from the Gwangju Institute of
Science and Technology, Gwangju, South Korea,
in 2019. He is currently working as a Com-
puter Vision Researcher with LG Electronics. His
research interests include multiobject tracking and
deep learning. 38072 VOLUME 8, 2020
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English
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Ethylene Signaling Facilitates Plant Adaption to Physical Barriers
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Frontiers in plant science
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cc-by
| 7,284
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Ethylene Signaling Facilitates Plant
Adaption to Physical Barriers Simu Liu1* and Hui Chen2* 1 Guangdong Provincial Key Laboratory for Plant Epigenetics, College of Life Sciences and Oceanography, Shenzhen
University, Shenzhen, China, 2 Guangdong Key Laboratory of Genome Instability and Human Disease, School of Medicine,
Shenzhen University, Shenzhen, China Reviewed by: Reviewed by:
Keith Lindsey,
Durham University, United Kingdom
Irina Ivanova Vaseva,
Bulgarian Academy of Sciences
(BAS), Bulgaria
Satoko Yoshida,
Nara Institute of Science
and Technology (NAIST), Japan
Brad M. Binder,
The University of Tennessee,
Knoxville, United States Reviewed by:
Keith Lindsey,
Durham University, United Kingdom
Irina Ivanova Vaseva,
Bulgarian Academy of Sciences
(BAS), Bulgaria
Satoko Yoshida,
Nara Institute of Science
and Technology (NAIST), Japan
Brad M. Binder,
The University of Tennessee,
Knoxville, United States Keywords: ethylene signaling, mechanical impedance, morphological changes, seedling emergence, root
elongation, parasitic plant invasion INTRODUCTION *Correspondence:
Simu Liu
liusm@szu.edu.cn
Hui Chen
huichen@szu.edu.cn;
huichen_0408@163.com In natural environments, the life cycle of terrestrial plants usually starts with the germination of
seeds buried under the soil. After germination, seedlings need to successfully fulfill emergence
from the soil to establish a photoautotrophic lifestyle, and the roots of seedling should forage
soil for mineral nutrients and water acquisition. During these processes, the shoots and roots
are subjected to mechanical stress from the soil. In addition, parasitic plants, whose survival
depends on the host plants, encounter the physical barrier provided by the host plants, thereby
they need to penetrate through the host tissues to access nutrients. To acclimate to these
physical barriers, plants adopt a developmental strategy by which they adjust their morphological
modifications in accordance with the mechanical impedance (Leivar et al., 2008; Yoshida et al.,
2016; Gommers and Monte, 2018; Vaseva et al., 2018), such as hypocotyl thickening, apical hook
formation, and root shortening occur within autotrophic plants and haustoria emergence within
parasitic plants. Specialty section:
This article was submitted to
Plant Physiology,
a section of the journal
Frontiers in Plant Science Specialty section:
This article was submitted to
Plant Physiology,
a section of the journal
Frontiers in Plant Science
Received: 20 April 2021
Accepted: 02 July 2021
Published: 29 July 2021 Received: 20 April 2021
Accepted: 02 July 2021
Published: 29 July 2021 It has been reported that mechanical impedance boosts the production of ethylene (Goeschl
et al., 1966; Zhong et al., 2014). As a gaseous plant hormone, ethylene diffuses rapidly and regulates
diverse developmental and physiological processes in plants such as seedling emergence, root
elongation, leaf senescence, fruit ripening, root nodulation, as well as biotic and abiotic stress
responses (Bleecker and Kende, 2000). It is not surprising that terrestrial plants employ ethylene
as the vital signal molecule to regulate the directional growth of plant organs since the ethylene The morphological changes are usually observed in the terrestrial plants to respond
to physical barriers. The phytohormone ethylene plays an essential role in the
morphological development of plants encountering exogenous mechanical impedance,
which enables plants to grow optimally in response to physical barriers. Ethylene is
shown to regulate these developmental processes directly or in concert with other
phytohormones, especially auxin. In this mini review, the involvement of ethylene action
in seedling emergence from the soil, root movement within the soil, and parasitic plant
invasion of the host plant are described. Edited by: Anna N. Stepanova,
North Carolina State University,
United States MINI REVIEW
published: 29 July 2021
doi: 10.3389/fpls.2021.697988 MINI REVIEW
published: 29 July 2021
doi: 10.3389/fpls.2021.697988 ETHYLENE SIGNALING PATHWAY To overcome the soil barrier and reach sunlight, seedlings
frequently adopt a developmental strategy dynamically adjusting
their morphological structures in accordance to soil depth
(Zhong et al., 2014). In Arabidopsis, the short and thick hypocotyl
is suitable for enhancing the lifting capacity of etiolated seedlings,
and the apical hook is an elegant structure for protecting the
delicate shoot apical meristem against the mechanical damage
when seedlings emerge from the soil. In rice, the coleoptile
and mesocotyl are the two essential structures of seedlings
responsible for moving toward the soil surface (Lee et al., 2017;
Xiong et al., 2017). The coleoptile, which wraps the emergent
shoot, safeguards the plumule against mechanical injures. The
mesocotyl, a structure between the coleoptile node and the basal
part of the seminal root in etiolated monocot seedlings, pushes
the buds out of the soil. It has been reported that the soil-imposed
mechanical impedance stimulates the production of ethylene
(Goeschl et al., 1966; Zhong et al., 2014; Xiong et al., 2017), which
acts as a key signal linking plant responses to the environmental
cues (Sarquis et al., 1991; He et al., 1996). In 1901, Dimitry Neljubov described the effects of ethylene
on the growth of etiolated pea seedlings, a morphology that
by 1913 was termed as the “triple response” in some dicots
showing shorter and thicker hypocotyl, an exaggerated apical
hook, and a shorter and swelling root (Bakshi et al., 2015). This response was utilized to genetically screen the mutants
with altered ethylene response in dicotyledonous Arabidopsis
thaliana (Bleecker et al., 1988; Guzmán and Ecker, 1990). The
main components of ethylene signaling have been identified,
including five endoplasmic reticulum (ER)-localized receptors,
namely, ethylene response 1 (ETR1), ethylene response sensor
1 (ERS1), ETR2, ERS2, and ethylene insensitive 4 (EIN4), the
negative regulator, namely, constitutive triple response 1 (CTR1),
the central mediator EIN2, and the master transcription factors,
namely, ethylene insensitive 3 (EIN3) and EIN3-like 1 (EIL1). The ethylene signaling pathway in plants is well characterized
(Figure 1A). In the absence of ethylene, the active receptors
recruit CTR1 to phosphorylate EIN2 and thus lead to EIN2
degradation mediated by the 26S proteasome, which represses
the downstream signaling pathway. In the presence of ethylene,
the receptors are inactivated, reducing CTR1-mediated EIN2
phosphorylation and consequently resulting in EIN2 C-terminal
end (EIN2-C) cleavage from ER-bound EIN2-N portion (Ju et al.,
2012; Qiao et al., 2012). ETHYLENE SIGNALING PATHWAY The EIN2-C enters the cytoplasmic
P-body, where it represses the translation of EIN3-degrading
factors, namely, EIN3-binding F-box proteins 1 and 2 (EBF1/2)
and stabilizes EIN3/EIL1 proteins (Li et al., 2015; Merchante et al.,
2015). EIN2-C is also able to translocate from the cytoplasm to
the nucleus, where it associates with a histone binding protein for
epigenetically facilitating EIN3/EIL1-dependent transcriptional
regulation (Zhang et al., 2017). EIN3/EIL1 can function as a
transcriptional activator or repressor in response to ethylene
(Wang et al., 2020). Citation: Citation:
Liu S and Chen H (2021) Ethylene
Signaling Facilitates Plant Adaption
to Physical Barriers. Front. Plant Sci. 12:697988. doi: 10.3389/fpls.2021.697988 Citation:
Liu S and Chen H (2021) Ethylene
Signaling Facilitates Plant Adaption
to Physical Barriers. Front. Plant Sci. 12:697988. doi: 10.3389/fpls.2021.697988 July 2021 | Volume 12 | Article 697988 1 Frontiers in Plant Science | www.frontiersin.org Ethylene Regulation on Mechanical Stresses Liu and Chen stress (Hattori et al., 2009). These findings suggest a pivotal
role of ethylene in modulating plant developmental processes to
acclimate to ever-changing environments. signaling system existed prior to the plant colonization of land
(Ju et al., 2015). In this mini review, we aimed to summarize
current advances regarding ethylene regulation of morphological
changes in facilitating autotrophic plant adaption to soil-
provided physical barriers, as well as parasitic plant conquest to
host plant-created physical barriers. Frontiers in Plant Science | www.frontiersin.org ETHYLENE SUPPRESSES HYPOCOTYL
ELONGATION IN RESPONSE TO SOIL
BARRIER (B) Ethylene facilitates Arabidopsis adaption to soil
barriers. Ethylene regulates apical hook development by two different pathways. On the one hand, ethylene reinforces asymmetric auxin distribution within the apical
hypocotyl by inducing the expression of AUX1 and PINs, and by repressing the expression of WDL4 that influences PIN protein trafficking (Vandenbussche et al.,
2010; Zádníková et al., 2010; Deng et al., 2021). On the other hand, ethylene regulates differential growth of apical hypocotyl through EIN3/EIL1 transcriptional
regulation of HLS1 expression (Shen et al., 2016). Other endogenous phytohormones including gibberellic acid, jasmonic acid, and salicylic acid and light signal also
modulate ethylene-regulated apical hook development by influencing the EIN3/EIL1-HLS1 module (An et al., 2012; Zhang et al., 2014; Shi et al., 2016a; Huang
et al., 2020). Ethylene inhibits hypocotyl elongation by activation of ERF1 pathway, upregulation of WDL5 expression, and downregulation of PGX3 expression via
EIN3-dependent signaling pathway (Zhong et al., 2014; Sun et al., 2015; Wu et al., 2020). Ethylene inhibits root elongation by induction of auxin biosynthesis through
upregulating WEI8/TAA1 and ASA1 expression in EIN3- and ARR1-dependent and ERF1-dependent manner, respectively (Stepanova et al., 2005, 2008; Mao et al.,
2016; Yan et al., 2017). (C) Ethylene facilitates rice adaption to soil barriers. In monocot rice seedlings, ethylene promotes both coleoptile and mesocotyl elongation
by repression of GY1 expression via OsEIL2-dependent signaling pathway (Xiong et al., 2017). Ethylene inhibits root growth by promoting auxin biosynthesis through
inducing OsYUC8 expression in an OsEIL1-dependent manner (Qin et al., 2017). (D) Ethylene signaling is required for the parasitic plant to invade the host plant. In
the parasitic plant Phtheirospermum japonicum, the PjETR1 and PjEIN2, homologs of Arabidopsis ETR1 and EIN2, are required for intrusive cell development in
response to ethylene produced by the host plants (Cui et al., 2020). beneficial for EIN3-phytochrome interacting factor 3 (PIF3)-
modulated preassembly of photosynthetic machinery, which
is necessary for the seedling transition from dark to light
(Zhong et al., 2012, 2014). phases of seedling germination, a gravity-stimulated and PIN2-
directed asymmetric auxin distribution in the root is able to
extend into hypocotyl and thus initiates the establishment of
asymmetric auxin distribution in this zone (Zhu et al., 2019). Ethylene-induced expression of AUX1 and PINs within the apical
hypocotyl may further reinforce this initial auxin asymmetry
(Vandenbussche et al., 2010; Zádníková et al., 2010). ETHYLENE SUPPRESSES HYPOCOTYL
ELONGATION IN RESPONSE TO SOIL
BARRIER In Arabidopsis, the subterranean hypocotyl growth during soil
emergence is an important phase for seedling establishment and
survival. Under the soil, ethylene employs diverse pathways to
suppress the hypocotyl elongation and increase the hypocotyl
strength (Figure 1B), hence improves the capacity of hypocotyl
response to soil barrier. Ethylene activates the ethylene response
factor 1 (ERF1) pathway in an EIN3-dependent manner to
slow down hypocotyl elongation and thus enables seedlings to
penetrate through the soil layers more easily (Zhong et al., 2014). Ethylene is also able to inhibit hypocotyl cell elongation by
upregulating wave-dampened2-like 5 (WDL5) gene expression
through the EIN3-dependent signaling pathway (Sun et al., 2015). WDL5 encodes a microtubule-associated protein that stabilizes
the microtubule of cortical cells in the etiolated hypocotyl. In addition, ethylene suppresses pectin degradation and thus
enhances cell wall stiffness by EIN3-mediated downregulation
of polygalacturonase involved in expansion 3 (PGX3) gene
expression (Wu et al., 2020). This EIN3-PGX3 regulatory
module contributes to the inhibition of hypocotyl elongation
as well as the facilitation of seedling soil emergence. Although
ethylene inhibition of hypocotyl elongation may delay seedlings
reaching the sunlight, a longer period of etiolated growth is Although the linear ethylene signaling pathway is conserved
between monocotyledonous rice and dicotyledonous Arabidopsis
(Yang et al., 2015a), the exogenous application of ethylene inhibits
root growth but promotes coleoptile growth of etiolated rice
seedlings (Ma et al., 2013). Different from Arabidopsis EIN3 and
EIL1, which regulate both shoot and root responses to ethylene,
the rice EIN3 transcription factors, namely, OsEIL1 and OsEIL2,
mediate the inhibition of root elongation and promotion of
coleoptile elongation, respectively (Yang et al., 2015b). Unlike
rice, ethylene inhibits coleoptile elongation in other monocots,
including maize, wheat, sorghum, and Brachypodium distachyon
(Yang et al., 2015a). Considering that rice is cultivated mainly
in the frequently flooded river deltas, ethylene-promoted tissue
elongation via gibberellin may contribute to diminish flooding July 2021 | Volume 12 | Article 697988 Frontiers in Plant Science | www.frontiersin.org 2 Ethylene Regulation on Mechanical Stresses Liu and Chen FIGURE 1 | Ethylene signaling regulates the growth and development of plants encountering physical barriers. (A) The linear model of the ethylene signaling
pathway in Arabidopsis. Ethylene signaling involves ethylene receptors [ethylene response 1 (ETR1), ethylene response sensor 1 (ERS1), ETR2, ethylene insensitive 4
(EIN4), and ERS2], the negative regulator [constitutive triple response 1 (CTR1)], the central mediator (EIN2), and the master transcription factors [EIN3/EIN3-like 1
(EIL1)]. ETHYLENE SUPPRESSES HYPOCOTYL
ELONGATION IN RESPONSE TO SOIL
BARRIER Moreover,
ethylene represses the expression of WDL4 via the EIN3-
dependent signaling pathway, which influences PIN protein
trafficking and auxin asymmetric distribution (Figure 1B; Deng
et al., 2021). Then, the transmembrane kinase 1 (TMK1)-
mediated auxin response pathway translates differential cellular
auxin to differential cell growth during apical hook formation
(Cao et al., 2019). A recent finding also supports that PIN-
facilitated polar auxin transport followed by TMK-mediated
auxin signaling pathway is required for mechanically induced
apical hook formation (Baral et al., 2021). ETHYLENE SUPPRESSES HYPOCOTYL
ELONGATION IN RESPONSE TO SOIL
BARRIER The linear ethylene signaling pathway is conserved between rice and Arabidopsis (Yang et al., 2015a). (B) Ethylene facilitates Arabidopsis adaption to soil
barriers. Ethylene regulates apical hook development by two different pathways. On the one hand, ethylene reinforces asymmetric auxin distribution within the apical
hypocotyl by inducing the expression of AUX1 and PINs, and by repressing the expression of WDL4 that influences PIN protein trafficking (Vandenbussche et al.,
2010; Zádníková et al., 2010; Deng et al., 2021). On the other hand, ethylene regulates differential growth of apical hypocotyl through EIN3/EIL1 transcriptional
regulation of HLS1 expression (Shen et al., 2016). Other endogenous phytohormones including gibberellic acid, jasmonic acid, and salicylic acid and light signal also
modulate ethylene-regulated apical hook development by influencing the EIN3/EIL1-HLS1 module (An et al., 2012; Zhang et al., 2014; Shi et al., 2016a; Huang
et al., 2020). Ethylene inhibits hypocotyl elongation by activation of ERF1 pathway, upregulation of WDL5 expression, and downregulation of PGX3 expression via
EIN3-dependent signaling pathway (Zhong et al., 2014; Sun et al., 2015; Wu et al., 2020). Ethylene inhibits root elongation by induction of auxin biosynthesis through
upregulating WEI8/TAA1 and ASA1 expression in EIN3- and ARR1-dependent and ERF1-dependent manner, respectively (Stepanova et al., 2005, 2008; Mao et al.,
2016; Yan et al., 2017). (C) Ethylene facilitates rice adaption to soil barriers. In monocot rice seedlings, ethylene promotes both coleoptile and mesocotyl elongation
by repression of GY1 expression via OsEIL2-dependent signaling pathway (Xiong et al., 2017). Ethylene inhibits root growth by promoting auxin biosynthesis through
inducing OsYUC8 expression in an OsEIL1-dependent manner (Qin et al., 2017). (D) Ethylene signaling is required for the parasitic plant to invade the host plant. In
the parasitic plant Phtheirospermum japonicum, the PjETR1 and PjEIN2, homologs of Arabidopsis ETR1 and EIN2, are required for intrusive cell development in
response to ethylene produced by the host plants (Cui et al., 2020). FIGURE 1 | Ethylene signaling regulates the growth and development of plants encountering physical barriers. (A) The linear model of the ethylene signaling
pathway in Arabidopsis. Ethylene signaling involves ethylene receptors [ethylene response 1 (ETR1), ethylene response sensor 1 (ERS1), ETR2, ethylene insensitive 4
(EIN4), and ERS2], the negative regulator [constitutive triple response 1 (CTR1)], the central mediator (EIN2), and the master transcription factors [EIN3/EIN3-like 1
(EIL1)]. The linear ethylene signaling pathway is conserved between rice and Arabidopsis (Yang et al., 2015a). ETHYLENE SIGNALING IS REQUIRED
FOR ROOT ADAPTION TO SOIL
BARRIER In most cases, the shoots encounter soil barriers only before they
emerge from the soil, whereas the roots have to face persistent
soil barriers in the whole life cycle. Thus, the roots need to
continuously adjust their morphological structures to respond to
ever-changing local soil environments. The physically impeded
roots display diverse morphological alterations, such as inhibition
of root elongation and increase of radial dimension, resembling
the morphological changes observed when roots are grown in
the presence of ethylene (Jacobsen et al., 2021). Consistent with
these observations, when ethylene biosynthesis or signaling is
repressed, the degree of root response to mechanical impedance
is significantly alleviated (Santisree et al., 2011). Interestingly,
the loss of function of WDL5 gene, which is a target of EIN3,
impairs mechanical stress-inhibited elongation of both hypocotyl
and root (Sun et al., 2015; Okamoto et al., 2021), suggesting that
a general mechanism regulating cell elongation should be crucial
for ethylene regulation of morphological changes in response to
mechanical impedance. Upon seedling reaching the soil surface, the mechanical force-
stimulated production of ethylene decreases, whereas the light
exposure increases. The light represses COP1 nuclear localization
and thus releases EBF1/2 for EIN3 degradation (Shi et al., 2016a). Meanwhile, the light activates the photoreceptor phytochrome
B (PhyB) and promotes its translocation into the nucleus,
where it enhances EBF1/2-stimulated EIN3/EIL1 degradation
and disrupts HLS1 oligomer formation as well (Shi et al.,
2016b; Lyu et al., 2019). Therefore, the light-regulated EIN3/EIL1
reduction inactivates ethylene signaling, which attenuates hook
curvature but facilitates hook opening and cotyledon expansion
to initiate photomorphogenic growth programs. The root cap, which is located at the apex of the root
and protects the root apical meristem, is able to sense
mechanical stimulus and control the direction of root growth
(Dreyer and Edelmann, 2018). Removal of root cap disturbs
ethylene-inhibited root growth and affects root exploration and
penetration in the soils, indicating that the root cap should be
a potential site of the ethylene-regulated root–soil interaction
(Hahn et al., 2008). When in contact with the soil, the root cap
needs to evaluate the resistance strength of the soil and then takes
the decision to penetrate through the soil layers or avoidance
of obstacles. Charles and Francis Darwin have described this
intelligent root cap in their book “The Power of Movement
of Plants” (Darwin and Darwin, 1881). ETHYLENE SIGNALING IS REQUIRED
FOR ROOT ADAPTION TO SOIL
BARRIER Ethylene emission and
accumulation around the root tip tissues may be crucial for root
caps to choose the above two different strategies according to the
soil conditions. Ethylene-promoted increased diameter of roots
may lead to favorable mechanical strength to penetrate through
the penetrable soil layers; alternatively, ethylene-inhibited root
elongation may facilitate avoidance of compacted soils. In
agreement with this view, compacted soil-restricted ethylene
diffusion triggers a rapid and sustained increase of EIN3-green
fluorescent protein in cell nuclei at the root elongation zone,
indicating that ethylene signaling is strongly activated in this
zone. Consistently, the roots of Osein2 and Oseil1 mutants ETHYLENE REGULATES APICAL HOOK
DEVELOPMENT DURING SOIL
EMERGENCE The differential distribution of auxin across the apical hypocotyl
region and accumulation on the concave (inner) is essential
for regulating asymmetric cell elongation between concave
and convex (outer) and consequently leading to apical hook
formation (Raz and Ecker, 1999; Béziat and Kleine-Vehn, 2018). Auxin distribution is facilitated by the auxin influx carrier auxin-
resistant 1 (AUX1) and the efflux carrier pin-formed (PIN)
family proteins (Petrásek and Friml, 2009). During the early Besides depending on the auxin pathway, ethylene regulates
apical hook development through EIN3/EIL1 transcriptional July 2021 | Volume 12 | Article 697988 Frontiers in Plant Science | www.frontiersin.org 3 Ethylene Regulation on Mechanical Stresses Liu and Chen regulation of hookless 1 (HLS1) expression (EIN3/EIL1-HLS
module) (Figure 1B; Shen et al., 2016). HLS1 encodes a
putative N-acetyltransferase, and the loss of function of the
HLS1 gene completely disrupts apical hook curvature (Lehman
et al., 1996), indicating an essential role of HLS1 in apical
hook formation. Although the biochemical nature of HLS1
protein remains mysterious, the formation of HLS1 oligomer
is required for its function during apical hook formation (Lyu
et al., 2019). Other endogenous phytohormones and light
signal also modulate apical hook development by influencing
the EIN3/EIL1-HLS1 module. In darkness, the gibberellin-
repressed DELLA, jasmonic acid-activated MYC2, or salicylic
acid-activated NPR1 can directly interact with and alter the
transcriptional activity of EIN3/EIL1 (An et al., 2012; Zhang
et al., 2014; Huang et al., 2020). The photomorphogenic
central repressor constitutive photomorphogensis 1 (COP1)
is
also
able
to
stabilize
EIN3/EIL1
protein
by
directly
ubiquitinating EBF1/2 for degradation (Shi et al., 2016a). It
is still unknown how HLS1 regulates asymmetric cell growth
during apical hook formation. A potential explanation may
be
that
HLS1
mediates
ethylene-repressed
accumulation
of auxin response factor 2 (ARF2) protein, which is an
auxin
response
transcription
factor
negatively
regulating
differential auxin response during apical hook formation
(Li et al., 2004). longer coleoptile and longer mesocotyl of etiolated rice seedlings
in the presence of ethylene (Ma et al., 2013), indicating a
major role of ethylene in promoting coleoptile and mesocotyl
elongation in rice seedlings. Other phytohormones such as
brassinosteroids and strigolactones are also involved in regulating
mesocotyl growth (Sun et al., 2018; Zheng et al., 2020). How
ethylene coordinately with other hormones modulates mesocotyl
elongation during rice seedling emergence from the soil needs
more detailed studies. Frontiers in Plant Science | www.frontiersin.org ETHYLENE PROMOTES ELONGATION
OF COLEOPTILE AND MESOCOTYL IN
RICE In Arabidopsis seedlings, thickened hypocotyl and apical hook are
the key morphological structures facilitating the soil emergence,
while in rice seedlings they are coleoptile and mesocotyl. Ethylene-promoted coleoptile and mesocotyl elongation are
mediated by transcription factor OsEIL2 in rice seedlings
(Figure 1C). OsEIL2 directly represses transcription of the
gaoyao 1 (GY1), which encodes a phospholipase that functions
at the initial step of jasmonic acid biosynthesis (Xiong et al.,
2017). Consistent with this, ethylene emission increases while
jasmonic acid content decreases in the rice seedlings with
increasing seed-sowing depth of soil, suggesting that ethylene
stimulates coleoptile and mesocotyl elongation to facilitate
seedling emergence by inhibition of jasmonic acid biosynthesis
(Xiong et al., 2017). Overexpression of OsEIN2 also leads to a Frontiers in Plant Science | www.frontiersin.org July 2021 | Volume 12 | Article 697988 4 Ethylene Regulation on Mechanical Stresses Liu and Chen laboratory to expand our understanding of communication
between parasitic and host plants. that are insensitive to ethylene penetrate compacted soil more
effectively than wild-type’s roots (Pandey et al., 2021). In screening for mutants that display ethylene insensitivity
in roots of Arabidopsis, mutants related to auxin biosynthesis,
auxin transport, and auxin signaling were identified (Stepanova
et al., 2007). In Arabidopsis, the main auxin is indole-3-acetic
acid (IAA), which is mainly synthesized by the indole-3-pyruvic
acid (IPA) pathway using tryptophan (Trp) as a precursor (Zhao,
2012). Upon perception of auxin, transport inhibitor response
1 (TIR1) and auxin signaling F-box proteins (AFBs) physically
interact with auxin or IAA (Aux/IAA) transcriptional repressors
and promote the degradation of them, which release ARF
transcription factors to activate diverse auxin-responsive genes
(Chapman and Estelle, 2009). After germination, Orobanchaceae parasitic plants initiate
haustorium formation since they recognize active haustorium-
inducing factors (HIFs) such as 2,6-dimethoxy-p-benzoquinone
(DMBQ), produced by the potential hosts. Upon contact to
host roots, a haustorium initiates invasion using the intrusive
cells potentially differentiated from epidermal cells at the
apex. In T. versicolor, the production of ethylene increases in
response to DMBQ, and the blockage of ethylene biosynthesis
or signaling almost completely suppresses haustorium formation,
suggesting that ethylene is involved in haustorium development
(Tomilov et al., 2005). To uncover the genetic programs of
haustorium development, a genetic screening was performed
using P. japonicum, and the mutation of PjETR1 and PjEIN2,
encoding a homolog of Arabidopsis ETR1 and EIN2, respectively,
were identified (Figure 1D). Both mutants displayed elongated
haustorium but failed host invasion. CONCLUSION AND PERSPECTIVES As a plant hormone, ethylene promotes the environmental
adaption
of
plants
by
regulating
diverse
morphological
development. In this mini review, we described the role of
ethylene in facilitating plant adaption to diverse physical barriers
for survival. To establish a photoautotrophic lifestyle, ethylene
promotes hypocotyl shortening and apical hook formation in
Arabidopsis and both coleoptile and mesocotyl elongation in
rice to support them to move through the soils more effectively
and safely. To uptake water and mineral nutrients from the
soils, ethylene modulates root growth for optimal response to
soil barriers. To establish a successful parasitic relationship,
ethylene promotes host invasion by stimulating intrusive cell
development at the haustorium apex. ETHYLENE PROMOTES ELONGATION
OF COLEOPTILE AND MESOCOTYL IN
RICE Further observations suggest
that parasitic plants initiate haustorium development when they
detect HIFs and maintain haustorium elongation until they sense
host-derived ethylene, which inhibits haustorium elongation but
promotes differentiation of haustorium apex cells into intrusive
cells for host invasion (Cui et al., 2020). These findings indicate
that ethylene signaling is essential for parasitic plants to overcome
physical barriers provided by host plants. p
The weak ethylene insensitive 2/anthranilate synthase alpha
1 (WEI2/ASA1) and WEI7/anthranilate synthase beta 1 (ASB1)
genes encode rate-limiting enzyme of Trp biosynthesis. Ethylene
inhibits root elongation by upregulation of WEI2/ASA1 and
WEI7/ASB1 to lead to auxin accumulation in the root tip, and
ERF1 is shown to physically bind to the ASA1 promoter and
induces its expression (Stepanova et al., 2005; Mao et al., 2016). The WEI8/tryptophan aminotransferase of Arabidopsis 1 (TAA1)
and Yucca (YUC) encode two key enzymes in the IPA pathway
and catalyze the conversion of Trp to IPA and IPA to IAA,
respectively (Zhao, 2012). EIN3 regulates WEI8/TAA1 expression
by interacting with and enhancing response regulator 1 (ARR1)
transcriptional activity (Figure 1B; Stepanova et al., 2008; Yan
et al., 2017). In rice, OsEIL1 is able to directly target the OsYUC8
promoter to regulate its expression (Figure 1C; Qin et al., 2017). In addition, reduced expression of rice auxin receptor genes
OsTIR1 and OsAFB2 cause root ethylene insensitive (Chen et al.,
2018). Furthermore, loss of function of soil-surface rooting 1
(SOR1), an E3 ubiquitin ligase modulating protein stability of
OsIAA26 and OsIAA9, also leads to defective of root ethylene
response (Chen et al., 2018). A recent finding shows that auxin
is involved in root-obstacle avoidance and especially the PIN-
mediated polar auxin transport facilitates this process (Lee et al.,
2020). These findings indicate that ethylene largely employs the
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roots. Mol. Plant 1, 359–367. doi: 10.1093/mp/ssm027 Béziat,
C.,
and
Kleine-Vehn,
J. (2018). The
road
to
auxin-dependent
growth
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10.1104/pp.112.2.463 Bleecker, A. B., Estelle, M. A., Somerville, C., and Kende, H. (1988). Insensitivity
to ethylene conferred by a dominant mutation in Arabidopsis thaliana. FUNDING Characterizing the role of ethylene in facilitating plant
adaption to physical barriers at the levels of molecular biology,
biochemistry, and cellular biology is therefore important not
only to expand our understanding of the plant–soil/plant–
plant interaction but also to breed crops that can grow
optimally
in
unfavorable
lands
with
compact
soils
and
develop a useful strategy to control parasitic plant-caused
cereal yield losses. This study was supported by a grant from the National Natural
Science Foundation of China (31800213). ETHYLENE SIGNALING IS REQUIRED
FOR THE PARASITIC PLANT TO INVADE
THE HOST PLANT Although some targets regulated directly by EIN3/EIL1
have been identified (Figure 1), it remains largely unclear
how
mechanical
impedance
triggers
ethylene
action. Mechanosensitive ion channels are a common mechanism
for sensing mechanical impedance. In Arabidopsis, the MID1-
complementing
activity
1
(MCA1)
is
a
Ca2+-permeable
mechanosensitive
channel
enabling
roots
to
overcome
mechanical barriers (Okamoto et al., 2021), and the piezo1
(PZO1) is another ion channel that affects Ca2+ transients in
response to mechanical stimulation and is required for roots
to penetrate hard agar (Mousavi et al., 2021). The ethylene
is biosynthesized by two dedicated enzymatic reactions. The
substrate S-adenosyl-L-methionine is converted to the ethylene
precursor 1-aminocyclopropane-1-carboxylic acid (ACC) by the
ACC synthase (ACS), and then the ACC is converted to ethylene Unlike most of plants whose shoots and roots mainly encounter
soil-caused physical barriers, parasitic plants majorly experience
another type of physical barrier organized by the host plants
(Twyford, 2018). The parasitic plants need to obtain all or
part of nutrients from host plants to support their survival, so
they form a special morphological structure termed haustoria to
attach to hosts, penetrate through the host tissues, and ultimately
establish the physiological conduit (Clarke et al., 2019). The
Orobanchaceae parasitic plants are root parasitic plants that
establish a connection with host plants by the formation of
haustorium. The Triphysaria versicolor and Phtheirospermum
japonicum belong to Orobanchaceae have been studied in the July 2021 | Volume 12 | Article 697988 5 Ethylene Regulation on Mechanical Stresses Liu and Chen ACKNOWLEDGMENTS We apologize to those whose work could not be cited due to
space limitations. AUTHOR CONTRIBUTIONS by the ACC oxidase (ACO) (Pattyn et al., 2021). In Vigna
radiata, the AIM-1 is a key ACS, whose mRNA levels dramatically
increase in the presence of mechanical stimulation (Botella et al.,
1995). In Arabidopsis, the ACS6 expression was induced by touch
(Arteca and Arteca, 1999). It will be interesting to study whether
and how mechano-sensitive ion channel-mediated increase of
cytoplasmic Ca2+ upregulates ACS gene expression and thus
triggers ethylene production and signaling. Both authors listed have made a substantial, direct and
intellectual
contribution
to
the
work,
and
approved
it
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signaling acts parallel to and additively with strigolactone signaling to regulate
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tpc.20.00123 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. 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. Frontiers in Plant Science | www.frontiersin.org July 2021 | Volume 12 | Article 697988 REFERENCES Any product that may be evaluated in
this article, or claim that may be made by its manufacturer, is not guaranteed or
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pnas.1402491111 Copyright © 2021 Liu and Chen. 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. Zhu, Q., Gallemi, M., Pospisil, J., Zadnikova, P., Strnad, M., and Benkova, E. (2019). Root gravity response module guides differential growth determining
both root bending and apical hook formation in Arabidopsis. Development
146:dev175919. July 2021 | Volume 12 | Article 697988 Frontiers in Plant Science | www.frontiersin.org 8
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First record of the invasive Hemidactylus mabouia (Moreau de Jonn&egrave;s, 1818) (Squamata, Gekkonidae), in the dry Chaco, Argentina
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Introduction Arnold (2006), which based on DNA sequences suggest
the presence of H. mabouia in the new world at least 500
years ago. In Argentina, was reported for the first time
by Williams (1988), from an urban area in Buenos Aires
city, although misidentified as Hemidactylus turcicus (see
Baldo et al. 2008). Later on, the presence of H. mabouia
was described for Iguazu National Park, Misiones
province (Genise and Montanelli 1991), and successive
reports indicate the presence of this taxon in Chaco, Cor
rientes, and Formosa provinces (Federico and Cacivio
2000, Alvarez et al. 2002, 2009, Baldo et al. 2008). The invasive Hemidactylus mabouia is one of the most
widespread introduced species of reptiles (Hughes et al. 2015). It inhabits in many countries of Africa (including
several Seychelles islands), the Caribbean; in the state of
Florida (USA), on Madeira (Portugal), Central America
(Costa Rica, Honduras, Mexico, and Panama), and South
America (Argentina, Bolivia, Brazil, Colombia, Ecua
dor, French Guiana, Guyana, Paraguay, Peru, Suriname,
Venezuela and Uruguay) (Uetz et al. 2017). The species
inhabits natural anthropic impacted areas or urban cen
ters (Vanzolini 1978, Baldo et al. 2008), being reported
in some invasive Hemidactylus, the ability to displace
native species (Hanley et al. 1998, Rivas Fuenmayor et
al. 2005, Dame and Petren 2006). Key words Introduced species; Hemidactylus; gecko; biogeographic region. Academic editor: Raúl Maneyro | Received 4 April 2018 | Accepted 10 July 2018 | Published 3 August 2018 Citation: Torres PJ, Escalante O, Cardozo D (2018) First record of the invasive Hemidactylus mabouia (Moreau de Jonnès, 1818) (Squamata,
Gekkonidae), in the dry Chaco, Argentina. Check List 14 (4): 633–636. https://doi.org/10.15560/14.4.633 Check List 14 (4): 633–636
https://doi.org/10.15560/14.4.633
NOTES ON GEOGRAPHIC DISTRIBUTION Check List 14 (4): 633–636
https://doi.org/10.15560/14.4.633
NOTES ON GEOGRAPHIC DISTRIBUTION Copyright Torres 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. Check List 14 (4): 633–636
https://doi.org/10.15560/14.4.633 NOTES ON GEOGRAPHIC DISTRIBUTION First record of the invasive Hemidactylus mabouia (Moreau de
Jonnès, 1818) (Squamata, Gekkonidae), in the dry Chaco, Argentina Pablo Javier Torres, Orlando Escalante, Darío Cardozo Pablo Javier Torres, Orlando Escalante, Darío Cardozo Laboratorio de Genética Evolutiva, Instituto de Biología Subtropical (IBS, UNaM-CONICET), Feliz de Azara 1552, Posadas, CP 3300, Misiones,
Argentina. Corresponding author: Darío Cardozo, darcardz@gmail.com Laboratorio de Genética Evolutiva, Instituto de Biología Subtropical (IBS, UNaM-CONICET), Feliz de Azara 1552, Posadas, C
Argentina Laboratorio de Genética Evolutiva, Instituto de Biología Subtropical (IBS, UNaM-CONICET), Feliz de Azara 1552, Posadas, CP 3300, Misiones,
Argentina. C
di
th
D í C d
d
d @
il g
Corresponding author: Darío Cardozo, darcardz@gmail.com Corresponding author: Darío Cardozo, darcardz@gmail.com Abstract The invasive Hemidactylus mabouia (Moreau de Jonnès, 1818) is one of the most widespread introduced species of
reptiles, being present in the New World at least 500 years ago. In this work, we report for the first time, the presence
of the invasive gecko H. mabouia in the dry Chaco, a biogeographic region included in the Gran Chaco Sudamericano. We collected 3 individuals in an urban zone at Las Lomitas, Patiño department, Formosa Province, Argentina. This
new record extends the distribution range of this introduced species by nearly 300 km (in a straight line) from Formosa
city, the nearest point previously reported. Hemidactylus mabouia (Moreau de Jonnès, 1818) New record. Argentina: Formosa Province, Patiño
department: Las Lomitas city (24°42ʹ26ʺ S, 060°35ʹ40ʺ
W, WGS 84, 130 m a.s.l.), collected by D. Cardozo, P. Torres and O. Escalante on 29 September 2017 (3 juve
nile specimens, LGE 20047–9) (Fig. 1). Acknowledgements DC thanks PICT 2014-1343 for financial support. Authors thanks to anonymous reviewers for improving
the manuscript. Identification. Specimens were identified following
Kluge (1969), Hoogmoed (1973) and Avila-Pires (1995)
by the lamellae under the fourth toe, shape of the pupil,
number of supralabial and infra labial scales (Fig. 2). Authors’ Contributions All authors collected the data, made identifications, wrote
the manuscript; and made the figures. Methods Three juvenile specimens were collected during a field
trip in austral spring 2017. The collected specimens were
euthanized with Pentothal Sodium injection, fixed in The biogeographic pattern and colonization from
Africa to the New world was study by Carranza and Check List 14 (4) Figure 1. Distribution map of H. mabouia in Argentina. The blue solid circle indicates the new record. Red solid squares indicate previous
records. The light yellow shaded area corresponds to the dry Chaco biogeographic region. Figure 1. Distribution map of H. mabouia in Argentina. The blue solid circle indicates the new record. Red solid squares indicate previous
records. The light yellow shaded area corresponds to the dry Chaco biogeographic region. 10% formalin, and preserved in 70% alcohol. Voucher
specimens have been deposited in the herpetological col
lection of the Laboratorio de Genetica Evolutiva (LGE),
Instituto de Biología Subtropical (CONICET-UNaM),
Posadas city, Misiones province, Argentina. conservation of fauna. As was previously mentioned,
some Hemidactylus species share the ability of displacing
native fauna (Hanley et al. 1998, Dame and Petren 2006,
Rivas Fuenmayor et al. 2005), which suggests the need to
carry out a greater survey of the fauna present in the dry
Chaco and the potential threats to the conservation of the
native fauna. This record extends the distribution range of
H. mabouia by nearly 300 kilometers (in a straight line)
from Formosa city, the nearest point previously reported
(Alvarez et al. 2009). Discussion The new report is the first record for the invasive species
H. mabouia in the dry Chaco, a biogeographic region
included in the Gran Chaco Sudamericano (Naumann
2006). The presence of H. mabouia in this region, which
is characterized by the presence of multiple endemism
(Szumik et al. 2012), represents a potential problem for References Alvarez BB, Aguirre RH, Céspedez JA, Hernando AB, Tedesco ME
(2002) Atlas de Anfibios y Reptiles de las Provincias de Corrien
tes, Chaco y Formosa (Argentina) I. (Anuros, Cecílidos, Saurios,
Amphisbénidos y Serpientes). Corrientes: Universidad Nacional
del Nordeste. 156 p. Alvarez BB, Aguirre RH, Céspedez JA, Hernando AB, Tedesco ME
(2002) Atlas de Anfibios y Reptiles de las Provincias de Corrien
tes, Chaco y Formosa (Argentina) I. (Anuros, Cecílidos, Saurios,
Amphisbénidos y Serpientes). Corrientes: Universidad Nacional
del Nordeste. 156 p. 635 Torres et al. | Hemidactylus mabouia in Dry Chaco Figure 2. Specimens of H. mabouia LGE 20047. A. Dorsal view of the body. Scale bar = 5 mm. B. Distance of the lamellae from the base of
the foot. C. Lateral view of the head. Scale bar = 1 mm. Figure 2. Specimens of H. mabouia LGE 20047. A. Dorsal view of the body. Scale bar = 5 mm. B. Distance of the lamellae from the base of
the foot. C. Lateral view of the head. Scale bar = 1 mm. Figure 2. Specimens of H. mabouia LGE 20047. A. Dorsal view of th
the foot. C. Lateral view of the head. Scale bar = 1 mm. Figure 2. Specimens of H. mabouia LGE 20047. A. Dorsal view of the body. Scale bar = 5 mm. B. Distance of the lamellae from the base of
the foot. C. Lateral view of the head. Scale bar = 1 mm. Alvarez BB, Ruiz García A, Céspedez JA, Hernando A, Zaracho V,
Calamante C, Aguirre R (2009) Herpetofauna, provinces of Chaco
and Formosa, Chaco Oriental region, north-eastern Argentina. Check List 5 (1): 074–082. http://doi.org/10.15560/5.1.74 of the competitive displacement of a native gecko by an invading
gecko: no role for parasites. Oecologia 115: 196–205. http://doi. org/10.1007/s004420050508 Hoogmoed MS (1973) Notes on the herpetofauna of Surinam IV. The
lizards and amphisbaenians of Surinam. Biogeographia 4: 419. Avila-Pires TCS (1995) Lizards of Brazilian Amazonia (Reptilia:
Squamata). Zoologische Verhandelingen 299, Rijksmuseum van
Natuurliike Historie, Leiden, The Netherlands, 706 pp. Hughes DF, Meshaka WE Jr, Van Buurt G (2015) The superior colo
nizing gecko Hemidactylus mabouia on Curaçao: conservation
implications for the native gecko Phyllodactylus martini. Journal
of Herpetology 49 (1): 60–63. http://doi.org/10.1670/13–16 Baldo D, Borteiro C, Brusquetti F, García JE, Prigioni C (2008). Rep
tilia, Gekkonidae, Hemidactylus mabouia, Tarentola mauritanica:
distribution extension and anthropogenic dispersal. Check List 4
(4): 434–438. http://doi.org/10.15560/4.4.434 of Herpetology 49 (1): 60–63. org/10.1111/j.1096- 0031.2011.00385.x
Uetz P, Freed P, Hošek J (Eds) (2017) The Reptile Database. http://
www.reptile-database.org. Accessed on: 2018-2-10.
Vanzolini PE (1978) On South American Hemidactylus (Sauria, Gek
konidae). Papéis Avulsos de Zoologia 31 (20): 307–343. Lizarralde M, Molina A, Mollerach M, Navarro F, Nomdedeu
S, Panizza A, Pereyra VV, Sandoval M, Scrocchi G, Zuloaga
FO (2012) Detecting areas of endemism with a taxonomically
diverse data set: plants, mammals, reptiles, amphibians, birds,
and insects from Argentina. Cladistics 28 (3): 317–329. http://doi. Lizarralde M, Molina A, Mollerach M, Navarro F, Nomdedeu
S, Panizza A, Pereyra VV, Sandoval M, Scrocchi G, Zuloaga
FO (2012) Detecting areas of endemism with a taxonomically
diverse data set: plants, mammals, reptiles, amphibians, birds,
and insects from Argentina. Cladistics 28 (3): 317–329. http://doi. References http://doi.org/10.1670/13–16 Kluge AG (1969) The evolution and geographical origin of the New
World Hemidactylus mabouia–brookii complex (Gekkonidae,
Sauria). Miscellaneous Publications, Museum of Zoology, Uni
versity of Michigan 138: 78. Carranza S, Arnold EN (2006) Systematics, biogeography, and evolution
of Hemidactylus geckos (Reptilia: Gekkonidae) elucidated using
mitochondrial DNA sequences. Molecular Phylogenetics and Evo
lution 38: 531–545. http://doi.org/10.1016/j.ympev.2005.07.012 Naumann M (2006) Atlas del Gran Chaco Sudamericano. Sociedad
Alemana de Cooperación Técnica (GTZ), ErreGé & Asoc. Buenos
Aires, 92 pp. Dame EA, Petren K (2006) Behavioural mechanisms of invasion and dis
placement in Pacific island geckos (Hemidactylus). Animal Behav
iour 71: 1165–1173. http://doi.org/10.1016/j.anbehav.2005.10.009 Rivas Fuenmayor G, Ungueto GN, Bauer AM, Barros T, Manzanilla
J (2005) Expansion and natural history of a successful coloni
zing gecko in Venezuela (Reptilia: Gekkonidae: Hemidactylus
mabouia) and the discovery of H. frenatus in Venezuela. Herpeto
logical Review 36 (2): 121–125. iour 71: 1165–1173. http://doi.org/10.1016/j.anbehav.2005.10.009 Federico L, Cacivio PM (2000) Hemidactylus mabouia. Herpetologi
cal Review 31 (1): 53. Genise JF, Montanelli SB (1991) Primer hallazgo de Hemidactylus
mabouia (Moreau de Jones) en la Argentina. Boletín de la Asocia
ción Herpetológica Argentina 7 (1): 23. Szumik C, Aagesen L, Casagranda D, Arzamendia V, Baldo D,
Claps LE, Cuezzo F, Díaz Gómez JM, Di Giacomo A, Giraudo
A, Goloboff P, Gramajo C, Kopuchian C, Kretzschmar S, Hanley KA, Petren K, Case TJ (1998) An experimental investigation Check List 14 (4) Check List 14 (4)
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Konflikty zbrojne a prawa kobiet w świetle praktyki Komitetu ONZ ds. Likwidacji Dyskryminacji Kobiet
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BEZPIECZEÑSTWO LUDZKIE ECZEÑSTWO LUDZKIE BEZPIECZEÑSTWO LU Przegl¹d Strategiczny 2013, nr 1 Katarzyna SÊKOWSKA-KOZ£OWSKA
Poznañskie Centrum Praw Cz³owieka
Instytut Nauk Prawnych PAN 3 Zgodnie z art. 1 konwencji: „W rozumieniu niniejszej konwencji okreœlenie «dyskryminacja
kobiet» oznacza wszelkie zró¿nicowanie, wy³¹czenie lub ograniczenie ze wzglêdu na p³eæ, które po-
woduje lub ma na celu uszczuplenie albo uniemo¿liwienie kobietom, niezale¿nie od ich stanu cywil-
nego, przyznania, realizacji b¹dŸ korzystania na równi z mê¿czyznami z praw cz³owieka oraz
podstawowych wolnoœci w dziedzinach ¿ycia politycznego, gospodarczego, spo³ecznego, kulturalne-
go, obywatelskiego i innych”. 5 Nale¿y jednak¿e zwróciæ uwagê, ¿e wiele pañstw, w szczególnoœci muzu³mañskich, z³o¿y³o
zastrze¿enia, które mog¹ godziæ w przedmiot i cel traktatu. Zob. szerzej m.in.: H. B. Schöpp-Schil-
ling, Reservations to the Convention on the Elimination of All Forms of Discrimination against Wo-
men: an Unresolved Issue or (No) New Developments?, w: Reservations to Human Rights Treaties
and the Vienna Convention Regime. Conflict, Harmony or Reconciliation, ed. I. Ziemele, Leiden–Bo-
ston 2004; R. J. Cook, Reservations to the Convention on the Elimination of All Forms of Discrimina-
tion against Women, „Virginia Journal of International Law” 1990, No. 3. p y ¹p
j
(
,
, p
)
2 Termin „prawa cz³owieka kobiet” (women’s human rights) jest stosowany w literaturze w celu
podkreœlenia, ¿e prawa te nie s¹ osobn¹, wydzielon¹ kategori¹ praw pewnej grupy, co móg³by sugero-
waæ termin „prawa kobiet”, ale stanowi¹ integralny element praw cz³owieka i odnosz¹ siê do korzy-
stania przez kobiety ze wszystkich praw i wolnoœci cz³owieka. Por.: Ch. Bunch, S. Frost, Prawa
kobiet w kontekœcie praw cz³owieka, w: Prawa kobiet w dokumentach ONZ, red. A. Grzybek, Warsza-
wa 1998, s. 9–20. U¿ywane w dalszej czêœci artyku³u, powszechne na gruncie j. polskiego sfor-
mu³owanie „prawa kobiet” oznacza de facto „prawa cz³owieka kobiet”.
3 g ,
y
g
y
4 Dane za: http://treaties.un.org/Pages/ViewDetails.aspx?src=TREATY&mtdsg_no=IV-8&chap-
ter=4&lang=en (27.06.2012).
5 1 Polska przyst¹pi³a do Konwencji w 1980 r. (Dz. U. 1982, Nr 10, poz. 71).
2 Termin „prawa cz³owieka kobiet” (women’s human rights) jest stosowany w literaturze w celu
podkreœlenia, ¿e prawa te nie s¹ osobn¹, wydzielon¹ kategori¹ praw pewnej grupy, co móg³by sugero-
waæ termin „prawa kobiet”, ale stanowi¹ integralny element praw cz³owieka i odnosz¹ siê do korzy-
stania przez kobiety ze wszystkich praw i wolnoœci cz³owieka. Por.: Ch. Bunch, S. Frost, Prawa
kobiet w kontekœcie praw cz³owieka, w: Prawa kobiet w dokumentach ONZ, red. A. Grzybek, Warsza-
wa 1998, s. 9–20. U¿ywane w dalszej czêœci artyku³u, powszechne na gruncie j. polskiego sfor-
mu³owanie „prawa kobiet” oznacza de facto „prawa cz³owieka kobiet”.
3 Zgodnie z art. 1 konwencji: „W rozumieniu niniejszej konwencji okreœlenie «dyskryminacja
kobiet» oznacza wszelkie zró¿nicowanie, wy³¹czenie lub ograniczenie ze wzglêdu na p³eæ, które po-
woduje lub ma na celu uszczuplenie albo uniemo¿liwienie kobietom, niezale¿nie od ich stanu cywil-
nego, przyznania, realizacji b¹dŸ korzystania na równi z mê¿czyznami z praw cz³owieka oraz
podstawowych wolnoœci w dziedzinach ¿ycia politycznego, gospodarczego, spo³ecznego, kulturalne-
go, obywatelskiego i innych”.
4 Dane za: http://treaties.un.org/Pages/ViewDetails.aspx?src=TREATY&mtdsg_no=IV-8&chap-
ter=4&lang=en (27.06.2012).
5 Nale¿y jednak¿e zwróciæ uwagê, ¿e wiele pañstw, w szczególnoœci muzu³mañskich, z³o¿y³o
zastrze¿enia, które mog¹ godziæ w przedmiot i cel traktatu. Zob. szerzej m.in.: H. B. Schöpp-Schil-
ling, Reservations to the Convention on the Elimination of All Forms of Discrimination against Wo-
men: an Unresolved Issue or (No) New Developments?, w: Reservations to Human Rights Treaties
and the Vienna Convention Regime. Conflict, Harmony or Reconciliation, ed. I. Ziemele, Leiden–Bo-
ston 2004; R. J. Cook, Reservations to the Convention on the Elimination of All Forms of Discrimina-
tion against Women, „Virginia Journal of International Law” 1990, No. 3. 1 Polska przyst¹pi³a do Konwencji w 1980 r. (Dz. U. 1982, Nr 10, poz. 71).
2 KONFLIKTY ZBROJNE A PRAWA KOBIET
W ŒWIETLE PRAKTYKI KOMITETU ONZ
DS. LIKWIDACJI DYSKRYMINACJI KOBIET Uchwalona pod auspicjami ONZ w 1979 r. Konwencja w sprawie likwidacji wszel-
kich form dyskryminacji kobiet (dalej: konwencja)1 stanowi najwa¿niejsz¹ umowê
miêdzynarodow¹ dotycz¹c¹ praw cz³owieka kobiet2. Jest to kompleksowy traktat,
który zobowi¹zuje pañstwa-strony do likwidacji dyskryminacji kobiet3, istniej¹cej za-
równo na p³aszczyŸnie prawa, jak i faktycznej, wystêpuj¹cej w wielu dziedzinach ¿ycia
spo³ecznego – m.in. w dostêpie do funkcji publicznych, na rynku pracy, w edukacji,
w sferze ¿ycia ma³¿eñskiego i rodzinnego czy w dostêpie do ochrony zdrowia. Do kon-
wencji przyst¹pi³o do dnia dzisiejszego 187 stron4, czyli ok. 97% pañstw ONZ, co spra-
wia ¿e jest jednym z nielicznych traktatów z dziedziny praw cz³owieka, które uzyska³y
niemal powszechn¹ ratyfikacjê5. Celem niniejszego artyku³u jest odpowiedŸ na pytanie czy i w jakim zakresie prze-
pisy konwencji chroni¹ prawa kobiet podczas konfliktów zbrojnych. Problem ten wy-
maga g³êbszej analizy, bowiem w przeciwieñstwie do traktatów z dziedziny prawa 4 Dane za: http://treaties.un.org/Pages/ViewDetails.aspx?src=TREATY&mtdsg_no=IV-8&chap-
ter=4&lang=en (27.06.2012). 5 Katarzyna SÊKOWSKA-KOZ£OWSKA 232 humanitarnego, konwencja nie zawiera postanowieñ odnosz¹cych siê bezpoœrednio do
konfliktów zbrojnych. To, i¿ konwencja milczy, jeœli chodzi o sytuacjê kobiet podczas
dzia³añ wojennych i innych zdarzeñ o podobnym charakterze, np. konfliktów wewnê-
trznych, nie oznacza, ¿e nie znajduje zastosowania w sytuacjach tego typu. Jak pokazu-
je dorobek Komitetu ds. Likwidacji Dyskryminacji Kobiet, postanowienia konwencji
nak³adaj¹ na pañstwa szereg zobowi¹zañ w sferze zapewnienia kobietom ich praw pod-
czas konfliktów zbrojnych i po ich zakoñczeniu. 6 Szerzej nt. dzia³alnoœci tego organu: K. Sêkowska-Koz³owska, Komitet ONZ ds. Likwidacji
Dyskryminacji Kobiet – ustrój, kompetencje, funkcjonowanie, Toruñ 2011. y
y
j
j,
p
j , f
j
,
7 Treœæ wszystkich zaleceñ ogólnych komitetu dostêpna jest na stronie http://www2.ohchr.org/
english/bodies/cedaw/comments.htm (27.06.2012). 6 Szerzej nt. dzia³alnoœci tego organu: K. Sêkowska-Koz³owska, Komitet ONZ ds. Likwidacji
Dyskryminacji Kobiet – ustrój, kompetencje, funkcjonowanie, Toruñ 2011.
7 Treœæ wszystkich zaleceñ ogólnych komitetu dostêpna jest na stronie http://www2.ohchr.org/ 6 Szerzej nt. dzia³alnoœci tego organu: K. Sêkowska-Koz³owska, Komitet ONZ ds. Likwidacji
Dyskryminacji Kobiet – ustrój, kompetencje, funkcjonowanie, Toruñ 2011.
7 Treœæ wszystkich zaleceñ ogólnych komitetu dostêpna jest na stronie http://www2.ohchr.org/
english/bodies/cedaw/comments.htm (27.06.2012). y
y
j
j,
p
j , f
j
7 Treœæ wszystkich zaleceñ ogólnych komitetu do
english/bodies/cedaw/comments.htm (27.06.2012). KOMITET DS. LIKWIDACJI DYSKRYMINACJI KOBIET
I JEGO KOMPETENCJE Komitet ds. Likwidacji Dyskryminacji Kobiet (dalej: komitet, CEDAW) jest usta-
nowionym przez art. 17 konwencji organem kontrolnym czuwaj¹cym nad realizacj¹
postanowieñ tego traktatu przez pañstwa-strony6. W jego sk³ad wchodzi 23 niezale¿-
nych ekspertów, którzy obraduj¹ œrednio trzy razy do roku na kilkutygodniowych se-
sjach odbywaj¹cych siê w siedzibach ONZ w Genewie lub Nowym Jorku. Zgodnie
z art. 17, komitet zosta³ powo³any w celu „badania postêpu w stosowaniu Konwencji”
przez pañstwa-strony. Dorobek komitetu wypracowany podczas wykonywania powie-
rzonej mu funkcji kontrolnej stanowi jednoczeœnie wa¿ne Ÿród³o interpretacji konwen-
cji. Przed przejœciem do analizy praktyki komitetu zwi¹zanej z omawianym tematem,
nale¿y przybli¿yæ jego g³ówne kompetencje. y p y
y j g
g
p
j
G³ównym instrumentem kontroli, którym dysponuje komitet jest procedura badania
sprawozdañ pañstw. Ka¿de z pañstw-stron jest zobowi¹zane do przed³o¿enia komiteto-
wi, zazwyczaj co cztery lata, sprawozdania, w którym prezentuje stan przestrzegania
praw zawartych w konwencji. Komitet dokonuje oceny sprawozdania, korzystaj¹c tak-
¿e z informacji otrzymanych z innych Ÿróde³ (w szczególnoœci nades³anych przez orga-
nizacje pozarz¹dowe) oraz uzyskanych w trakcie dialogu prowadzonego podczas sesji
z delegacj¹ pañstwa. Efektem koñcowym procedury badania sprawozdañ jest uchwale-
nie przez komitet dokumentu zwanego uwagami koñcowymi, w którym przedstawia on
swoj¹ ocenê stanu przestrzegania przez pañstwo praw zawartych w konwencji, wska-
zuje g³ówne problemy oraz formu³uje rekomendacje adresowane do pañstwa. Od uwag
koñcowych, które stanowi¹ rekomendacje kierowane indywidualnie do pañstwa, nale-
¿y odró¿niæ zalecenia ogólne, które równie¿ s¹ tworzone w oparciu o praktykê komite-
tu zwi¹zan¹ z badaniem sprawozdañ. Zalecenia ogólne s¹ kierowanymi do ogó³u
pañstw-stron konwencji uchwa³ami, które stanowi¹ wyk³adniê wybranej materii kon-
wencyjnej po³¹czonej z rekomendacjami adresowanymi do pañstw. Dotychczas
uchwalono 28 zaleceñ ogólnych, spoœród których dla omawianego tematu szczególne
znaczenie maj¹ zalecenia poœwiêcone przemocy wobec kobiet oraz zdrowiu kobiet7. Konflikty zbrojne a prawa kobiet w œwietle praktyki Komitetu ONZ 233 Nale¿y dodaæ, ¿e obecnie trwaj¹ prace nad zaleceniami ogólnymi poœwiêconymi sytu-
acji kobiet podczas konfliktów zbrojnych i po ich zakoñczeniu8. j
p
j y
p
Komitet dysponuje równie¿ mo¿liwoœci¹ zwrócenia siê do pañstwa o przed³o¿enie
sprawozdania poza regularnym cyklem sprawozdawczym. Sprawozdania te, okreœlane
mianem sprawozdañ nadzwyczajnych, s¹ jednym z nielicznych instrumentów kontroli
doraŸnej, którymi dysponuje komitet. Kompetencja ta odgrywa szczególne znaczenie
w przypadku pañstw ogarniêtych konfliktami zbrojnymi, pozwala bowiem komitetowi
zwróciæ siê poza regularnym cyklem sprawozdawczym o przedstawienie przez pañ-
stwo informacji dotycz¹cej stanu przestrzegania praw kobiet. Procedura ta zosta³a za-
inicjowana w 1993 r. 8 Decyzja 47/I, Sprawozdanie z 47 sesji komitetu, 4–22.10. 2010, A/66/38.
9 Punkt 732–757, Sprawozdanie nadzwyczajne Boœni i Hercegowiny, 1994, CEDAW A/49/38.
10 Sprawozdanie nadzwyczajne Federalnej Republiki Jugos³awii (Serbii i Czarnogóry), 1993,
CEDAW/C/YUG/SP.
11 Sprawozdanie nadzwyczajne Chorwacji, 1994, CEDAW/C/CRO/SP.1.
12 Punkt 301–331, Sprawozdanie nadzwyczajne Rwandy, 1996, A/51/38.
13 Punkt 344–351, Sprawozdanie nadzwyczajne Zairu, 1997, A/52/38/Rev. 1.
14 S. Cartwright, The Committee on the Elimination of Discrimination against Women, w: Women
and International Human Rights Law, Volume 2, eds. K. D. Askin, D. M. Koenig, Ardsley–New York
2000, s. 172.
15 Decyzja 33/II, Sprawozdanie z 33 sesji komitetu, 5–22.07.2005, A/60/38.
16 Decyzja 36/II, Sprawozdanie z 36 sesji komitetu, 7–25.08.2006, A/61/38. 16 Decyzja 36/II, Sprawozdanie z 36 sesji komitetu, 7–25.08.2006, A/61/38. 15 Decyzja 33/II, Sprawozdanie z 33 sesji komitetu, 5–22.07.2005, A/60/38.
16 KOMITET DS. LIKWIDACJI DYSKRYMINACJI KOBIET
I JEGO KOMPETENCJE W praktyce komitet nie jest przygoto-
wany do wykonywania takich zadañ, zw³aszcza w sytuacjach tak ekstremalnych, jak
konflikt zbrojny. Zapewnienie bezpieczeñstwa cz³onkom delegacji przez ONZ, jak
i pañstwo, na terytorium którego odbywa siê misja, jest w sytuacji wojny niezmiernie
trudnym zadaniem. Równie¿ mo¿liwoœæ dokonania odpowiednich ustaleñ na miejscu
by³aby znacznie ograniczona, poniewa¿ komitet nie dysponuje takimi œrodkami czy
kompetencjami jak np. Miêdzynarodowy Czerwony Krzy¿. Jest zatem ma³o prawdopo-
dobne, by instrument postêpowania wyjaœniaj¹cego zosta³ wykorzystany przez komitet
w celu badania naruszeñ praw zawartych w konwencji podczas konfliktu zbrojnego. Bardziej realnym wydaje siê wykorzystanie jego potencja³u dla zbadania sytuacji ko-
biet w danym pañstwie ju¿ po zakoñczeniu konfliktu zbrojnego. Na mocy wspomnianego protoko³u fakultatywnego komitet otrzyma³ równie¿ kom-
petencjê do rozpatrywania skarg indywidualnych. Procedura ta stwarza zatem mo¿li-
woœæ dochodzenia przed komitetem swoich praw jednostce, której prawa zosta³y
naruszone przez pozwane pañstwo. Jak dotychczas komitet rozpatrzy³ co do istoty kil-
kanaœcie skarg, z których ¿adna nie odnosi³a siê do naruszeñ praw kobiet podczas kon-
fliktów zbrojnych. KOMITET DS. LIKWIDACJI DYSKRYMINACJI KOBIET
I JEGO KOMPETENCJE w zwi¹zku z wojn¹ tocz¹c¹ siê na terenie by³ej Jugos³awii. CEDAW zwróci³ siê wówczas o przedstawienie sprawozdañ nadzwyczajnych do Boœni
i Hercegowiny9, Federalnej Republiki Jugos³awii (Serbii i Czarnogóry)10 oraz Chorwa-
cji11. Nied³ugo potem rozpatrzone zosta³y sprawozdania nadzwyczajne Rwandy12
i ówczesnego Zairu13. Od kilkunastu lat procedura ta nie by³a ponownie wykorzysty-
wana. Jej skutecznoœæ wywo³uje spore w¹tpliwoœci, równie¿ wewn¹trz samego komi-
tetu14, bowiem wiadomym jest, ¿e pañstwo znajduj¹ce siê w stanie wojny czy ogarniête
innego typu klêsk¹ nie bêdzie w stanie przygotowaæ wyczerpuj¹cej informacji, a na-
³o¿enie na niego obowi¹zku przedstawienia sprawozdania (chocia¿by w formie ustne-
go raportu z³o¿onego przez przedstawiciela pañstwa podczas sesji) stanowi du¿e
obci¹¿enie. Powstaje tak¿e pytanie czy zwa¿ywszy na stan, w jakim znajduje siê pañ-
stwo, rekomendacje komitetu maj¹ jakiekolwiek szanse byæ wdro¿one w praktyce. Za-
inicjowanie tej procedury nale¿y raczej odczytywaæ jako gest solidarnoœci z kobietami,
w kraju ogarniêtym konfliktem i próbê zwrócenia uwagi spo³ecznoœci miêdzynarodo-
wej na ich sytuacjê. Podobn¹ funkcjê pe³ni¹ decyzje komitetu uchwalane w formie
okolicznoœciowych oœwiadczeñ maj¹cych na celu zwrócenie uwagi na powa¿ne naru-
szenia praw kobiet, równie¿ w sytuacji konfliktu zbrojnego. Przyk³adem takich decyzji
s¹ oœwiadczenie dotycz¹ce sytuacji kobiet w Iraku z 2005 r.15 czy oœwiadczenie w spra-
wie sytuacji kobiet na Bliskim Wschodzie z 2006 r.16 Kolejnym instrumentem kontroli ad hoc, którym dysponuje komitet jest mo¿liwoœæ
inicjowania postêpowania wyjaœniaj¹cego w przypadku otrzymania informacji o po-
wa¿nych lub systematycznych naruszeniach praw zawartych w konwencji. Postêpowanie
takie mo¿e obejmowaæ m.in. wizytê przedstawicieli komitetu na terytorium pañ-
stwa-strony w celu dokonania niezbêdnych ustaleñ, na podstawie których sporz¹dzane Katarzyna SÊKOWSKA-KOZ£OWSKA 234 jest sprawozdanie komitetu wraz z rekomendacjami kierowanymi do pañstwa. Kompeten-
cja ta zosta³a przyznana komitetowi na mocy postanowieñ protoko³u fakultatywnego
do konwencji, który wszed³ w ¿ycie w 2000 r.17 Od tego czasu komitet przeprowadzi³
postêpowanie wyjaœniaj¹ce dotycz¹ce masowych zabójstw kobiet w rejonie Ciudad Ju-
arez w Meksyku (2003 r.), a w 2011 r. zainicjowa³ dochodzenie w sprawie zabójstw
i zaginiêæ w Kanadzie kobiet aborygeñskiego pochodzenia. Teoretycznie spoœród
wszystkich œrodków, którymi dysponuje CEDAW to w³aœnie procedura dochodzenio-
wa stanowi najbardziej adekwatny instrument do podjêcia interwencji w sprawie
ochrony praw kobiet w pañstwie ogarniêtym konfliktem zbrojnym. Komitet mo¿e bo-
wiem z urzêdu, w relatywnie szybkim czasie zorganizowaæ wizytê, która umo¿liwi
zbadanie sytuacji na miejscu, w przeciwieñstwie do procedury sprawozdawczej, która
opiera siê na pisemnych lub ustnych przekazach. y
p
(
p
)
18 Przyk³adowo art. 2 ust. 3 uchwalonej w 2011 r. Konwencji Rady Europy w sprawie zapobiega-
nia i zwalczania przemocy wobec kobiet i przemocy domowej stwierdza explicite, ¿e postanowienia
tego traktatu znajduj¹ zastosowanie w czasie pokoju i w przypadku konfliktów zbrojnych. 17 Polska ratyfikowa³a protokó³ w 2003 r. (Dz. U. 2004, Nr 248, poz. 2484). 19 Komentarz Ogólny nr 28 z 2010 r., pkt 11. Wszystkie t³umaczenia zawarte w artykule, za
wyj¹tkiem tekstów aktów prawnych opublikowanych w Dzienniku Ustaw, pochodz¹ od autorki.
20 Punkt 243, Uwagi koñcowe do sprawozdania Izraela, 2005, A/60/38.
21 Zob.: punkt 391, Uwagi koñcowe do sprawozdania Rosji, 2002, A/57/38.
22 Punkt 309, Sprawozdanie nadzwyczajne Rwandy, 1996, A/51/38. OBOWI¥ZYWANIE KONWENCJI
PODCZAS KONFLIKTÓW ZBROJNYCH Jak ju¿ wspomniano, w konwencji w przeciwieñstwie do niektórych umów miêdzy-
narodowych z zakresu praw cz³owieka, nie zawarto postanowieñ dotycz¹cych jej obo-
wi¹zywania podczas konfliktów zbrojnych18. Nie zawiera ona tak¿e, w odró¿nieniu od
np. Miêdzynarodowego Paktu Praw Obywatelskich (Osobistych) i Politycznych, klau-
zuli umo¿liwiaj¹cej derogacjê pewnych praw w sytuacji stanu wyj¹tkowego. Nale¿y
zatem przyj¹æ, ¿e pañstwo nie ma mo¿liwoœci zawieszenia stosowania konwencji lub
jej wybranych postanowieñ na czas wojny czy te¿ konfliktu wewnêtrznego. Interpreta- Konflikty zbrojne a prawa kobiet w œwietle praktyki Komitetu ONZ 235 cjê tê potwierdza wyk³adnia dokonana przez komitet w Zaleceniach ogólnych nr 28
dotycz¹cych charakteru zobowi¹zañ na³o¿onych na pañstwa przez konwencjê. Jak
stwierdzi³ CEDAW: „zobowi¹zania Pañstw-Stron nie wygasaj¹ w czasie konfliktów
zbrojnych lub stanów nadzwyczajnych wywo³anych przyczynami politycznymi lub
klêskami ¿ywio³owymi. Sytuacje takie wywieraj¹ silny wp³yw i rodz¹ powa¿ne konse-
kwencje w sferze równego posiadania i korzystania przez kobiety z ich podstawowych
praw. Pañstwa-strony powinny uchwaliæ strategie i podj¹æ œrodki nakierowane na
szczególne potrzeby kobiet w czasie konfliktów zbrojnych lub stanów nadzwyczaj-
nych”19. Nale¿y tak¿e podkreœliæ, ¿e na pañstwie ci¹¿y obowi¹zek wype³niania zobo-
wi¹zañ konwencyjnych w odniesieniu do kobiet zamieszkuj¹cych terytoria okupowane,
co zosta³o potwierdzone przez komitet przy okazji rozpatrywania sprawozdania
Izraela20. ZOBOWI¥ZANIA PAÑSTW DOTYCZ¥CE OCHRONY PRAW KOBIET
PODCZAS KONFLIKTÓW ZBROJNYCH
– ANALIZA PRAKTYKI KOMITETU Przystêpuj¹c do analizy praktyki komitetu odnosz¹cej siê do omawianej problema-
tyki, nale¿y zauwa¿yæ, ¿e z racji specyfiki instrumentów kontrolnych, którymi dyspo-
nuje ten organ, ustalenia przez niego dokonywane odnosz¹ siê przede wszystkim do
sytuacji maj¹cych miejsce po zakoñczeniu konfliktu zbrojnego. Z regu³y dopiero wte-
dy pañstwo jest w stanie z³o¿yæ sprawozdanie do komitetu, chyba ¿e tocz¹cy siê kon-
flikt obejmuje jedynie czêœæ kraju, czego przyk³adem mo¿e byæ wojna w Czeczenii21. Wyj¹tek stanowi¹ wspomniane wczeœniej sprawozdania nadzwyczajne, które umo¿li-
wiaj¹ podjêcie kontroli w czasie trwania konfliktu. Jest rzecz¹ oczywist¹, i¿ dzia³ania zbrojne, w szczególnoœci te, których ofiarami jest
ludnoœæ cywilna, naruszaj¹ elementarne prawa cz³owieka, w tym prawo do ¿ycia. Podczas
ich trwania zniszczeniu ulega czêsto ca³a infrastruktura, a pañstwo nie jest w stanie wykony-
waæ swoich podstawowych funkcji. Bez przywrócenia prawid³owego funkcjonowania
pañstwa, które umo¿liwi zaspokojenie choæby minimum potrzeb jego obywateli, trudno
jest mówiæ o pe³nym zapewnieniu praw cz³owieka, w tym praw kobiet. Cz³onkowie
CEDAW naturalnie zdaj¹ sobie sprawê z istniej¹cych przeszkód, co nie oznacza i¿ wyra-
¿aj¹ zgodê na taki stan rzeczy. Odnosz¹c siê do sytuacji w Rwandzie, komitet zidentyfiko-
wa³ nastêpuj¹ce czynniki utrudniaj¹ce lub wrêcz uniemo¿liwiaj¹ce realizacjê konwencji:
zbyt s³aby aparat pañstwowy uniemo¿liwiaj¹cy skuteczne podtrzymanie procesu pokojo-
wego, trudny przebieg rekoncyliacji, za³amanie infrastruktury pañstwa oraz sektora prywat-
nego, du¿a liczba uchodŸców i tzw. uchodŸców wewnêtrznych, du¿a liczba uzbrojonych
cz³onków armii oraz cywilów, upadek gospodarki oraz ekstremalna bieda22. Bêd¹c œwia- Katarzyna SÊKOWSKA-KOZ£OWSKA 236 domym istniej¹cych trudnoœci, komitet k³adzie jednoczeœnie nacisk na potrzebê
uwzglêdnienia praw i potrzeb kobiet. Komentuj¹c proces odbudowy powojennej w Bo-
œni i Hercegowinie, komitet zauwa¿y³, i¿: „bêd¹c œwiadomym trudnoœci napotykanych
przez Pañstwo Stronê wynikaj¹cych z konsekwencji konfliktu zbrojnego, a tak¿e od-
bywaj¹cej siê politycznej, gospodarczej i spo³ecznej transformacji, Komitet jest za-
niepokojony faktem, i¿ korzystanie przez kobiety z ich praw cz³owieka w okresie
powojennym napotyka na przeszkody wywo³ane takimi czynnikami jak: brak
uwzglêdniaj¹cej perspektywê p³ci (gender) analizy konsekwencji konfliktu zbrojnego,
nieobecnoϾ kobiet w procesie przywracania pokoju i transformacji oraz brak analizy
w tym zakresie uwzglêdniaj¹cych perspektywê kobiet”23. Nale¿y zauwa¿yæ, ¿e podejœcie CEDAW cechuje d¹¿enie do upodmiotowienia ko-
biet i w³¹czenia ich we wszystkie dziedziny ¿ycia, tak by mia³y one wp³yw na ota-
czaj¹c¹ je rzeczywistoœæ, staj¹c siê aktywnymi aktorami, nie zaœ ofiarami lub biernymi
beneficjentami przyznawanych przywilejów. St¹d te¿, kieruj¹c ka¿dorazowo uwagi do
pañstwa dotkniêtego konfliktem lub bêd¹cego na etapie przywracania pokoju, komitet
podkreœla koniecznoœæ aktywnego uczestnictwa kobiet w negocjacjach pokojowych
i procesie odbudowy pañstwa. 23 Punkt 116, Uwagi koñcowe do sprawozdania Boœni i Herzegowiny, 2006, CEDAW/C/BIH/
CO/3. 23 Punkt 116, Uwagi koñcowe do sprawozdania Boœni i Herzegowiny, 2006, CEDAW/C/BIH/
CO/3.
24 Punkt 756, Sprawozdanie nadzwyczajne Boœni i Hercegowiny, 1994, CEDAW A/49/38. Zob.
równie¿: punkt 776, Sprawozdanie nadzwyczajne Federalnej Republiki Jugos³awii (Serbii i Cza-
rnogóry), 1993, CEDAW/C/YUG/SP.
25 Zob. m.in.: punkt 72, Uwagi koñcowe do sprawozdania Indii, 2000, A/55/38; punkt 50, Uwagi
koñcowe do sprawozdania Burundi, 2001, A/56/38; punkt 176, Uwagi koñcowe do sprawozdania
Gwatemali, 2002, A/57/38; punkt 203, Uwagi koñcowe do sprawozdania Nepalu, 2004, A/59/38.
26 Punkt 156, Uwagi koñcowe do sprawozdania Ugandy, 2002, A/57/38.
27 Rezolucja 1325(2000) z 31 paŸdziernika 2000.
28 Zob. np.: punkt 172, Uwagi koñcowe do sprawozdania Libanu, 2008, CEDAW/C/LBN/CO/3;
oraz Oœwiadczenie Komitetu z okazji 10-lecia uchwalenia rezolucji; Decyzja 46/III, Sprawozdanie
z 46 sesji Komitetu, 12–30.07.2010, A/66/38. 24 Punkt 756, Sprawozdanie nadzwyczajne Boœni i Hercegowiny, 1994, CEDAW A/49/38. Zob.
równie¿: punkt 776, Sprawozdanie nadzwyczajne Federalnej Republiki Jugos³awii (Serbii i Cza-
rnogóry), 1993, CEDAW/C/YUG/SP.
25 Zob. m.in.: punkt 72, Uwagi koñcowe do sprawozdania Indii, 2000, A/55/38; punkt 50, Uwagi
koñcowe do sprawozdania Burundi, 2001, A/56/38; punkt 176, Uwagi koñcowe do sprawozdania
Gwatemali, 2002, A/57/38; punkt 203, Uwagi koñcowe do sprawozdania Nepalu, 2004, A/59/38.
26 Punkt 156, Uwagi koñcowe do sprawozdania Ugandy, 2002, A/57/38. j
(
)
p
28 Zob. np.: punkt 172, Uwagi koñcowe do sprawozdania Libanu, 2008, CEDAW/C/LBN/CO/3;
oraz Oœwiadczenie Komitetu z okazji 10-lecia uchwalenia rezolucji; Decyzja 46/III, Sprawozdanie
z 46 sesji Komitetu, 12–30.07.2010, A/66/38. ZOBOWI¥ZANIA PAÑSTW DOTYCZ¥CE OCHRONY PRAW KOBIET
PODCZAS KONFLIKTÓW ZBROJNYCH
– ANALIZA PRAKTYKI KOMITETU W przypadku ogarniêtych wojn¹ pañstw by³ej Jugo-
s³awii, stanowisko komitetu, wyra¿ane zwykle w doœæ powœci¹gliwy sposób, przybra³o
wrêcz formê odezwy: „Komitet […] wzywa wszystkie kobiety w Boœni i Hercegowi-
nie, by nie pozostawa³y bierne. Kobiety musz¹ staæ siê widzialne, zarówno na szczeblu
rz¹dowym, jak i pozarz¹dowym. Komitet ma nadziejê, ¿e w ten sposób kobiety
wzbudz¹ wolê polityczn¹ niezbêdn¹ dla dokonania zmian i szybkiego zakoñczenia
wojny”24. Zazwyczaj jednak¿e komitet poprzestaje na wystosowanej pod adresem pañ-
stwa rekomendacji w³¹czenia kobiet w proces przywracania pokoju25, uwzglêdniaj¹c
czasami specyfikê konfliktu, przyk³adowo w przypadku Ugandy podkreœli³, i¿ w pro-
ces ten powinny byæ w³¹czone kobiety ze wszystkich grup etnicznych26. W swoich za-
leceniach komitet odwo³uje siê równie¿ do rezolucji Rady Bezpieczeñstwa z 2000 r. dotycz¹cej uczestnictwa kobiet w procesie przywracania pokoju27, która zawiera sze-
reg szczegó³owych rekomendacji pod adresem pañstw28. Nale¿y podkreœliæ, ¿e intencj¹ komitetu nie jest umiejscowienie kobiety jedynie
w roli mediatora, który doprowadzi do pokojowego rozwi¹zania konfliktu. Uczestnic-
two kobiet we wszelkich dzia³aniach politycznych ma prowadziæ do ich uw³asnowol-
nienia (ang. empowerment), a tym samym przyczyniaæ siê do realizacji celu konwencji, Konflikty zbrojne a prawa kobiet w œwietle praktyki Komitetu ONZ 237 czyli likwidacji ich dyskryminacji. Zgodnie z art. 7 konwencji, na pañstwach ci¹¿y
obowi¹zek podjêcia wszelkich kroków w celu likwidacji dyskryminacji kobiet w ¿yciu
publicznym i politycznym pañstwa, m.in. poprzez umo¿liwienie uczestniczenia
w kszta³towaniu polityki pañstwa, zajmowania stanowisk publicznych i wykonywania
wszelkich funkcji publicznych na wszystkich szczeblach zarz¹dzania. Z tej te¿ przy-
czyny, analizuj¹c sytuacjê w pañstwach znajduj¹cych siê w procesie transformacji po-
wojennej, CEDAW zwraca uwagê na liczbê kobiet sprawuj¹cych wszelkiego rodzaju
funkcje publiczne29. Warto zaznaczyæ, i¿ jak zauwa¿y³ komitet w Zaleceniach ogól-
nych nr 23, art. 7 konwencji obejmuje równie¿ pe³en udzia³ kobiet w si³ach zbrojnych
pañstw30. Ponadto, zgodnie z art. 8 konwencji, który odnosi siê do uczestnictwa kobiet
w stosunkach miêdzynarodowych oraz organizacjach miêdzynarodowych, kobiety
powinny braæ udzia³ we wszelkiego rodzaju miêdzynarodowych strukturach wojsko-
wych, w tym równie¿ na stanowiskach dowódczych31. W zwi¹zku z powy¿szym, za-
strze¿enia pañstw, w œwietle których przepisy konwencji nie maj¹ wp³ywu na
postanowienia prawa krajowego ograniczaj¹cego lub wy³¹czaj¹cego udzia³ kobiet
w armii s¹ przedmiotem krytyki komitetu32, natomiast wycofanie takich zastrze¿eñ od-
notowywane jest z aprobat¹33. Kolejn¹ kwesti¹, która jest sta³ym przedmiotem uwagi CEDAW w przypadku bada-
nia sytuacji w pañstwach ogarniêtych wojn¹ lub znajduj¹cych siê w okresie powojen-
nym jest problem przemocy wobec kobiet. Sama problematyka przemocy nie zosta³a
bezpoœrednio ujêta w przepisach konwencji. 29 Punkt 92, Uwagi koñcowe do sprawozdania Republiki Serbii, 2007, CEDAW/C/SCG/CO/1.
30 Punkt 31, Zalecenia Ogólne nr 23, Sprawozdanie z 17 sesji komitetu, 1997, A/52/38/Rev. 1.
31 Punkt 37, op. cit.
32 Zob. np.: punkt 43, Uwagi koñcowe. do sprawozdania Cypru, 1996, CEDAW, A/51/38.
33 Zob. np.: punkt 219, Uwagi koñcowe do sprawozdania Austrii, 2000, A/55/38.
34 Punkt 6, Zalecenia ogólne nr 19, Sprawozdanie z 11 sesji komitetu, 1992, A/47/38.
35 Problematyka ta jest rozwijana przez Komitet w toku jego dalszej praktyki, w szczególnoœci
orzeczniczej (zob. m.in. A.T. p. Wêgrom, decyzja z 26 stycznia 2005, skarga nr 2/2003; Goecke p. Au-
strii, decyzja z 6 sierpnia 2007, skarga nr 5/2005; V.K. p. Bu³garii, decyzja z 25 lipca 2011, skarga nr
20/2008). Dorobek Komitetu przyczyni³ siê niew¹tpliwie do uznania przemocy wobec kobiet za akt
dyskryminacji równie¿ na p³aszczyŸnie regionalnych systemów ochrony praw cz³owieka – w ramach
systemu interamerykañskiego ju¿ w 1994 r. uchwalono Konwencjê w sprawie zapobiegania, karania
i likwidacji przemocy wobec kobiet (tzw. konwencja z Belém do Pará), natomiast w ramach systemu
europejskiego kwestia ta by³a przedmiotem orzeczeñ Europejskiego Trybuna³u Praw Cz³owieka
(zw³aszcza prze³omowy wyrok w sprawie Opuz p. Turcji, skarga nr 33401/02, wyrok z 9 czerwca
2001), zaœ w 2011 r. z uchwalono Konwencjê Rady Europy w sprawie zapobiegania i zwalczania
przemocy wobec kobiet i przemocy domowej. Zob. szerzej: K. Sêkowska-Koz³owska, Zakres zobo-
wi¹zañ stron Konwencji Rady Europy w sprawie zapobiegania i zwalczania przemocy wobec kobiet
i przemocy domowej, „Studia Prawnicze” 2012, nr 1. 29 Punkt 92, Uwagi koñcowe do sprawozdania Republiki Serbii, 2007, CEDAW/C/SCG/CO/1.
30 Punkt 31, Zalecenia Ogólne nr 23, Sprawozdanie z 17 sesji komitetu, 1997, A/52/38/Rev. 1.
31 Punkt 37, op. cit. p
2 Zob. np.: punkt 43, Uwagi koñcowe. do sprawozdania Cypru, 1996, CEDAW, A/51/38.
3 Z b
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d
d
i
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ii 2000 A/55/38 0 Punkt 31, Zalecenia Ogólne nr 23, Sprawozdanie z 17 sesji komitetu, 1997, A/52/38/Rev. 1
1 Punkt 37, op. cit.
2 9 Punkt 92, Uwagi koñcowe do sprawozdania Republiki Serbii, 2007, CEDAW/C/SCG/CO/1.
0
k
l
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d
k
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/ 3 Zob. np.: punkt 219, Uwagi koñcowe do sprawozdania Austrii, 2000, A/55/38.
4 u
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9, Sp awo danie
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99 ,
/ 7/38.
35 Problematyka ta jest rozwijana przez Komitet w toku jego dalszej praktyki, w szczególnoœci
orzeczniczej (zob. m.in. A.T. p. Wêgrom, decyzja z 26 stycznia 2005, skarga nr 2/2003; Goecke p. Au-
strii, decyzja z 6 sierpnia 2007, skarga nr 5/2005; V.K. p. Bu³garii, decyzja z 25 lipca 2011, skarga nr
20/2008). Dorobek Komitetu przyczyni³ siê niew¹tpliwie do uznania przemocy wobec kobiet za akt
dyskryminacji równie¿ na p³aszczyŸnie regionalnych systemów ochrony praw cz³owieka – w ramach
systemu interamerykañskiego ju¿ w 1994 r. uchwalono Konwencjê w sprawie zapobiegania, karania
i likwidacji przemocy wobec kobiet (tzw. konwencja z Belém do Pará), natomiast w ramach systemu
europejskiego kwestia ta by³a przedmiotem orzeczeñ Europejskiego Trybuna³u Praw Cz³owieka
(zw³aszcza prze³omowy wyrok w sprawie Opuz p. Turcji, skarga nr 33401/02, wyrok z 9 czerwca
2001), zaœ w 2011 r. z uchwalono Konwencjê Rady Europy w sprawie zapobiegania i zwalczania
przemocy wobec kobiet i przemocy domowej. Zob. szerzej: K. Sêkowska-Koz³owska, Zakres zobo-
wi¹zañ stron Konwencji Rady Europy w sprawie zapobiegania i zwalczania przemocy wobec kobiet
i przemocy domowej, „Studia Prawnicze” 2012, nr 1. ZOBOWI¥ZANIA PAÑSTW DOTYCZ¥CE OCHRONY PRAW KOBIET
PODCZAS KONFLIKTÓW ZBROJNYCH
– ANALIZA PRAKTYKI KOMITETU W uchwalonych w 1992 r. Zaleceniach
ogólnych nr 19 dotycz¹cych przemocy wobec kobiet, komitet dokona³ natomiast inter-
pretacji poszczególnych przepisów konwencji, dowodz¹c, ¿e przemoc, która staje siê
udzia³em kobiet tylko dlatego, ¿e s¹ kobietami lub dotyka ich w sposób nieproporcjo-
nalny stanowi formê dyskryminacji ze wzglêdu na p³eæ34. Zalecenia te mia³y prze³omo-
we znaczenie dla uznania na p³aszczyŸnie prawa miêdzynarodowego przemocy wobec
kobiet, a w szczególnoœci przemocy domowej, za formê dyskryminacji kobiet, a tym
samym naruszenie praw cz³owieka35. 238 Katarzyna SÊKOWSKA-KOZ£OWSKA W przypadku konfliktów zbrojnych uwagê komitetu zwraca przede wszystkim pro-
blem przemocy seksualnej wobec kobiet. Jak zauwa¿y³ komitet w odniesieniu do spra-
wozdania Indii, „na obszarach objêtych powstaniem zbrojnym kobiety s¹ nara¿one na
wysokie ryzyko przemocy, gwa³tów, wykorzystywania seksualnego i tortur”36. Wswoich
uwagach koñcowych wystosowanych m.in. do Rosji i Sri Lanki CEDAW odniós³ siê do
informacji o gwa³tach pope³nianych na kobietach przez ¿o³nierzy lub cz³onków innych
formacji bezpieczeñstwa, wzywaj¹c pañstwa do ochrony ofiar i ukarania winnych37. Choæ jest oczywistym, ¿e gwa³t, zarówno pope³niony w czasie pokoju, jak i wojny, stano-
wi przestêpstwo, ca³y czas istnieje potrzeba walki ze stereotypowym postrzeganiem
przemocy seksualnej wobec kobiet jako „skutku ubocznego” dzia³añ wojennych38. Od-
nosz¹c siê w 1994 r. do sprawozdania nadzwyczajnego Federacyjnej Republiki Ju-
gos³awii (Serbii i Czarnogóry) CEDAW surowo oceni³ stwierdzenie reprezentantki (sic!)
rz¹du, ¿e „odbiegaj¹ce od normy i nacechowane przemoc¹ zachowania seksualne s¹ cha-
rakterystyczne nie tylko dla wojny w by³ej Boœni i Hercegowinie; zachowania takie jak
dot¹d mia³y miejsce podczas wszystkich wojen”, uznaj¹c je za „niedopuszczalne, nie-
moralne i zatrwa¿aj¹ce”39. Komentuj¹c sytuacjê na Ba³kanach komitet odniós³ siê rów-
nie¿ do zjawiska wykorzystywania gwa³tu jako „orê¿a wojennego” i narzêdzia czystek
etnicznych40. Obserwacje komitetu w tej materii znalaz³y potwierdzenie w dalszym roz-
woju prawa miêdzynarodowego – w 1998 r. w precedensowym wyroku w sprawie Akay-
esu, Miêdzynarodowy Trybuna³ Karny dla Rwandy uzna³ gwa³t za formê ludobójstwa41. Na mocy zobowi¹zañ na³o¿onych przez konwencjê, pañstwa s¹ zobowi¹zane zarów-
no do zapewnienia bezpieczeñstwa kobietom poprzez ochronê przed ró¿nymi formami
przemocy, w tym szczególnoœci przed przemoc¹ seksualn¹, jak i do pomocy ofiarom,
które takiej przemocy doœwiadczy³y. W przypadku kobiet-ofiar gwa³tów wojennych
problem ten ma najczêœciej charakter masowy i w zwi¹zku z tym wymaga podjêcia od-
powiednich kroków o charakterze strukturalnym, w³¹czaj¹c w to rozwi¹zania praw-
ne42. 36 Punkt 71, Uwagi koñcowe do sprawozdania Indii, 2000, A/55/38.
37 Punkt 286, Uwagi koñcowe do sprawozdania Sri Lanki, 2002, A/57/38; punkt 191, Uwagi koñ-
cowe do sprawozdania Federacji Rosyjskiej, 2002, A/57/381.
38 Warto zauwa¿yæ, ¿e art. 5 konwencji uznaje stereotypowe postrzeganie ról kobiet, w tym prze-
œwiadczenie o ni¿szoœci kobiet za Ÿród³o ich dyskryminacji i nak³ada na pañstwa obowi¹zek przeciw-
dzia³ania takim stereotypom.
39 Punkt 768, Sprawozdanie nadzwyczajne Federalnej Republiki Jugos³awii (Serbii i Czarno-
góry), 1993, CEDAW/C/YUG/SP.
40 Punkt 586, Sprawozdanie nadzwyczajne Chorwacji, 1994, A/50/38.
41 Prokurator v. Jean-Paul Akayesu, ICTR-96-4-T, wyrok z 2 paŸdziernika 1998 r. Zob. równie¿
postanowienia rezolucji Rady Bezpieczeñstwa 1820 (2008) z 19 czerwca 2008.
42 Punkt 218, Uwagi koñcowe. do sprawozdania Konga, 2000, A/55/38.
43 Zob. równie¿: punkt 16, Zalecenia ogólne nr 24, Sprawozdanie z 20 sesji komitetu, 1999,
A/54/38/Rerv. 1..
44 Punkt 218, Uwagi koñcowe do sprawozdania Konga, op. cit.; punkt 54, Uwagi koñcowe do sprawoz-
daniaBurundi,2001,A/56/38;punkt205,UwagikoñcowedosprawozdaniaGwatemali,2002, A/57/38. 44 Punkt 218, Uwagi koñcowe do sprawozdania Konga, op. cit.; punkt 54, Uwagi koñcowe do sprawoz-
daniaBurundi,2001,A/56/38;punkt205,UwagikoñcowedosprawozdaniaGwatemali,2002, A/57/38. 37 Punkt 286, Uwagi koñcowe do sprawozdania Sri Lanki, 2002, A/57/38; punkt 191, U
cowe do sprawozdania Federacji Rosyjskiej, 2002, A/57/381.
38 Punkt 768, Sprawozdanie nadzwyczajne Federalnej Republiki Jugos³awii (Serbii i Czarno-
1993, CEDAW/C/YUG/SP. 38 Warto zauwa¿yæ, ¿e art. 5 konwencji uznaje stereotypowe postrzeganie ról kobiet, w tym prze-
œwiadczenie o ni¿szoœci kobiet za Ÿród³o ich dyskryminacji i nak³ada na pañstwa obowi¹zek przeciw-
dzia³ania takim stereotypom.
39 36 Punkt 71, Uwagi koñcowe do sprawozdania Indii, 2000, A/55/38.
37 40 Punkt 586, Sprawozdanie nadzwyczajne Chorwacji, 1994, A/50/38. g
p
g
43 Zob. równie¿: punkt 16, Zalecenia ogólne nr 24, Sprawozdanie z 20 sesji komitetu, 1999,
A/54/38/Rerv. 1.. 36 Punkt 71, Uwagi koñcowe do sprawozdania Indii, 2000, A/55/38.
37 Punkt 286, Uwagi koñcowe do sprawozdania Sri Lanki, 2002, A/57/38; punkt 191, Uwagi koñ-
cowe do sprawozdania Federacji Rosyjskiej, 2002, A/57/381. 42 Punkt 218, Uwagi koñcowe. do sprawozdania Konga, 2000, A/55/38.
43 41 Prokurator v. Jean-Paul Akayesu, ICTR-96-4-T, wyrok z 2 paŸdziernika 1998 r. Zob. równie¿
postanowienia rezolucji Rady Bezpieczeñstwa 1820 (2008) z 19 czerwca 2008.
42 45 Punkt 761, Sprawozdanie nadzwyczajne Federalnej Republiki Jugos³awii (Serbii i Czarno-
góry), 1993, CEDAW/C/YUG/SP.
46 Punkt 591, Sprawozdanie nadzwyczajne Chorwacji, 1994, A/50/38.
47 Punkt 472, Sprawozdanie nadzwyczajne Boœni i Hercegowiny, 1994, CEDAW A/49/38.
48 Punkt 321, Sprawozdanie nadzwyczajne Rwandy, 1996, A/51/38.
49 Punkt 320, Sprawozdanie nadzwyczajne Rwandy, op. cit.; punkt 436, Uwagi koñcowe do spra-
wozdania Kolumbii, 2007, CEDAW/C/COL/CO/6.
50 Punkt 340, Uwagi koñcowe do sprawozdania Nigerii, 2008, CEDAW/C/NGA/CO/6; punkt
298–299, Uwagi koñcowe do sprawozdania Sri Lanki, 2002, A/57/38.
51 Punkt 305, Sprawozdanie nadzwyczajne Rwandy, op. cit.; punkt 358, Uwagi koñcowe do spra-
wozdania Konga, op. cit. ZOBOWI¥ZANIA PAÑSTW DOTYCZ¥CE OCHRONY PRAW KOBIET
PODCZAS KONFLIKTÓW ZBROJNYCH
– ANALIZA PRAKTYKI KOMITETU W³adze pañstw powinny przede wszystkim zapewniæ ofiarom gwa³tów pomoc
psychologiczn¹ maj¹c¹ na celu przezwyciê¿enie doznanej traumy43, a tak¿e odpowied-
nie wsparcie o charakterze gospodarczo-spo³ecznym, które umo¿liwi takim osobom
ponown¹ integracjê w spo³eczeñstwie44. Krokom tym musz¹ towarzyszyæ dzia³ania 44 Punkt 218, Uwagi koñcowe do sprawozdania Konga, op. cit.; punkt 54, Uwagi koñcowe do sprawoz-
daniaBurundi,2001,A/56/38;punkt205,UwagikoñcowedosprawozdaniaGwatemali,2002, A/57/38. Konflikty zbrojne a prawa kobiet w œwietle praktyki Komitetu ONZ 239 maj¹ce na celu ukaranie winnych i zapewnienie ofiarom zadoœæuczynienia o charakte-
rze finansowym. Mo¿na zauwa¿yæ, ¿e œciganie i ukaranie, czy to na szczeblu krajowym
czy miêdzynarodowym, sprawców przemocy seksualnej wobec kobiet stanowi obo-
wi¹zek pañstw, który bywa bagatelizowany i lekcewa¿ony. Przyk³adowo w 1994 r. przedstawicielka Federacyjnej Republiki Jugos³awii (Serbii i Czarnogóry) argumento-
wa³a, ¿e „zamiast szacowaæ liczbê zgwa³conych kobiet, próbuj¹c udowodniæ, która
strona doœwiadczy³a wiêkszych cierpieñ, w¹tpi¹c w ich œwiadectwa i u¿ywaj¹c ich do
politycznej manipulacji, by³oby lepiej pomóc zgwa³conym kobietom i umo¿liwiæ rein-
tegracjê w spo³eczeñstwie”45. Jak jednak¿e podkreœli³ komitet w odniesieniu do spra-
wozdania Chorwacji „w pierwszym rzêdzie nale¿y przerwaæ milczenie dotycz¹ce
nadu¿yæ seksualnych i przemocy, którymi ofiarami s¹ kobiety, zidentyfikowaæ winne
strony i postawiæ je przed s¹dami krajowymi i miêdzynarodowymi, a tak¿e przyznaæ
ofiarom finansow¹ rekompensatê”46. Przedmiotem uwagi CEDAW s¹ tak¿e inne kon-
sekwencje przemocy seksualnej, takie jak problem dostêpu ofiar do zabiegów prze-
rywania ci¹¿y i ochrony dzieci urodzonych na skutek gwa³tu47, a tak¿e epidemia
HIV/AIDS i innych chorób48. Do nastêpstw konfliktu zbrojnego, do których odnosi siê komitet nale¿¹ tak¿e bie-
da, a w konsekwencji m.in. brak nale¿ytej ochrony zdrowia49, do której pañstwa s¹
zobowi¹zane w œwietle art. 12 konwencji oraz prostytucja, do której odnosi siê art. 6
konwencji. Szczególnie nara¿one na naruszenia praw zawartych w konwencji s¹ kobie-
ty-uchodŸcy oraz tzw. uchodŸcy-wewnêtrzni (ang. internally displaced persons) prze-
bywaj¹ce w trudnych warunkach bytowych w obozach, w których ich udzia³em staj¹
siê takie zjawiska jak zwiêkszone ryzyko przemocy seksualnej lub innych form prze-
mocy, brak dostêpu do opieki zdrowotnej czy edukacji50. Zniszczenie przez wojnê
wszelkiej infrastruktury przyczynia siê tak¿e, w szczególnoœci w przypadku krajów
najbiedniejszych, takich jak niektóre pañstwa afrykañskie, do pog³êbienia dyskrymina-
cji kobiet w dostêpie do edukacji51. * * * Choæ przepisy Konwencji w sprawie likwidacji wszelkich form dyskryminacji ko-
biet nie zawieraj¹ bezpoœredniego odniesienia do kwestii bezpieczeñstwa kobiet oraz
przestrzegania ich praw w czasie konfliktów zbrojnych, interpretacja tego traktatu do-
konana przez Komitet ds. Likwidacji Dyskryminacji Kobiet wskazuje, ¿e nak³ada on
w tej materii wiele zobowi¹zañ na pañstwa. ZOBOWI¥ZANIA PAÑSTW DOTYCZ¥CE OCHRONY PRAW KOBIET
PODCZAS KONFLIKTÓW ZBROJNYCH
– ANALIZA PRAKTYKI KOMITETU W ramach tego nurtu komitet koncentruje siê przede
wszystkim na tych naruszeniach bêd¹cych udzia³em kobiet, które ³¹cz¹ siê bezpoœred-
nio z ich p³ci¹. Jest to przede wszystkim przemoc seksualna. Komitet k³adzie
szczególny nacisk na obowi¹zek œcigania i ukarania sprawców gwa³tów, zapewnienia
ofiarom nale¿ytego zadoœæuczynienia oraz wsparcia, w tym pomocy psychologicznej
oraz wdro¿enia programów, umo¿liwiaj¹cych reintegracjê w spo³eczeñstwie. Wœród
innych problemów bêd¹cych nastêpstwami konfliktów zbrojnych, na które zwraca
uwagê komitet znajduj¹ siê m.in. ubóstwo, problem dostêpu do opieki zdrowotnej, pro-
stytucja czy sytuacja kobiet-uchodŸców oraz uchodŸców wewnêtrznych, a tak¿e dostêp
do edukacji. Dokonuj¹c interpretacji materii konwencyjnej pod k¹tem zobowi¹zañ na³o¿onych
na pañstwa w sytuacji konfliktu zbrojnego oraz na po jego zakoñczeniu, komitet ak-
tywnie w³¹cza siê w kszta³towanie miêdzynarodowych standardów w odniesieniu do
bezpieczeñstwa kobiet i ochrony ich praw podczas dzia³añ wojennych. Kolejnym wa¿-
nym krokiem bêdzie przedstawienie kompleksowej wyk³adni tej problematyki w zale-
ceniach ogólnych, które s¹ obecnie przedmiotem prac tego organu. ZOBOWI¥ZANIA PAÑSTW DOTYCZ¥CE OCHRONY PRAW KOBIET
PODCZAS KONFLIKTÓW ZBROJNYCH
– ANALIZA PRAKTYKI KOMITETU Nale¿y podkreœliæ, ¿e konwencja obo- 240 Katarzyna SÊKOWSKA-KOZ£OWSKA wi¹zuje tak¿e w czasie konfliktu zbrojnego, a pañstwa nie maj¹ mo¿liwoœci derogowa-
nia jej postanowieñ. Prowadzenie dzia³añ wojennych nie zwalnia zatem z obowi¹zku
przestrzegania praw zawartych w konwencji. Ponadto pañstwa s¹ zobowi¹zane do re-
spektowania tego traktatu w odniesieniu do terytoriów okupowanych. Jak wskazano, instrumenty którymi dysponuje komitet umo¿liwiaj¹ przede wszyst-
kim dokonanie kontroli po zakoñczeniu konfliktu zbrojnego. Z tego te¿ powodu re-
komendacje komitetu koncentruj¹ siê przede wszystkim na zapewnieniu kobietom
realizacji ich praw w procesie odbudowy pañstwa oraz zadoœæuczynienia za krzywdy
doznane podczas wojny. Analizuj¹c dorobek CEDAW mo¿na wyodrêbniæ dwa g³ówne
nurty bêd¹ce przedmiotem zainteresowania tego organu. Pierwszy z nich mo¿na okre-
œliæ jako „kobieta – uczestnik procesu pokojowego”. W ramach tego nurtu komitet akcen-
tuje aktywn¹ rolê kobiet i rekomenduje pañstwom ich w³¹czenie w proces rozwi¹zania
konfliktu i odbudowy powojennej. Pañstwa s¹ zobowi¹zane zapewniæ kobietom na
równi z mê¿czyznami dostêp do uczestnictwa w ¿yciu publicznym, w tym zajmowanie
decyzyjnych stanowisk, tak¿e w ramach armii. Drugi nurt mo¿na opisaæ jako „kobieta Jak wskazano, instrumenty którymi dysponuje komitet umo¿liwiaj¹ przede wszyst-
kim dokonanie kontroli po zakoñczeniu konfliktu zbrojnego. Z tego te¿ powodu re-
komendacje komitetu koncentruj¹ siê przede wszystkim na zapewnieniu kobietom
realizacji ich praw w procesie odbudowy pañstwa oraz zadoœæuczynienia za krzywdy
doznane podczas wojny. Analizuj¹c dorobek CEDAW mo¿na wyodrêbniæ dwa g³ówne
nurty bêd¹ce przedmiotem zainteresowania tego organu. Pierwszy z nich mo¿na okre-
œliæ jako „kobieta – uczestnik procesu pokojowego”. W ramach tego nurtu komitet akcen-
tuje aktywn¹ rolê kobiet i rekomenduje pañstwom ich w³¹czenie w proces rozwi¹zania
konfliktu i odbudowy powojennej. Pañstwa s¹ zobowi¹zane zapewniæ kobietom na
równi z mê¿czyznami dostêp do uczestnictwa w ¿yciu publicznym, w tym zajmowanie
decyzyjnych stanowisk, tak¿e w ramach armii. Drugi nurt mo¿na opisaæ jako „kobieta
– ofiara dzia³añ wojennych”. W ramach tego nurtu komitet koncentruje siê przede
wszystkim na tych naruszeniach bêd¹cych udzia³em kobiet, które ³¹cz¹ siê bezpoœred-
nio z ich p³ci¹. Jest to przede wszystkim przemoc seksualna. Komitet k³adzie
szczególny nacisk na obowi¹zek œcigania i ukarania sprawców gwa³tów, zapewnienia
ofiarom nale¿ytego zadoœæuczynienia oraz wsparcia, w tym pomocy psychologicznej
oraz wdro¿enia programów, umo¿liwiaj¹cych reintegracjê w spo³eczeñstwie. Wœród
innych problemów bêd¹cych nastêpstwami konfliktów zbrojnych, na które zwraca
uwagê komitet znajduj¹ siê m.in. ubóstwo, problem dostêpu do opieki zdrowotnej, pro-
stytucja czy sytuacja kobiet-uchodŸców oraz uchodŸców wewnêtrznych, a tak¿e dostêp
do edukacji. y yj y
g
p
j
– ofiara dzia³añ wojennych”. STRESZCZENIE Celem artyku³u jest odpowiedŸ na pytanie, jakie obowi¹zki nak³adaj¹ na pañstwa postano-
wienia Konwencji w sprawie likwidacji wszelkich form dyskryminacji kobiet uchwalonej pod
auspicjami ONZ w 1979 r. w odniesieniu do ochrony praw kobiet podczas konfliktów zbrojnych
i procesu pokojowego. Przedstawione badania opieraj¹ siê na analizie praktyki Komitetu ONZ
ds. Likwidacji Dyskryminacji Kobiet, niezale¿nego organu kontrolnego powo³anego przez Kon-
wencjê w celu monitorowania stanu przestrzegania przez pañstwa-strony praw w niej zawar-
tych. Analiza dorobku komitetu zosta³a poprzedzona przegl¹dem przys³uguj¹cych temu
organowi narzêdzi kontrolnych pod k¹tem ich przydatnoœci do weryfikacji realizacji obo-
wi¹zków konwencyjnych przez pañstwa w szczególnej sytuacji, jak¹ jest konflikt zbrojny. W³aœciwa czêœæ prezentowanych badañ opiera siê natomiast na analizie uchwa³ tego organu
– w szczególnoœci zaleceñ ogólnych i uwag koñcowych kierowanych do pañstw-stron. 241 Konflikty zbrojne a prawa kobiet w œwietle praktyki Komitetu ONZ ABSTRACT ABSTRACT Presented paper aims to answer the question what are obligations imposed on states by the
UN Convention on the Elimination of All Forms of Discrimination against Women (1979) in re-
lation to protection of women’s rights during the armed conflicts and peace process. Presented
research is based on analysis of the practice of the UN Committee on the Elimination of Discrim-
ination against Women, an independent control body established by the Convention to monitor
implementation of its provisions by the state parties. The analysis of the Committee’s output is
proceeded by the review of its control tools in order to assess their usefulness in process of verifi-
cation how the states perform duties imposed on them by the Convention in such specific situa-
tion as armed conflict. The core part of presented research is based on analysis of decisions of
this body, particularly the general recommendations and concluding observations addressed to
the state parties.
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Assessing vocal performance in complex birdsong: a novel approach
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BMC biology
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Abstract Background: Vocal performance refers to the ability to produce vocal signals close to physical limits. Such motor
skills can be used by conspecifics to assess a signaler’s competitive potential. For example it is difficult for birds to
produce repeated syllables both rapidly and with a broad frequency bandwidth. Deviation from an upper-bound
regression of frequency bandwidth on trill rate has been widely used to assess vocal performance. This approach is,
however, only applicable to simple trilled songs, and even then may be affected by differences in syllable complexity. Results: Using skylarks (Alauda arvensis) as a birdsong model with a very complex song structure, we detected another
performance trade-off: minimum gap duration between syllables was longer when the frequency ratio between the
end of one syllable and the start of the next syllable (inter-syllable frequency shift) was large. This allowed us to
apply a novel measure of vocal performance - vocal gap deviation: the deviation from a lower-bound regression of
gap duration on inter-syllable frequency shift. We show that skylarks increase vocal performance in an aggressive
context suggesting that this trait might serve as a signal for competitive potential. Conclusions: We suggest using vocal gap deviation in future studies to assess vocal performance in songbird
species with complex structure. Keywords: Aggressive signaling, Alauda arvensis, Birdsong, Complex song, Contextual variation, Production limits precise central nervous control of respiration, modulation
of the sound-producing organ, the syrinx, and vocal tract
modulation [5,6]. Different traits of birdsong could be
subject to performance limits, such as the consistency in
performing recurring vocal elements, termed syllables [7];
song density - the proportion of sound per unit of time
[8]; low-pitched vocalizations [9]; or song amplitude
[10,11]. One song trait that has received particular atten-
tion is trill performance (reviewed in [4,12,13]). In trilled
song components, a given syllable type is repeated several
times. In such components, trill rate might be limited by
itself and in addition, evidence from several species sug-
gests that there is a structural trade-off between the fre-
quency bandwidth and the rate at which syllables are
repeated (reviewed in [4]). A candidate mechanism for this
trade-off is the vocal tract, which has to be tuned to the
fundamental frequency produced by the syrinx in order to
suppress energy in higher harmonics; the movements re-
quired for tuning the vocal tract are constrained by the
speed of the involved motor systems [5,14]. RESEARCH ARTICLE Open Access * Correspondence: geberzahn@gmail.com
1Centre National de la Recherche Scientifique, Centre de Neuroscience Paris
Sud, UMR 8195, 91405 Orsay, France
2Université Paris Sud, Equipe Communications Acoustiques/CNPS, Bat. 446,
91405 Orsay, France
Full list of author information is available at the end of the article Abstract Additionally,
the frequency control mechanism in the syrinx itself could
be subject to speed limits although birds might overcome © 2014 Geberzahn and Aubin; licensee BioMed Central. 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 use,
distribution, and reproduction in any medium, provided the original work is properly credited. 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. Assessing vocal performance in complex
birdsong: a novel approach Nicole Geberzahn1,2,3* and Thierry Aubin1,2 Nicole Geberzahn1,2,3* and Thierry Aubin1,2 Geberzahn and Aubin BMC Biology 2014, 12:58
http://www.biomedcentral.com/1741-7007/12/58 Geberzahn and Aubin BMC Biology 2014, 12:58
http://www.biomedcentral.com/1741-7007/12/58 Geberzahn and Aubin BMC Biology 2014, 12:58 Background Displays used in animal communication are often subject
to physical constraints, such as biomechanical limits arising
during their production, and only highly skilled individuals
should be capable of performing challenging displays. Consequently, display traits linked to production limits
may indicate a signaler’s quality or motivational state in
a variety of contexts such as mate choice, male-male
competition, or during predator-prey interaction. Exam-
ples are prey animals performing displays of agility or
speed indicating their ability to escape [1,2], bird species
performing acrobatic aerial displays to attract females
[3], or male songbirds using their vocal skills in male-
male vocal interactions or female attraction [4]. Birdsong is a fruitful model for studying how perform-
ance limits affect signal properties: vocal production requires Geberzahn and Aubin BMC Biology 2014, 12:58
http://www.biomedcentral.com/1741-7007/12/58 Page 2 of 9 such limits at the sound source by switching between the
different sides of the syrinx (for example, see [15]). Finally,
respiratory requirements certainly play a role as well, be-
cause the duration of silent gaps between syllables is likely
to be regulated by respiratory needs. Canaries (Serinus
canaria), for instance, use a mini-breath respiratory
pattern during song production in which volumes of
air expired during phonation are closely matched to those
inspired during silent gaps [16]. Thus, respiratory require-
ments may also limit an increase in trill rate. up-sweeping syllables, a syllable might begin at the same
frequency as the preceding one ended (for example, house
wrens, Troglodytes aedon [19,20]; dark-eyed junco, Junco
hyemalis [21]; compare with figure one A in [4]). Thus,
males may be able to shorten gaps between such syllables
more easily as the vocal tract does not need to be largely
reconfigured. This may allow them to increase the rate of
syllable production more easily. In addition to such variation
with the structure of trills, performance difficulty might also
be affected by other parameters such as frequency or ampli-
tude modulations within syllables or the degree of tonality. Podos et al. [4] suggested that performance difficulty
may vary with the structure of trills: If trills consist for
instance mainly of down-sweeping syllables (or mainly of
up-sweeping syllables), a subsequent syllable will often
begin at a different frequency as the previous syllable
ended (for example, banded wren, Thryothorus pleurostic-
tus [17]; swamp sparrows, Melospiza georgiana [18]; com-
pare with figure one B in [4]). Longer syllables are not associated with larger
inter-syllable frequency shifts Longer syllables could be associated with higher inter-
syllable frequency ratios as they have more time to Figure 2 Gap duration plotted against inter-syllable frequency
ratio. Blue line: lower-bound regression line. Red diamonds: syllables
produced in response to territorial playbacks. Black triangles: syllables
produced in spontaneous song (both contexts together n = 6,910
syllables of 16 subjects). Blue crosses: data points used to calculate
the lower-bound regression line assessed by the equal samples per
bin method [40]. Vocal gap deviation is measured as the minimum
orthogonal distance of each data point to the lower-bound regression
line; an example is shown for one data point (black dotted line). Minimum area convex polygon is given in red for reactive and in
grey for spontaneous singing. For statistics see text. We recently reported [8] that skylarks increase the
sound density of their song, as compared to baseline
singing, in an aggressive context and they seem to do so
by switching to a different set of syllables for which the
subsequent gap durations are smaller. This new and
context-specific set of syllables might be associated with
smaller
inter-syllable
frequency
shifts
allowing
for
smaller gap durations. Therefore, our second hypothesis
was that skylarks decrease gap durations in reactive
singing by producing unique, context-specific syllables
that have syllable transitions with smaller inter-syllable
frequency shifts compared to the context-specific sylla-
bles produced uniquely in spontaneous singing. We pre-
dicted that inter-syllable frequency shifts would be
significantly smaller in syllables performed in response
to the territorial challenge and in particularly in those
that were unique to this context. Third, we hypothesized
that skylarks sing closer to the performance limit when
challenged by a territorial playback. If this is the case, Figure 2 Gap duration plotted against inter-syllable frequency
ratio. Blue line: lower-bound regression line. Red diamonds: syllables
produced in response to territorial playbacks. Black triangles: syllables
produced in spontaneous song (both contexts together n = 6,910
syllables of 16 subjects). Blue crosses: data points used to calculate
the lower-bound regression line assessed by the equal samples per
bin method [40]. Vocal gap deviation is measured as the minimum
orthogonal distance of each data point to the lower-bound regression
line; an example is shown for one data point (black dotted line). Minimum area convex polygon is given in red for reactive and in
grey for spontaneous singing. For statistics see text. Background Accordingly, the vocal tract
will need to be largely reconfigured during the silent gaps
between two consecutive syllables. This might strongly
limit the trill rate as such reconfiguration needs some
time. Contrarily, in songbird species singing trills mainly
consisting of an alternating sequence of down- and Assessing vocal performance in songbird species with
a complex song thus requires novel measures that are
independent of such differences. We here applied such
a measure, which we call ‘vocal gap deviation’, to assess
modulation of vocal performance in an aggressive con-
text in skylarks, a songbird with an extremely complex
and versatile song. Vocal gap deviation is based on the
assumption that the gap duration between two successive
syllables is traded off against the inter-syllable frequency
shift (assessed as ratio of frequency from the end of one
syllable to the start of the next syllable, Figure 1C). This
should impede the production of syllable transitions with Figure 1 Spectrograms of subsequent syllables produced by a male skylark in response to a territorial playback. (A,B) Examples with
small gaps. (C,D) Examples with large gaps. Red stars indicate peak frequency of the end of the first syllable and the start of the subsequent
syllable. Inter-syllable frequency ratio was calculated by dividing the larger peak frequency (vertical bar ‘F’) by the smaller one (vertical bar ‘f’). The
horizontal bar indicates gap duration. Figure 1 Spectrograms of subsequent syllables produced by a male skylark in response to a territorial playback. (A,B) Examples with
small gaps. (C,D) Examples with large gaps. Red stars indicate peak frequency of the end of the first syllable and the start of the subsequent
syllable. Inter-syllable frequency ratio was calculated by dividing the larger peak frequency (vertical bar ‘F’) by the smaller one (vertical bar ‘f’). The
horizontal bar indicates gap duration. Geberzahn and Aubin BMC Biology 2014, 12:58
http://www.biomedcentral.com/1741-7007/12/58 Page 3 of 9 Geberzahn and Aubin BMC Biology 2014, 12:58
http://www.biomedcentral.com/1741-7007/12/58 the deviation from a lower-bound regression should be
smaller in reactive than in spontaneous singing. both large inter-syllable frequency shifts and very short
gaps between syllables. Vocal gap deviation assesses how
closely a male is performing at this limit and is thus related
to ‘vocal deviation’, which assesses the speed of frequency
modulation in a trill [22]. Background Vocal gap deviation is, however,
more broadly applicable as it is not restricted to a given
succession of trills and allows estimation of vocal per-
formance in a species with a complex song structure. both large inter-syllable frequency shifts and very short
gaps between syllables. Vocal gap deviation assesses how
closely a male is performing at this limit and is thus related
to ‘vocal deviation’, which assesses the speed of frequency
modulation in a trill [22]. Vocal gap deviation is, however,
more broadly applicable as it is not restricted to a given
succession of trills and allows estimation of vocal per-
formance in a species with a complex song structure. Relationship between gap duration and inter-syllable
frequency shift When plotting gap duration against inter-syllable frequency
ratio for all syllables measured, we revealed a triangular dis-
tribution (Figure 2). The lower-bound regression, which
represents the putative performance limit, had a significant
positive slope (y = 6.3963x + 6.9766; linear regression:
F1,13 = 4.93, r2 = 0.27, P = 0.045; Figure 2). In the current study, we reanalyzed a dataset published
earlier [8] in which skylarks sang in response to a terri-
torial playback (reactive singing) and in baseline condi-
tion (spontaneous singing). We measured gap duration
and related it to inter-syllable frequency shift in corre-
sponding syllables. Our first hypothesis was that gap
duration is traded off against inter-syllable frequency
shift in the song of skylarks. Because males should be
easily capable of singing with short gaps when inter-
syllable frequency shifts are small but should require
longer gaps to produce large inter-syllable frequency
shifts, we expected to find a triangular distribution of
data points when plotting these two parameters against
each other with a significant lower-bound regression. Constraints are often reflected in such triangular distri-
butions, because data falls only on one side of a bound-
ary [23]. An alternative explanation for a trade-off
between gap duration and inter-syllable frequency shifts
could be that larger inter-syllable frequency shifts are as-
sociated with longer syllables (because longer syllables
could have more time to modulate to extreme frequen-
cies). In this case, longer gaps could simply be a conse-
quence of larger respiration requirements rather than
motor constraints caused by vocal tract reconfiguration. To assess this possibility, we tested for a relationship
between syllable duration and inter-syllable frequency
shifting. Longer syllables are not associated with larger
inter-syllable frequency shifts Figure 2 Gap duration plotted against inter-syllable frequency g
p
p
g
y
q
y
ratio. Blue line: lower-bound regression line. Red diamonds: syllables
produced in response to territorial playbacks. Black triangles: syllables
produced in spontaneous song (both contexts together n = 6,910
syllables of 16 subjects). Blue crosses: data points used to calculate
the lower-bound regression line assessed by the equal samples per
bin method [40]. Vocal gap deviation is measured as the minimum
orthogonal distance of each data point to the lower-bound regression
line; an example is shown for one data point (black dotted line). Minimum area convex polygon is given in red for reactive and in
grey for spontaneous singing. For statistics see text. Geberzahn and Aubin BMC Biology 2014, 12:58
http://www.biomedcentral.com/1741-7007/12/58 Page 4 of 9 Page 4 of 9 the inter-syllable frequency ratios, revealed that some
of the syllables sung in the baseline condition (spon-
taneous song) were far above the lower-bound regres-
sion line, suggesting that males were singing far below
their performance capacities as far as the trade-off be-
tween inter-syllable frequency shift and gap duration is
concerned. At the same time, some of the syllables
produced in reactive song seemed to be closer to the
lower-bound regression line. In fact, when taking into
account all syllables, vocal gap deviation was signifi-
cantly smaller when birds were singing in response to
the territorial playback than when singing spontan-
eously (spontaneous singing: mean ± SD 4.61 ± 0.76;
reactive singing: 4.15 ± 0.55; paired t-test, t = -2.76,
df = 15, P = 0.01; Figure 3). modulate to extreme frequencies. Thus, longer gaps fol-
lowing higher inter-syllable frequency ratios could simply
be a consequence of larger respiration requirements to re-
plenish air exhaled during long syllables. However, our
data did not reveal such relationships between syllable
duration and inter-syllable frequency ratios: only 12 out of
32 subject-wise correlation tests had positive coefficients,
only one of which was significant (corresponding to the
expected proportion of false positive findings, Table 1). Inter-syllable frequency shift alone does not vary with the
context Skylarks did not shift the frequency between syllables to
a lesser degree in reactive than in spontaneous singing:
we could not detect a significant contextual difference in
the inter-syllable frequency ratio when comparing all sylla-
bles (spontaneous singing: mean ± standard deviation (SD)
1.43 ± 0.04; reactive singing: 1.42 ± 0.04; paired t-test,
t = -0.85, degrees of freedom (df) = 15, P = 0.41) nor
when considering only context-specific syllable types
(spontaneous singing: mean ± SD 1.43 ± 0.04; reactive
singing: 1.42 ± 0.05; paired t-test, t = -0.26, df = 15, P = 0.8). To understand how skylarks arrive at singing closer to
this performance limit, we examined contextual vari-
ation in two subsets of syllable types: context-specific
syllable types and context-independent syllable types
(the later termed ‘shared syllables’ in [8]). Vocal gap
deviation was significantly smaller when considering
only context-specific syllable types (spontaneous sing-
ing: mean ± SD 4.68 ± 0.8; reactive singing: 4.2 ± 0.65;
paired t-test, t = -2.21, df = 15, P = 0.04). We observed
the same trend for context-independent syllable types
(spontaneous singing: mean ± SD, 4.54 ± 0.93; reactive
singing: 4.24 ± 1.01; Wilcoxon signed rank test, V = 33,
P = 0.07). Structure-dependent variation of vocal performance
h
b
h
d
l
d Structure-dependent variation of vocal performance
The observation that gap duration is related to inter-
syllable frequency shift in the complex song of skylarks
supports the hypothesis that it takes males more time to
pass from one syllable to the next if the change in fre-
quency between the end of the preceding syllable and
the start of the subsequent syllable is large. Likewise, it
seems to be easier for them to sing with shorter gaps if
the inter-syllable frequency ratio is small. A candidate
mechanism proposed for this trade-off is the temporal
requirements of the motor system to tune the vocal tract
to the fundamental frequency in order to suppress en-
ergy in higher harmonics [5,14]. This interpretation is
supported by the pure-tone quality of skylark song. The
necessity to replenish air supply and thus to ensure oxy-
gen delivery certainly also constraints the brevity of silent
gaps in between syllables in the skylark, a continuous
singer that sings during flight. In this species, gap duration
correlates with preceding syllable duration [24], suggesting
that skylarks exhale more air when producing longer sylla-
bles and thus require longer gaps for inhaling. We could
not, however, find an association between inter-syllable
frequency ratios and syllable durations. We can therefore
rule out that the reported trade-off (that is, between gap
duration and inter-syllable frequency ratio) arises simply
because syllables with larger frequency shifts are the ones
with longer syllable durations that need longer subsequent
gaps. Skylarks sing closer to the performance limit when
challenged by a territorial playback Visual examination of the distribution of all syllables in
Figure 2, in which the gap durations are plotted against Table 1 No relationship between syllable duration and
inter-syllable frequency ratios
Subject
Spontaneous singing
Reactive singing
rho
P
rho
P
1
-0.08
0.35
-0.16
0.04
2
-0.06
0.52
-0.14
0.10
3
0.07
0.60
0.15
0.09
4
-0.10
0.32
-0.10
0.29
5
0.20
0.01
0.09
0.18
6
-0.02
0.82
-0.14
0.13
7
-0.01
0.93
0.01
0.91
8
-0.17
0.11
-0.05
0.56
9
-0.13
0.25
0.06
0.54
10
-0.09
0.33
-0.19
0.08
11
-0.001
0.99
0.08
0.42
12
0.03
0.70
0.14
0.16
13
-0.08
0.51
0.003
0.98
14
0.12
0.20
0.10
0.35
15
-0.06
0.56
-0.04
0.63
16
-0.11
0.24
-0.15
0.10
Spearman’s rank rho is given for correlations between syllable duration and
inter-syllable frequency ratios of each of the 16 subjects in spontaneous and
reactive singing. Tests are based on mean values per syllable type. Significant
P-values are given in bold. Table 1 No relationship between syllable duration and
inter-syllable frequency ratios Figure 3 Vocal gap deviation was smaller when skylarks were
singing in response to a territorial playback. This indicates that
they were singing closer to their performance limit when challenged
than when singing spontaneously. Average values of all syllable types
are shown for each of 16 subjects. For statistics see text. Figure 3 Vocal gap deviation was smaller when skylarks were
singing in response to a territorial playback. This indicates that
they were singing closer to their performance limit when challenged
than when singing spontaneously. Average values of all syllable types
are shown for each of 16 subjects. For statistics see text. Geberzahn and Aubin BMC Biology 2014, 12:58
http://www.biomedcentral.com/1741-7007/12/58 Page 5 of 9 syllables in which the inter-syllable frequency shift is
small. Indeed, studies on species with trilled song compo-
nents containing mainly either up- or down-sweeps have
found a relationship between vocal performance and the
aggressive motivation or quality of males (for example,
swamp sparrows [25], banded wren, Thryophilus pleuros-
tictus [26]) whereas most studies on species with trill
components also made up by chevron-shaped syllables or
consisting of an alternating sequence of down- and up-
sweeping syllables so far failed to find such relationships
(for example, house wren [19,20], dark-eyed junco [21],
but see [27]). We here provide clear empirical evidence
supporting the hypothesis that performance constraints
vary with the structure of syllable sequences of birdsong. Discussion We found a triangular relationship between gap duration
and inter-syllable frequency shift with a significant lower-
bound regression in the song of skylarks. This relationship
cannot be explained by a correlation of inter-syllable fre-
quency shift with syllable duration, which might lead to
longer gap durations due to respiratory requirements. Thus, gap duration seems to be traded-off against inter-
syllable frequency shift, which suggests a performance
limit that skylarks face during song production. Contrary
to our expectation, inter-syllable frequency shift was not
significantly smaller when males were singing in response
to a playback of conspecific song simulating a territorial
intrusion. However, vocal gap deviation (the orthogonal
distance of data points to the lower-bound regression) was
significantly smaller when singing reactively than when
singing spontaneously, indicating that skylarks performed
closer to the performance limit when challenged. No context-dependent modulation of inter-syllable
frequency shift In a previous study, we found that gaps following those
syllables that skylarks produced uniquely in an aggressive
context were shorter than for syllables produced uniquely
in baseline condition [8]. This suggests that skylarks might
selectively choose such syllable transitions in which inter-
syllable frequency shifting is small in order to allow for
small gap durations and consequently higher sound dens-
ity. However, this hypothesis is not supported by our data
as we failed to detect contextual modulation of inter-
syllable frequency shift. Actually, skylarks may be limited
in the freedom to recombine syllables. This might be due
to, for example, the function of sequential organization in
neighbor-stranger discrimination in this species [28]: more
than 75% of the song is organized in recurring sequences
of syllables called phrases. Phrases that neighbors have in
common with each other convey information on group
membership and allow for reduced aggression between
established neighbors [28,29]. On a more proximate level,
recombination of syllables might be restricted by the song
learning process as sequential information might be in-
cluded in this process as already shown for nightingales
(Luscinia megarhynchos [30,31]). Skylarks sing closer to their performance limit when
challenged Vocal gap deviation was smaller when skylarks were sing-
ing in response to a territorial playback than when singing
in baseline condition. Thus, males performed closer to the
vocal production limit, which seemed to keep them from
increasing the inter-syllable frequency shift and decreasing
gap duration at the same time. When considering only
context-specific syllable types we cannot rule out that this
finding simply reflects the fact that gap duration alone
varies with the context. We have previously shown that
syllable types produced uniquely in reactive singing are
followed by smaller gaps than those produced uniquely in
spontaneous singing [8]. Perhaps the difference in vocal Our findings are consistent with the idea that perform-
ance difficulties vary with the structure - a hypothesis
originally proposed by Podos et al. [4]. These authors
suggested that the production of syllables in which the
end of one syllable is produced at a different frequency
than the start of the subsequent syllable might be more
likely to be subject to performance constraints than Geberzahn and Aubin BMC Biology 2014, 12:58
http://www.biomedcentral.com/1741-7007/12/58 Page 6 of 9 Page 6 of 9 assumption as the widely used ‘vocal deviation’, namely
that modulating the configuration of the vocal apparatus
in order to track changes in the fundamental frequency
requires a minimum amount of time. It assumes that
both the production of broadband trills with very high
trill rate as well as large inter-syllable frequency shifts
with very short gap durations should be physically chal-
lenging. The advantage of vocal gap deviation as a meas-
ure is that it can be applied to non-trilled components
of song and to birdsong with a very complex structure. In addition, this measure can assess vocal performance
independent of variation in other song features such as
frequency or amplitude modulations within syllables or
the degree of tonality. Using this measure on skylarks,
we showed that males decreased vocal gap deviation and
thus increased vocal performance when being challenged
by a simulated territorial intrusion, suggesting that this
trait might serve as a signal for aggressive motivation in
skylarks. gap deviation is mainly driven by this difference in gap
duration. Recording methods and playback experiments Song recordings were made at a sample rate of 44.1 kHz
using a Sennheiser ME62/K6 omnidirectional microphone
(frequency response: 20 Hz to 20 kHz ± 1 dB) mounted on
a Telinga Universal parabola (diameter: 50 cm, Telinga,
Tobo, Sweden) and connected to a Marantz PMD 670
solid-state recorder (Marantz, Kanagawa, Japan). We re-
corded songs between 0800 and 1300 hours. Spontaneous Study site, subjects and their song y
j
g
The study was conducted on sixteen male skylarks at
nine different locations in the agricultural fields sur-
rounding the University of Paris 11, France, during the
2011 (N = 9 subjects, 9 May to 1 July) and 2012 (N = 7
subjects, 3 May to 20 June) breeding seasons. Birds were
not individually marked as we did not succeed in catch-
ing them. However, we are confident that we were able
to identify individual subjects by carefully observing pos-
ition and behavior, especially the conspicuous flight
song, repeatedly displayed at a given location. Further-
more, site fidelity is very strong in breeding skylarks [35]
and boundaries between adjoining territories are stable
once territories are established [36]. Skylarks have a
large repertoire of different syllable types (>300 [28])
and perform their syllables in a continuous fashion with
short inter-syllable gaps. Furthermore, song is predomin-
antly produced during flight. Songs produced during
flight last on average for 261 s [37] but can last up to
1 h (NG, personal observation). Syllables are either sung
with ‘immediate variety’, males switching to a new syl-
lable type with each syllable produced, or with ‘eventual
variety’, a given syllable type being repeated several times
to constitute trilled components of the song. Searcy and Beecher [34] postulated three criteria that
should be met to establish that a given singing behavior
is an aggressive signal. The signal value should increase
in aggressive contexts (context criterion). The signal
should predict aggressive escalation by the signaler (pre-
dictive criterion). And differential signal values should
elicit differential responses (response criterion). In the
current study, we showed that vocal performance - mea-
sured as vocal gap deviation - meets the context criter-
ion and might therefore give a signal for competitive
potential in skylarks. It would be very interesting to test
in the future whether the predictive and the response
criterion are met as well in skylarks, and whether vocal
gap deviation is related to quality, condition, reproduct-
ive success, or age. Skylarks sing closer to their performance limit when
challenged However, the picture changes when consider-
ing all syllable types: here the newly introduced measure
of vocal performance allowed us to capture contextual
variation that could not be observed in the two parame-
ters going into its computation: we could neither detect
contextual variation in gap duration alone [8] nor in
inter-syllable frequency shifting alone (this study). Vocal
gap deviation, thus, provides a better biological explan-
ation of the overall variation observed in the song of
skylarks. Singing close to a performance limit has been well docu-
mented for different songbird species in the context of
female attraction (for example, canaries [32]) and male-
male competition (for example, swamp sparrows [25]). Studies on species with trilled song structure document a
relationship between high vocal performance and male
quality, condition, age, or aggressive motivation (for ex-
ample [26,33]). In skylarks, contextual variation in vocal
gap deviation was most pronounced when analyzing all
syllable types together; differences were less pronounced
(but still significant) for context-specific syllable types
alone and became marginally non-significant for context-
independent syllable types. Thus, we cannot draw conclu-
sions on which type of syllables might drive the differences,
and further studies will be needed to understand how
skylarks arrive at lower vocal gap deviations during re-
active singing. In addition, vocal gap deviation might be
just one of multiple different song features that can in-
dicate male skylarks’ aggressive motivation and/or qual-
ity. Thus, we cannot rule out that syllables performed
below the capacities concerning the trade-off between
gap duration and inter-syllable frequency shift might
nevertheless indicate the competitive potential of a male
in terms of other acoustic features. Song analysis
I
h In the current paper we reanalyzed a dataset published
earlier [8] using Avisoft SASLAB Pro. For this dataset we
had previously selected one song recording with the high-
est signal-to-noise ratio for each context and each subject,
and we had analyzed the first 40 s of the song correspond-
ing approximately to the ascending phase of the flight
[38]. Songs had been high-pass filtered (cut-off frequency:
1.4 kHz). For the current study, we created spectrograms
to measure peak frequency at the end and the start of
syllables (Fast Fourier transformation (FFT) length 1,024;
frame 100%; overlap 75%, Hamming window, frequency
resolution 43 Hz, temporal resolution of 5.8 ms) using the
Automatic Parameter Measurements setup based on
manual delineations of on- and offsets of syllables in oscil-
lograms. Likewise, we measured gap and syllable durations
from the same delineations directly on oscillograms. We
defined a syllable as a continuous trace on the sound spec-
trogram or a group of continuous traces spaced out by less
than 25 ms in all their renditions. Classification of syllables
into syllable types was based on visual comparison of over-
all frequency modulation shapes in spectrograms by one
observer (a subset of 947 syllables was controlled by a
second observer who agreed in 94% of classifications). To examine whether smaller gap durations in reactive
singing were related to a lesser degree in inter-syllable
frequency
shifting,
we
compared
the
inter-syllable
frequency ratios in all syllables and in context-specific
syllables (termed ‘unshared syllables’ in [8]) between the
spontaneous and reactive song. Context-specific syllables
are unique to one of the contexts, that is, they can be
found only either in spontaneous song (and not in react-
ive song) or only in reactive song (but not in spontan-
eous song). When comparing all syllables, we considered
those context-specific syllables as well as those syllables
that occurred both in spontaneous and in reactive song. To examine whether skylarks sing closer to a perform-
ance limit when challenged by a territorial playback, we
calculated the ‘vocal gap deviation’ of all syllables as the
minimum orthogonal distance to the lower-bound regres-
sion line (Figure 2). Note that due to properties of similar
triangles, the horizontal and vertical distance of a given
syllable to the lower-bound regression line are both
proportional, and then correlated, to the orthogonal dis-
tance. Thus, analyzing horizontal and vertical instead of
the orthogonal distance would give the same results. Conclusion
d We introduced a new measure of vocal performance, the
‘vocal gap deviation’. This measure is based on the same Page 7 of 9 Page 7 of 9 Geberzahn and Aubin BMC Biology 2014, 12:58
http://www.biomedcentral.com/1741-7007/12/58 [22]: we calculated frequency ratios instead of linear
frequency differences, used the equal samples per bin
method rather than equally sized bins, and plotted the
temporal parameter against the frequency parameter
rather than the reverse and thus used lower- instead of
upper-bound regression. The later was done as inter-
syllable frequency ratio is more likely to limit gap duration
than the reverse. Using the traditional upper-bound re-
gression method (on a plot of linear frequency differences
against gap duration) instead of our modified approach
did not change the results (see Additional files 1, 2 and 3). singing was defined as singing without any indication of in-
teractions with a conspecific. We conducted playback ex-
periments to simulate a territorial intrusion. Such
experiments elicited territorial responses consisting of
singing behavior, as well as approaching the simulated
intruder, landing in its vicinity, or flying low over it. We
recorded the song produced by the tested subject during
the playback and in the 10 min after the end of the presen-
tation of the playback stimulus (for further details see [8]). Song analysis
I
h Values of vocal gap deviation that fell below the lower-
bound regression line were assigned a negative deviation
(compare [20,41]). Greater values of vocal gap deviation
represent lower performance and smaller (or negative)
values of deviation reflect higher performance. When com-
paring parameters (inter-syllable frequency ratio, vocal gap
deviation) between spontaneous and reactive singing, we cal-
culated the average value for each subject based on average
values for each given syllable type that a male produced. g
We assessed ‘inter-syllable frequency shift’ as the ratio
between the end peak frequency of a given syllable and
the start peak frequency of the subsequent syllable. We
used frequency ratios because linear frequency differ-
ences
could
give
biased
results
by
overestimating
changes in the higher- relative to lower-frequency samples
(compare with [39]). To this end, we divided the peak
frequency with the higher value (either the end of the first
or the start of the subsequent syllable) by the smaller peak
frequency such that the minimum ratio was equal to 1 in
the case where both peak frequencies adopted the same
value. We then related gap duration between syllables to
inter-syllable frequency ratio (Figure 1). To assess whether
gap duration is traded-off against inter-syllable frequency
ratio, we conducted a lower-bound regression analysis fol-
lowing methods established by Podos et al. [22]. To avoid
sampling biases, we binned measurements of inter-syllable
ratio according to the equal samples per bin method [40]. We used a modified version of the traditional upper-
bound regression method established by Podos et al. References 1. Cresswell W: Song as a pursuit-deterrent signal, and its occurrence
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behavior in Golden-collared Manakins. J Ornithol 2012, 153:119–124. behavior in Golden collared Manakins. J Ornithol 2012, 153:119 124. 4. Podos J, Lahti DC, Moseley DL: Vocal performance and sensorimotor
learning in songbirds. Adv Study Behav 2009, 40:159–195. 4. Podos J, Lahti DC, Moseley DL: Vocal performance and sensorimotor
learning in songbirds. Adv Study Behav 2009, 40:159–195. 5. Riede T, Suthers RA, Fletcher NH, Blevins WE: Songbirds tune their vocal
tract to the fundamental frequency of their song. Proc Natl Acad Sci 2006
103:5543–5548. 6. Riede T, Goller F: Peripheral mechanisms for vocal production in
birds - differences and similarities to human speech and singing. Brain Lang 2010, 115:69–80. 7. Botero CA, Rossman RJ, Caro LM, Stenzler LM, Lovette IJ, De Kort SR,
Vehrencamp SL: Syllable type consistency is related to age, social status,
and reproductive success in the tropical mockingbird. Anim Behav 2009,
77:701–706. 7. Botero CA, Rossman RJ, Caro LM, Stenzler LM, Lovette IJ, De Kort SR,
Vehrencamp SL: Syllable type consistency is related to age, social status,
and reproductive success in the tropical mockingbird. Anim Behav 2009,
77:701–706. 8. Geberzahn N, Aubin T: How a songbird with a continuous singing style
modulates its song when territorially challenged. Behav Ecol Sociobiol 2014,
68:1–12. 8. Geberzahn N, Aubin T: How a songbird with a continuous singing style
modulates its song when territorially challenged. Behav Ecol Sociobiol 2014,
68:1–12. 9. Geberzahn N, Goymann W, Muck C, Ten Cate C: Females alter their song
when challenged in a sex-role reversed bird species. Behav Ecol
Sociobiol 2009, 64:193–204. Statistical analysis Statistical analyses were conducted using R2.13.0 [42]. We tested for normality using Shapiro-Wilk tests. In
cases of normal distributions, we used a paired t-test to
compare parameters in spontaneous and reactive sing-
ing. Where data were not normally distributed, we used
paired samples Wilcoxon tests. Similarly, we used linear
regressions to examine the relationship between inter-
syllable frequency ratio and gap duration and Spearman’s
rank correlation tests to check for relationships between
inter-syllable frequency ratio and syllable duration. Page 8 of 9 Page 8 of 9 Geberzahn and Aubin BMC Biology 2014, 12:58
http://www.biomedcentral.com/1741-7007/12/58 Geberzahn and Aubin BMC Biology 2014, 12:58
http://www.biomedcentral.com/1741-7007/12/58 Geberzahn and Aubin BMC Biology 2014, 12:58
http://www.biomedcentral.com/1741-7007/12/58 Additional files Scientific Research, France, and the Max Planck Society, Germany (GDRE
CNRS-MPG Neuroscience grant) to NG. Additional file 1: Spectrograms of subsequent syllables produced
by a male skylark in response to a territorial playback and
illustration of measures taken to apply the traditional upper-bound
regression method. (A, B) Examples with small gaps. (C, D) Examples
with large gaps. Red stars indicate peak frequency of the end of the first
syllable, and the start frequency of the subsequent syllable, the horizontal
bar indicates gap duration, the vertical bar indicates inter-syllable frequency
shift that was assessed by calculating the absolute difference of the end
peak frequency of a given syllable type and the start peak frequency of the
subsequent syllable. Additional file 1: Spectrograms of subsequent syllables produced
by a male skylark in response to a territorial playback and
illustration of measures taken to apply the traditional upper-bound Additional file 2: Inter-syllable frequency shift plotted against gap
duration using the traditional upper-bound regression method. g
pp
g
Black dotted line: upper-bound regression line. Red diamonds: syllables
produced in response to territorial playbacks. Black dots: syllables
produced in spontaneous song. Red diamonds and black dots belonging
to the same bin of gap duration are displayed slightly out of alignment
for better visibility. Black crosses: data points (assessed by applying
equally sized bins [40]) used to calculate the upper-bound regression
line). We binned measurements of gap duration on spectrograms into
2.9 ms increments - corresponding to the resolution of the FFT used for
these measurements (FFT length 512; frame 100%; overlap 75%, Hamming
window). The bin of gap durations with the highest inter-syllable frequency
shift for the whole dataset was the 52 ms bin. As there should be no
performance limit at gaps larger than this bin we considered all bins of
gap durations less than or equal to 52 ms for the regression analysis but
ignored bins of larger gap durations. We selected the maximum
inter-syllable frequency shift for each subsequent 2.9 ms bin of gap
duration (less than or equal to 52 ms) and calculated a linear regression
through these maximum values (black crosses). Vocal gap deviation was
measured as the minimum orthogonal distance of each data point to
the upper-bound regression line; an example is shown for one data
point (blue dotted line). Minimum area convex polygon is given in red
for reactive and in black for spontaneous singing. Note that for smaller
bins of gap durations (less than or equal to 52 ms) many data points lay
on top of each other. The upper-bound regression has a significant
positive slope (y = 85.455x + 629.68, linear regression: F1,13 = 44.86,
r2 = 0.78, P = 0.00001). References Additional file 3: Using the traditional upper-bound regression
method revealed that vocal gap deviation was smaller when skylarks
were singing in response to a territorial playback. This indicates that
they were singing closer to their performance limit when challenged
than when singing spontaneously. Average values of all syllable types
are shown for each of 16 subjects. Spontaneous singing: mean ± SD
41.53 ± 4.98; reactive singing: 38.85 ± 3.64; paired t-test, t = -2.56,
df = 15, P = 0.02. 10. Forstmeier W, Kempenaers B, Meyer A, Leisler B: A novel song parameter
correlates with extra-pair paternity and reflects male longevity. Proc Biol
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melodia), and its relationship to age, morphology and song complexity. Master’s Thesis: University of Western Ontario, Biology Department; 2013. Competing interests 15. Suthers RA, Zollinger SA: Producing song: the vocal apparatus. Ann N Y
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evidence for mini-breaths. J Comp Physiol A 1989, 165:15–26. Abbreviations df: degrees of freedom; FFT: Fast Fourier transformation; SD: standard
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receiver response in the banded wren. Proc Biol Sci 2006,
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1 1Centre National de la Recherche Scientifique, Centre de Neuroscience Paris
Sud, UMR 8195, 91405 Orsay, France. 2Université Paris Sud, Equipe
Communications Acoustiques/CNPS, Bat. 446, 91405 Orsay, France. 3Current
address: Laboratoire Éthologie Cognition Développement, Université Paris
Ouest Nanterre La Défense, 200 Avenue de la République 92001, Nanterre
Cedex, France. Acknowledgements 19. Cramer ERA: Physically challenging song traits, male quality, and
reproductive success in house wrens. PLoS One 2013, 8:e59208. Many thanks to Juliette Linossier for help with the fieldwork and to the
farmers for allowing us to work on their land. We thank Hélène Courvoisier
and Alexis Devulder for discussions. We thank Penelope Austin, Hélène
Courvoisier, and four anonymous reviewers for constructive comments on
earlier versions of this paper. This work was supported by a grant of the
European Research Network in Neurosciences of the National Centre for 20. Cramer ERA: Vocal deviation and trill consistency do not affect male
response to playback in house wrens. Behav Ecol 2012, 24:412–420. 21. Cardoso GC, Atwell JW, Hu Y, Ketterson ED, Price TD: No correlation
between three selected trade-offs in birdsong performance and male
quality for a species with song repertoires. Ethology 2012, 118:584–593. Page 9 of 9 Page 9 of 9 Geberzahn and Aubin BMC Biology 2014, 12:58
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http://www.biomedcentral.com/1741-7007/12/58 22. Podos J: Correlated evolution of morphology and vocal signal structure
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32. Drăgănoiu TI, Nagle L, Kreutzer M: Directional female preference for an
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107:466–492. 36. Aubin T: Etude expérimentale du chant territorial de l’alouette des champs
(Alauda arvensis L.). Caractéristiques physiques, valeur sémantique et
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37. Hedenström A: Song flight performance in the skylark Alauda arvensis. J Avian Biol 2011, 26:337–342. 37. Hedenström A: Song flight performance in the skylark Alauda arvensis. J Avian Biol 2011, 26:337–342. 38. Linossier J, Rybak F, Aubin T, Geberzahn N: Flight phases in the song of
skylarks: impact on acoustic parameters and coding strategy. PLoS One 2013,
8:e72768. 38. Linossier J, Rybak F, Aubin T, Geberzahn N: Flight phases in the song of
skylarks: impact on acoustic parameters and coding strategy. PLoS One 2013,
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analysis of vocal performance constraints. Am Nat 2014, 183:214–228. 41. Ballentine B: Vocal performance influences female response to male bird
song: an experimental test. Behav Ecol 2004, 15:163–168. 42. R Core Team: R: A language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing; 2013
[http://www.R-project.org/] doi:10.1186/s12915-014-0058-4
Cite this article as: Geberzahn and Aubin: Assessing vocal performance
in complex birdsong: a novel approach. BMC Biology 2014 12:58. doi:10.1186/s12915-014-0058-4
Cite this article as: Geberzahn and Aubin: Assessing vocal performance
in complex birdsong: a novel approach. BMC Biology 2014 12:58. Geberzahn and Aubin BMC Biology 2014, 12:58
http://www.biomedcentral.com/1741-7007/12/58 Acknowledgements Submit your next manuscript to BioMed Central
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and take full advantage of:
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Submit your manuscript at
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and take full advantage of:
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• Thorough peer review
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Submit your manuscript at
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https://openalex.org/W2727332199
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https://arpi.unipi.it/bitstream/11568/880805/1/galimberti%20bendamustina.pdf
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English
| null |
The Combination of Rituximab and Bendamustine as First-Line Treatment Is Highly Effective in the Eradicating Minimal Residual Disease in Follicular Lymphoma: An Italian Retrospective Study
|
Frontiers in pharmacology
| 2,017
|
cc-by
| 5,876
|
ORIGINAL RESEARCH
published: 29 June 2017
doi: 10.3389/fphar.2017.00413 Edited by:
Anna Rita Migliaccio,
Icahn School of Medicine at Mount
Sinai, United States 1 Section of Hematology, Department of Clinical and Experimental Medicine, University of Pisa, Pisa, Italy, 2 Doctoral School of
Genetics, Oncology and Clinical Medicine (GeNOMEC), University of Siena, Siena, Italy, 3 Department of Hematology,
University of Perugia, Perugia, Italy, 4 Department of Hematology, University of Firenze, Firenze, Italy, 5 Department of
Hematology and Oncology, Azienda Unità Sanitaria Locale di Piacenza, Piacenza, Italy, 6 Pathology, Azienda Ospedaliero
Universitaria Pisana, Pisa, Italy Reviewed by:
Francesca Palandri,
Università di Bologna, Italy
Liren Qian,
Navy General Hospital, China Reviewed by:
Francesca Palandri,
Università di Bologna, Italy
Liren Qian,
Navy General Hospital, China R-Bendamustine is an effective treatment for follicular lymphoma (FL). Previous large
trials demonstrated the prognostic role of the molecular minimal residual disease (MRD)
during the most frequently adopted chemotherapeutic regimens, but there are not yet
conclusive data about the effect of combination of rituximab (R) and bendamustine
in terms of MRD clearance. Thus, the aim of this retrospective study was to assess
if and in what extent the combination of rituximab and bendamustine would exert a
significant reduction of the molecular disease in 48 previously untreated FL patients. The molecular marker at baseline was found in the 62.5% of cases; no significant
differences were observed between patients with or without the molecular marker
in respect of the main clinical features. Moreover, the quantization of the baseline
molecular tumor burden showed a great variability: the median value was 1.4 × 10−2
copies, ranging from 3 × 10−5 to 4 × 104. The initial molecular tumor burden did
not correlate with clinical features and did not impact on the subsequent quality of
response. After treatment, 93% of cases became MRD-negative; the median reduction
of the BCL2/JH load was 4 logs. The 2-years PFS was 85%; it was significantly longer
for patients in complete than for those in partial response (91 vs. 57%; p = 0.002),
and for cases with lower FLIPI-2 score (88 vs. 60%; p = 0.004). On the contrary,
PFS did not differ between patients with or without the molecular marker at baseline;
a molecular tumor burden 15 times higher was observed in the relapsed subgroup
in comparison to the relapse-free one, but this difference did not change the PFS
length. The Combination of Rituximab and
Bendamustine as First-Line
Treatment Is Highly Effective in the
Eradicating Minimal Residual
Disease in Follicular Lymphoma: An
Italian Retrospective Study Sara Galimberti 1*, Elena Ciabatti 1, Giacomo Ercolano 1, Susanna Grassi 1, 2,
Francesca Guerrini 1, Nadia Cecconi 1, Martina Rousseau 1, Giulia Cervetti 1,
Francesco Mazziotta 1, Lorenzo Iovino 1, Franca Falzetti 3, Flavio Falcinelli 3, Alberto Bosi 4,
Luigi Rigacci 4, Sofia Kovalchuk 4, Daniele Vallisa 5, Lucia Macchia 1, Eugenio Ciancia 6 and
Mario Petrini 1 This study, even if retrospective *Correspondence:
Sara Galimberti
sara.galimberti@med.unipi.it Specialty section:
This article was submitted to
Cancer Molecular Targets and
Therapeutics,
a section of the journal
Frontiers in Pharmacology
Received: 28 April 2017
Accepted: 13 June 2017
Published: 29 June 2017 Specialty section:
This article was submitted to
Cancer Molecular Targets and
Therapeutics,
a section of the journal
Frontiers in Pharmacology The 2-years OS was 93.6%; the only variable that significantly impacted on
it was the FLIPI-2 score; the presence of the molecular marker at baseline or its
behavior after treatment did not impact on survival. This study, even if retrospective R-Bendamustine is an effective treatment for follicular lymphoma (FL). Previous large
trials demonstrated the prognostic role of the molecular minimal residual disease (MRD)
during the most frequently adopted chemotherapeutic regimens, but there are not yet
conclusive data about the effect of combination of rituximab (R) and bendamustine
in terms of MRD clearance. Thus, the aim of this retrospective study was to assess
if and in what extent the combination of rituximab and bendamustine would exert a
significant reduction of the molecular disease in 48 previously untreated FL patients. The molecular marker at baseline was found in the 62.5% of cases; no significant
differences were observed between patients with or without the molecular marker
in respect of the main clinical features. Moreover, the quantization of the baseline
molecular tumor burden showed a great variability: the median value was 1.4 × 10−2
copies, ranging from 3 × 10−5 to 4 × 104. The initial molecular tumor burden did
not correlate with clinical features and did not impact on the subsequent quality of
response. After treatment, 93% of cases became MRD-negative; the median reduction
of the BCL2/JH load was 4 logs. The 2-years PFS was 85%; it was significantly longer
for patients in complete than for those in partial response (91 vs. 57%; p = 0.002),
and for cases with lower FLIPI-2 score (88 vs. 60%; p = 0.004). On the contrary,
PFS did not differ between patients with or without the molecular marker at baseline;
a molecular tumor burden 15 times higher was observed in the relapsed subgroup
in comparison to the relapse-free one, but this difference did not change the PFS
length. The 2-years OS was 93.6%; the only variable that significantly impacted on
it was the FLIPI-2 score; the presence of the molecular marker at baseline or its
behavior after treatment did not impact on survival. Abbreviations: FL, follicular lymphoma; BM, bone marrow; MRD, minimal
residual disease; R, rituximab; PCR, polymerase chain reaction; PFS, progression-
free survival; OS, overall survival; FIL, fondazione italiana linfomi; CR, complete
remission; PR, partial remission; PET, positron-emission tomography; ESMO,
European Society for Medical Oncology; ROC, receiving operating characteristics;
FLIPI, follicular lymphoma international prognostic index. Citation: Galimberti S, Ciabatti E, Ercolano G,
Grassi S, Guerrini F, Cecconi N,
Rousseau M, Cervetti G, Mazziotta F,
Iovino L, Falzetti F, Falcinelli F, Bosi A,
Rigacci L, Kovalchuk S, Vallisa D,
Macchia L, Ciancia E and Petrini M
(2017) The Combination of Rituximab
and Bendamustine as First-Line
Treatment Is Highly Effective in the
Eradicating Minimal Residual Disease
in Follicular Lymphoma: An Italian
Retrospective Study. Front. Pharmacol. 8:413. doi: 10.3389/fphar.2017.00413 June 2017 | Volume 8 | Article 413 Frontiers in Pharmacology | www.frontiersin.org 1 Rituximab-Bendamustine and MRD in Follicular Lymphoma Galimberti et al. and conducted on a small series of patients, would represent a proof of concept that
R-bendamustine is able to so efficaciously eradicate MRD that it could be able to by-pass
the prognostic significance of MRD already demonstrated for other chemotherapeutic
regimens in FL. and conducted on a small series of patients, would represent a proof of concept that
R-bendamustine is able to so efficaciously eradicate MRD that it could be able to by-pass
the prognostic significance of MRD already demonstrated for other chemotherapeutic
regimens in FL. Keywords: MRD, follicular lymphoma, bendamustine, BCL2/IGH, rituximab, PCR INTRODUCTION In this context, few data about R-bendamustine and MRD in
FL are today available: Zohren and colleagues reported that 89.5%
of cases receiving R-bendamustine achieved the MRD negativity,
with a significant advantage in terms of PFS (Zohren et al., 2015). More recently, in the context of Gadolin trial, obinutuzumab
combined with bendamustine induced 82% of MRD negativity
vs. 43% of the bendamustine alone, with a significant longer PFS
for MRD-negative cases (Pott et al., 2015). In this context, few data about R-bendamustine and MRD in
FL are today available: Zohren and colleagues reported that 89.5%
of cases receiving R-bendamustine achieved the MRD negativity,
with a significant advantage in terms of PFS (Zohren et al., 2015). In the 2010, van Oers and colleagues demonstrated, in a
series of relapsed patients, that the presence of the BCL2/JH
at diagnosis had a negative impact on the outcome, whereas
the molecular status before rituximab maintenance did not
discriminate patients with worse outcome (van Oers et al., 2010). More recently, in the context of Gadolin trial, obinutuzumab
combined with bendamustine induced 82% of MRD negativity
vs. 43% of the bendamustine alone, with a significant longer PFS
for MRD-negative cases (Pott et al., 2015). More recently, two large studies conducted by the Fondazione
Italiana Linfomi (FIL) showed that after R-CHOP, R-CVP
(cyclophosphamide, vincristine, prednisone), R-FM (fludarabine
and mitoxantrone) and R-FND (fludarabine, mitoxantrone,
dexamethasone) the MRD status significantly conditioned the
PFS; indeed, patients in partial response (PR) but MRD-negative
showed longer PFS than those in complete remission (CR) but
still MRD-positive (Ladetto et al., 2013; Galimberti et al., 2014). With these premises, we decided to assess the impact of the
R-bendamustine on MRD in a cohort of 48 FL patients treated
in 4 different Italian centers. The aim of this retrospective study
was to measure both by qualitative and quantitative PCR the
molecular tumor burden before and 2 months after the end of
treatment with R-bendamustine. The presence and quantization
of the BCL2/JH rearrangement at baseline and its behavior after
induction was then compared with clinical features, response
rates, and 2-years survivals. More recently, the appearance in the scenario of the indolent
lymphomas of the new anti-CD20 monoclonal antibody,
obinutuzumab, prompted researchers to evaluate its value also in
the context of the molecular response. In the Gallium trial, where
CHOP, CVP or bendamustine were combined with rituximab or
obinutuzumab, the new antibody resulted more powerful than INTRODUCTION rituximab, offering 92.5% of MRD-negativity when combined
with bendamustine, 91.3% with CHOP, and 91.4% with CVP. Interestingly, the use of obinutuzumab in respect of rituximab
added 13.5% of good molecular responses in the cohort receiving
CHOP, 15.4% in those patients treated with CVP, but only 3% in
the bendamustine arm, thus suggesting that molecular clearance
offered by bendamustine was higher than that induced by the
other two chemotherapeutic regimens (Pott et al., 2016). The follicular lymphoma (FL) is the second more frequent
histotype of non-Hodgkin’s lymphomas in the Western world,
and represents one of the entities where the minimal residual
disease (MRD) has been more frequently studied. Indeed, the
history of MRD in FL is now 15 years old, starting with the
demonstration that rituximab (R) significantly reduced the
molecular tumor burden when administered after the CHOP
(Cyclophosphamide,
Doxorubicin,
Vincristine,
Prednisone)
regimen: in that setting, patients achieving MRD negativity
showed a significantly longer event-free survival (Rambaldi
et al., 2002). Then, many other works focused on the MRD in
the transplanted patients: cases MRD-negative after autologous
transplantation remained relapse-free and alive in a higher
percentage than those persistently MRD-positive (Galimberti
et al., 2003; Melillo et al., 2005). Then, MRD is still today a hot topic and often more frequently
it is introduced as aim in several phase-3 trials, as in the
FIL FOLL12 study (trial.gov identifier number: NCT02063685),
where the experimental arm is based on the PET and MRD
results: a de-intensified treatment was reserved to MRD- and
PET-negative cases, a consolidation with radio-immunotherapy
to patients still PET-positive after induction or a pre-emptive
therapy was adopted for PET-negative but MRD-positive cases. Nevertheless, MRD is still not a decisional tool in the routine
clinical practice: indeed, the ESMO guidelines recently edited,
even if clearly recognizing the prognostic role of the MRD,
state that the MRD “still cannot lead the therapeutic strategy”
(Dreyling et al., 2017). Subsequently,
when
yttrium-ibritumomab
tiuxetan
as
consolidation was compared to the observation only, it was
evident that radio-immunotherapy was able to rapidly eradicate
MRD in the majority of patients (Goffet al., 2009): after
this treatment 77% of patients became PCR-negative, with a
significant favorable impact on progression-free survival (PFS)
(Ibatici et al., 2014). Frontiers in Pharmacology | www.frontiersin.org RESULTS No significant differences were observed for the main
clinical features (age, sex, BM, infiltration, stage, histological
grade, FLIPI-2 score) between patients with or without
molecular marker detected by qualitative or quantitative PCR
(Table 2). Patients This retrospective observational study was conducted in 4 Italian
centers (Pisa, Firenze, Perugia, and Piacenza), in the context of
the routine clinical practice. Patients with newly diagnosed FL received bendamustine 90
mg/m2, days 1 and 2, once a month, plus rituximab 375 mg/m2,
for total 6 cycles. y
All patients signed a consent for leaving the leftover of
samples used for routine molecular analyses for further scientific June 2017 | Volume 8 | Article 413 Frontiers in Pharmacology | www.frontiersin.org 2 Rituximab-Bendamustine and MRD in Follicular Lymphoma Galimberti et al. TABLE 1 | Clinical characteristics of the enrolled patients. Characteristics
Number (%)
Number of patients
48
Median age, years
63
range
36-83
SEX
Male
24 (50%)
Female
24 (50%)
HISTOTYPE
Grade 1
17 (35%)
Grade 2
24 (50%)
Grade 3
7 (15%)
ANN ARBOR STAGE
II
8 (16%)
III
20 (42%)
IV
20 (42%)
ECOCG Performance Status >1
4 (8%)
FLIPI2
Low
9 (19%)
Intermediate
31 (65%)
High
8 (16%)
BM involvement
20 (42%) purposes. Forty-eight patients affected by FL were enrolled into
the study on the basis of the residual DNA availability; the
BCL2/JH rearrangement was evaluated on samples harvested at
diagnosis and 8–10 weeks after the end of induction. g
Samples have been collected between January 2012 and
December 2015. In addition to the physical examination and imaging
(total
body
computed
tomography
scan,
ultrasonography
or x-ray, according to physicians’ decision), at diagnosis
all
patients
underwent
bone
marrow
(BM)
biopsy
and
aspirate. In each center, BM biopsies were evaluated also
by immunohistochemistry (at least CD20, CD10, CD5, kappa,
and lambda clonality), in order to confirm the morphological
diagnosis of FL. Imaging was performed again for assessment of response at
treatment completion in addition to physical examination and
laboratory tests. Quality of response was defined according to the
standardized international criteria (Cheson et al., 2016). Statistical Analysis y
All statistical analyses were performed using the SPSS 21.0
software (SPSS Inc., Chicago, IL, USA). OS was calculated from
the date of diagnosis to death or last follow-up; PFS was measured
from the date of response to the induction treatment to the
last follow-up, disease progression or relapse. Survival curves
were calculated using the Kaplan-Meier method, and statistical
comparisons between curves were made using the log-rank test. Post-hoc comparisons were obtained using the Cox proportional
hazard regression method. The Chi squared test, Fisher’s exact
test and Kruskal-Wallis test were used to compare variables when
appropriate. For establishing a possible value of quantitative
BCL2/IGH rearrangement on relapse, a ROC curve was also
performed. All statistical comparisons were two-sided. The date
of the last molecular follow-up was October 2016. In 3 out of the 20 cases with microscopic BM infiltration (15%)
we did not find the BCL2/IGH rearrangement, probably for the
presence of some rare breakpoints in the BCL2 gene (not assessed
in this study). On the other hand, in 9 of the 28 cases without BM
infiltration (32%), the molecular marker was found, possibly due
to a submicroscopic involvement. In this study, the translocation between chromosome 14 and
18 (characteristic of FL) was not assessed. At baseline, the molecular tumor burden was also assessed
by quantitative PCR (QT-PCR) in the 28 cases with the MBR
breakpoint; it was quantified in 27 of them, because in one case
the BCL2/JH rearrangement resulted at the lowest limit of the test
sensitivity, and thus positive but not quantifiable. As expected,
measures showed a wide inter-patients variability: the median
value was 14 × 10−2 copies, but it ranged from 3 × 10−5 to 4
× 104 copies. Molecular Assays Qualitative BCL2/IGH rearrangement analysis was performed
at baseline and at the end of treatment. All qualitative and
quantitative analyses were centralized at the molecular laboratory
of the Hematology of the Pisa University, Italy. DNA was extracted from BM mononuclear cells by Wizard
Genomic DNA purification kit (Promega Madison WI, USA),
according to the suggestions by the FIL MRD Network (Mannu
et al., 2015). In order to amplify BCL2/IGH rearrangement,
nested PCR reactions were performed as previously described,
both for the MBR and mcr breakpoints (Galimberti et al., 2014). Their median age was 63 years (range 36–83), and half of them
were male. By microscope observation, BM resulted infiltrated in 20
cases; the 85% of them were scored as showing a grade 1 or
2 FL; 16% of our patients were in stage II, 42% in stage IV,
and more than 60% presented with intermediate FLIPI-2 risk
score. The sensitivity of the PCR assays was confirmed by testing
serial dilutions of DNA derived from the BCL2/IGH-positive
DOHH-2 cell line, achieving a limiting dilution of 1:10−5, either
for qualitative or quantitative tests. By qualitative PCR, BCL2/JH rearrangement at baseline was
found in 30 patients (62.5%). The breakpoint was MBR in 28
cases, and mcr in the remaining two. Clinical Response to Treatment Clinical Response to Treatment All 48 patients responded to treatment, 38 of them (79%)
achieving a complete response (CR). The achievement of CR was more frequently observed in cases
with low/intermediate than in those with high FLIPI-2 score (87
vs. 20%; p = 0.004). No other clinical features impacted on response rate or on the
quality of response. In particular, the presence of the molecular marker at baseline
did not correlate with the subsequent quality of response to
treatment: indeed, the CR rate was 45% in the subgroup of cases
with BCL2/JH detectable at baseline vs. 55% in the PCR-negative
cohort (p = 0.49). FIGURE 2 | Correlation between molecular tumor burden at baseline and
relapse rate: higher initial molecular burden was significantly correlated with a
higher probability of relapse/progression (mean of BCL2/JH copies in the
relapse-free subgroup: 9,411.8 ± 17,489.5 vs. 133,333.1 ± 230,940.1 in the
relapsed cases; p = 0.022). Also the molecular tumor burden (according to the median
value or the 1 or 4th quartile) did not impact on the response
rate. At
the
contrary,
a
higher
initial
molecular
burden
was significantly correlated with a higher probability of
relapse/progression (mean of BCL2/JH copies in the relapse-free
subgroup: 9,411.8 ± 17,489.5 vs. 133,333.1 ± 230,940.1 in the
relapsed cases; p = 0.022; Figure 2). Baseline: Patients Characteristics and
Molecular Marker Characteristics of the 48 patients enclosed into the study are
reported in the Table 1. June 2017 | Volume 8 | Article 413 Frontiers in Pharmacology | www.frontiersin.org 3 Rituximab-Bendamustine and MRD in Follicular Lymphoma Galimberti et al. FIGURE 1 | Kaplan-Meier analysis of progression-free survival of the whole
cohort. FIGURE 2 | Correlation between molecular tumor burden at baseline and
relapse rate: higher initial molecular burden was significantly correlated with a
higher probability of relapse/progression (mean of BCL2/JH copies in the
relapse-free subgroup: 9,411.8 ± 17,489.5 vs. 133,333.1 ± 230,940.1 in the
relapsed cases; p = 0.022). At
the
contrary,
a
higher
initial
molecular
burden
was significantly correlated with a higher probability of TABLE 2 | Comparison of the patients’ clinical characteristics according to the
presence/absence of the molecular marker at baseline. Characteristics
Patients with
molecular marker
number (%)
Patients without
molecular marker
number (%)
p
Number of patients
30
18
Median age, years
61
64
range
40-83
36-79
n.s. SEX
Male
16 (53%)
8 (45%)
Female
14 (47%)
10 (55%)
n.s. HISTOTYPE
Grade 1
11 (37%)
6 (33%)
n.s. Grade 2
15 (50%)
9 (50%)
Grade 3
4 (13%)
3 (17%)
ANN ARBOR STAGE
II
5 (17%)
3 (17%)
n.s. III
12 (40%)
8 (44%)
IV
13 (43%)
7 (39%)
ECOCG Performance Status >1
3 (10%)
1 (6%)
n.s. FLIPI2
Low
6 (20%)
3 (17%)
n.s. Intermediate
19 (63%)
12 (67%)
High
5 (17%)
3 (17%)
BM involvement
12 (40%)
8 (44%)
n.s. TABLE 2 | Comparison of the patients’ clinical characteristics according to the
presence/absence of the molecular marker at baseline. FIGURE 1 | Kaplan-Meier analysis of progression-free survival of the whole
cohort. FIGURE 1 | Kaplan-Meier analysis of progression-free survival of the whole
cohort. FIGURE 2 | Correlation between molecular tumor burden at baseline and
relapse rate: higher initial molecular burden was significantly correlated with a
higher probability of relapse/progression (mean of BCL2/JH copies in the
relapse-free subgroup: 9,411.8 ± 17,489.5 vs. 133,333.1 ± 230,940.1 in the
relapsed cases; p = 0.022). Frontiers in Pharmacology | www.frontiersin.org Overall and Progression-Free Survival In the whole series, the 2-years PFS was 85% (Figure 1); it
was significantly longer for patients in CR than for those
in partial remission (PR) (91 vs. 57%; p = 0.002) and for
cases with low vs. those with high FLIPI-2 score (88 vs. 60%;
p = 0.004). In the whole series, the 2-years PFS was 85% (Figure 1); it
was significantly longer for patients in CR than for those
in partial remission (PR) (91 vs. 57%; p = 0.002) and for
cases with low vs. those with high FLIPI-2 score (88 vs. 60%;
p = 0.004). Nevertheless, when we stratified patients according to the
median number of BCL2/JH copies (0.014) or according to
quartiles (75 or 25th percentile), no differences in terms of PFS
were observed (2-years PFS, 100% for cases with BCL2/JH ratio
< 0.014 vs. 81% of cases with higher molecular tumor burden;
p = 0.08). On the contrary, PFS did not differ between patients with
or without the molecular marker at baseline when BCL2/JH
rearrangement was assessed by qualitative PCR (2-years PFS,
83% for patients BCL2/JH-positive vs. 91% for those BCL2/JH-
negative; p = 0.25). When the PCR status at baseline, quality of response (CR vs. PR) and FLIPI-2 score (high/intermediate vs. low) were inserted June 2017 | Volume 8 | Article 413 Frontiers in Pharmacology | www.frontiersin.org 4 Rituximab-Bendamustine and MRD in Follicular Lymphoma Galimberti et al. as covariates in the multivariate analysis, all parameters lost their
statistical significance. as covariates in the multivariate analysis, all parameters lost their
statistical significance. This is probably due to several considerations: (1) different
studies proved that MRD-negative patients show a better
prognosis, either when receiving chemo-immunotherapy or
high-dose treatment. Nevertheless, these experiences have been
conducted or in the context of large phase-III multicenter
trials or, at the opposite, on small series of patients, and thus
many physicians consider those results not fully exportable to
their routine activity; (2) notwithstanding the standardization
performed by some cooperative groups (van der Velden et al.,
2003; Pott et al., 2013), the availability of a common, easily
performable and cheap molecular method for investigating MRD
is still lacking; (3) about MRD and R-bendamustine, few data are
still available, and thus further information on this specific setting
would be waited. The Impact of Treatment on MRD Our study is retrospective and involved a small number
of patients, but it represents the picture of that occurs to FL
patients in the real life. That statement is well supported by
the observation that our clinical results are superimposable to
those from other larger and pivotal trials: in particular, in our
series CR rate resulted 79%, 2-years PFS 85%, and 2-years OS
94%. These values result comparable to those recently reported
by Mondello et al. in 192 cases randomized to receive R-
bendamustine or R-CHOP (Mondello et al., 2016). Those authors
reported the superiority of R-bendamustine, with a significant
prolongation of the median PFS (152 vs. 132 months); in that
study, bendamustine offered 94% of ORR, and 63% of CR. Probably, the higher rate of overall response (100%) found
in our series could be explained by the retrospective nature
of this study (where enrolment was only based on the DNA
availability). After induction, all initially PCR-positive patients were re-
assessed by both qualitative and quantitative PCR: the qualitative
analysis became negative in all cases except two (93.3%); also in
the two patients with the mcr breakpoint, the molecular marker
disappeared. In addition, QT-PCR showed the achievement of a very deep
response in the majority of cases: 25 patients (92.6%) achieved the
MRD negativity (≤1 × 10−6 copies), and 2 resulted positive but
not quantifiable (molecular tumor burden between 1 × 10−5 and
1 × 10−6). Overall, the median reduction of BCL2/JH burden was
4 logs, with 6 cases showing a reduction around or higher than
5 logs. No significant differences in term of probability of relapse or
death nor in terms of PFS and OS length were observed in cases
with quantitative reductions higher or lower than the median of
4 logs. Nevertheless, even with the limitation of the number of our
cases, the present study could add some information about MRD
in the setting of R-bendamustine to those previously reported by
Zohren and colleagues in the contex of the StiL trial (Rummel
et al., 2013). Overall and Progression-Free Survival The 2-years OS for the whole series was 93.6% (Figure 3); the
only parameter that significantly impacted on survival was the
FLIPI-2 score, with 2-years OS of 97% for cases with low FLIPI-2
vs. 60% for those with high risk score (p = 0.001). Differently from the PFS, the quality of response was not
prognostic in terms of OS (2-years OS 57% for patients in CR
vs. 37% for those in PR; p = 0.55). Concerning the value of the presence of the molecular marker
or its load at baseline, the PCR status before treatment did not
significantly impact on OS. REFERENCES marrow predicts outcome in follicular lymphomas treated with a rituximab-
intensive program. Blood 122, 3759–3766. doi: 10.1182/blood-2013-06-507319 marrow predicts outcome in follicular lymphomas treated with a rituximab-
intensive program. Blood 122, 3759–3766. doi: 10.1182/blood-2013-06-507319 Cheson, B. D., Ansell, S., Schwartz, L., Gordon, L. I., Advani, R., Jacene, H. A.,
et al. (2016). Refinement of the Lugano Classification lymphoma response
criteria in the era of immunomodulatory therapy. Blood 128, 2489–2496. doi: 10.1182/blood-2016-05-718528 Mannu, C., Gazzola, A., Ciabatti, E., Fuligni, F., Cavalli, M., Della Starza, I., et al. (2015). Comparis on of different DNA extraction methods from peripheral
blood cells: advice from the Fondazione Italiana Linfomi Minimal Residual
Disease Network. Leuk. Lymphoma. 57, 1–11. doi: 10.3109/10428194.2014. 914199 Dreyling, M., Ghielmini, M., Rule, S., Salles, G., Vitolo, U., and Ladetto, M. (2017). Newly diagnosed and relapsed follicular lymphoma: ESMO clinical practice
guidelines for diagnosis, treatment and follow-up. Ann. Oncol. 25(Suppl. 3),
iii76–iii82. doi: 10.1093/annonc/mdx020 Melillo, L., Cascavilla, N., Lerma, E., Corsetti, M. T., and Carella, A. M. (2005). The significance of minimal residual disease in stem cell grafts and the role of
purging: is it better to purge in vivo or in vitro? Acta Haematol. 114, 206–213. doi: 10.1159/000088411 Galimberti, S., Guerrini, F., Morabito, F., Palumbo, G. A., Di Raimondo,
F., Papineschi, F., et al. (2003). Quantitative molecular evaluation in
autotransplant programs for follicular lymphoma: efficacy of in vivo purging by
Rituximab. Bone Marrow Transplant. 32, 57–63. doi: 10.1038/sj.bmt.1704102 Mondello, P., Steiner, N., Willenbacher, W., Wasle, I., Zaja, F., and Zambello,
R. (2016). Bendamustine plus rituximab versus R-CHOP as first-line
treatment for patients with indolent non-Hodgkin’s lymphoma: evidence
from a multicenter, retrospective study. Ann. Hematol. 95, 1107–1114. doi: 10.1007/s00277-016-2668-0 Galimberti, S., Luminari, S., Ciabatti, E., Grassi, S., Guerrini, F., Dondi,
A., et al. (2014). Minimal residual disease after conventional treatment
significantly impacts on progression-free survival of patients with follicular
lymphoma: the FIL FOLL05 trial. Clin. Cancer Res. 20, 6398–6405. doi: 10.1158/1078-0432.ccr-14-0407 Pott, C., Belada, D., Danesi, N., Fingerle-Rowson, G., Gribben, J., Harbron, C., et al. (2015). Analysis of minimal residual disease in follicular lymphoma patients
in gadolin, a Phase III study of obinutuzumab plus bendamustine versus
bendamustine in relapsed/refractory indolent non-Hodgkin Lymphoma. Blood
126, 3978. Goff, L., Summers, K., Iqbal, S., Kuhlmann, J., Kunz, M., Louton, T., et al. (2009). DISCUSSION Notwithstanding its long history, the MRD evaluation in FL and
its significance in the clinical practice are still matter of debate. The recent European guidelines affirm that MRD has really got
a predictive and prognostic significance, but that it is not still
suitable for driving treatment of FL patients (Dreyling et al.,
2017). Moreover, it is relevant to observe that our molecular data are
perfectly in line with those previously reported by other authors:
the BCL2/JH rearrangement at baseline has been detected in
62% of our patients; this is the same percentage reported in the
Gadolin trial (Pott et al., 2015) and in the StiL study (Zohren
et al., 2015), and slightly higher than that ourselves reported
in previous FIL trials (52% and 51%) (Ladetto et al., 2013;
Galimberti et al., 2014). FIGURE 3 | Kaplan-Meier analysis of overall survival of the whole cohort. Also the number of cases with BM involvement documented
by microscopic observation but without molecular marker at
baseline was analogous to that previously reported (14% in the
present study vs. 17% in the FOLL05 trial) (Galimberti et al.,
2014): this phenomenon could be surely related to a possible
poor BM sampling, but also to the emerging demonstration that
at least 8–10% of the “PCR-negative” cases present some “rare”
breakpoints in the BCL2 gene (these rearrangements have not be
evaluated in the present study). Thus, our study would suggest that R-bendamustine exerts a
so frequent and deep clearance of MRD that it would be able
to by-pass the negative prognostic impact usually played by the
MRD in other contexts, such as when R-CHOP, R-CVP, R-FM, or
R-FND were adopted as induction regimens. June 2017 | Volume 8 | Article 413 Frontiers in Pharmacology | www.frontiersin.org 5 Rituximab-Bendamustine and MRD in Follicular Lymphoma Galimberti et al. Indeed, in the present work the baseline molecular tumor
burden did not condition the quality of response, opposite to that
we observed in the FOLL05 study, where CR was achieved by the
half of the initially PCR-positive in respect of the PCR-negative
cases (Galimberti et al., 2014). In conclusion, our study suggests that the combination of
rituximab and bendamustine, by exerting a rapid and deep
clearance of the molecular disease, could represent a highly
effective therapeutic approach to FL, also in terms of molecular
disease. FUNDING The study was supported by the Associazione Italiana Linfomi,
Mielomi, Leucemie (AIL) Pisa. The study was supported by the Associazione Italiana Linfomi,
Mielomi, Leucemie (AIL) Pisa. ETHICS STATEMENT This study was carried out in accordance with the Declaration
of Helsinki. All patients gave the consent to leave the leftover of
the routine analyses for scientific purposes. this statement was
approved by the legal office of the AOUP. AUTHOR CONTRIBUTIONS All authors participated to the conception of the study. EC, GE,
SuGr, and FG performed PCR assays. SG, NC, MR, GC, FM,
LI, FF, AB, LR, SK, DV, and MP enrolled patients, performed
treatment and clinical follow-up. LM and EC performed the
microscopic analyses. SG and MP performed statistical analyses. All authors approved the final version of the manuscript. Interestingly, the rapidity of the molecular clearance offered
by bendamustine has been already well demonstrated in the
Gallium (Pott et al., 2016) and Gadolin (Pott et al., 2015) trials,
where bendamustine was combined with obinutuzumab: in both
trials, the rapid efficacy of the R-bendamustine also in molecular
terms has been clearly demonstrated. It is also relevant that
in the arm with R-bendamustine of the Gallium trial, 90% of
patients became MRD-negative, a percentage comparable to that
observed in our study (93%) and that in that trial, as in the present
study, the MRD status did not condition the PFS length. DISCUSSION The larger ongoing studies will show if the MRD would
be used as adjunctive tool for better selecting the more effective
treatment for FL patients. In the present study, relapsed patients presented an initial
molecular burden 15 times higher than that measured in not
relapsed/progressed cases, but this difference did not translate in
a significant advantage in terms of PFS. This could be probably
due to the small number of patients, but we cannot exclude that
it could be also the consequence of the more rapid and deeper
MRD clearance exerted by bendamustine: in our series, more
than 90% of cases achieved the MRD negativity, and quantitative
data showed that the majority of cases achieved a very deep
reduction of the molecular disease. The median reduction of 4
logs is encouraging in respect of the 2 logs reported after R-
CHOP (Galimberti et al., 2014) and the 2.5 logs reported after
yttrium-ibritumomab tiuxetan treatment (Ibatici et al., 2014). REFERENCES Quantitative PCR analysis for Bcl-2/IgH in a phase III study
of Yttrium-90 Ibritumomab Tiuxetan as consolidation of first remission
in patients with follicular lymphoma. J. Clin. Oncol. 27, 6094–6100. doi: 10.1200/jco.2009.22.6258 Pott, C., Brüggemann, M., Ritgen, M., van der Velden, V. H., van Dongen,
J. J., and Kneba, M. (2013). MRD detection in B-cell non-Hodgkin
lymphomas using Ig gene rearrangements and chromosomal translocations
as targets for real-time quantitative PCR. Methods Mol. Biol. 971, 175–200. doi: 10.1007/978-1-62703-269-8_10 Ibatici, A., Pica, G. M., Nati, S., Vitolo, U., Botto, B., Ciocchetto, C., et al. (2014). Safety and efficacy of (90) yttrium-ibritumomab-tiuxetan for untreated
follicular lymphoma patients. An Italian cooperative study. Br. J. Haematol. 164, 710–716. doi: 10.1111/bjh.12695 Pott, C., Hoster, E., Kehden, B., Unterhalt, M., Herold, M., van der Jagt, R. H., et al. (2016). Minimal residual disease in patients with follicular lymphoma treated
with obinutuzumab or rituximab as first-line induction Immunochemotherapy
and Maintenance in the Phase 3 GALLIUM Study. Blood 128, 613. Ladetto, M., Lobetti-Bodoni, C., Mantoan, B., Ceccarelli, M., Boccomini, C.,
Genuardi, E., et al. (2013). Persistence of minimal residual disease in bone June 2017 | Volume 8 | Article 413 Frontiers in Pharmacology | www.frontiersin.org 6 Rituximab-Bendamustine and MRD in Follicular Lymphoma Galimberti et al. Zohren,
F.,
Bruns,
I.,
Pechtel,
S.,
Schroeder,
T.,
Fenk,
R.,
Czibere,
A.,
et
al. (2015). Prognostic
value
of
circulating
Bcl-2/IgH
levels
in
patients
with
follicular
lymphoma
receiving
first-line
immunochemotherapy. Blood 126, 1407–1414. doi: 10.1182/blood-2015-03-6
30012 Rambaldi, A., Lazzari, M., Manzoni, C., Carlotti, E., Arcaini, L., Baccarani, M.,
et al. (2002). Monitoring of minimal residual disease after CHOP and rituximab
in previously untreated patients with follicular lymphoma. Blood 99, 856–862. doi: 10.1182/blood.V99.3.856 Rummel, M. J., Niederle, N., Maschmeyer, G., Banat, G. A., von Grunhagen,
U., Losen, C., et al. (2013). Bendamustine plus rituximab versus CHOP plus
rituximab as first-line treatment for patients with indolent and mantle-cell
lymphomas: an open-label, multicentre, randomised, phase 3 non-inferiority
trial. Lancet 381, 1203–1210. doi: 10.1016/S0140-6736(12)61763-2 Conflict of Interest Statement: 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. Conflict of Interest Statement: 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. van der Velden, V. Frontiers in Pharmacology | www.frontiersin.org June 2017 | Volume 8 | Article 413 REFERENCES H., Hochhaus, A., Cazzaniga, G., Szczepanski, T., Gabert, J., and
van Dongen, J. J. (2003). Detection of minimal residual disease in hematologic
malignancies by real-time quantitative PCR: principles, approaches, and
laboratory aspects. Leukemia 17, 1013–1034. doi: 10.1038/sj.leu.2402922 Copyright © 2017 Galimberti, Ciabatti, Ercolano, Grassi, Guerrini, Cecconi,
Rousseau, Cervetti, Mazziotta, Iovino, Falzetti, Falcinelli, Bosi, Rigacci, Kovalchuk,
Vallisa, Macchia, Ciancia and Petrini. 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) or licensor 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. van Oers, M. H., Tönnissen, E., Van Glabbeke, M., Giurgea, L., Jansen, J. H.,
Klasa, R., et al. (2010). BCL-2/IgH polymerase chain reaction status at the
end of induction treatment is not predictive for progression-free survival in
relapsed/resistant follicular lymphoma: results of a prospective randomized
EORTC 20981 phase III intergroup study. J. Clin. Oncol. 28, 2246–2252. doi: 10.1200/JCO.2009.25.0852 June 2017 | Volume 8 | Article 413 Frontiers in Pharmacology | www.frontiersin.org 7
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Design and Realization of Mongolian Syntactic Retrieval System Based on Dependency Treebank
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I.
INTRODUCTION Language Research Institute of Inner Mongolia University
has constructed a 1-million-word modern Mongolian corpus in
a span of eight years from 1984 to 1991 and expanded it twice
into what is now a 10-million-word corpus. The 1-million-
word corpus contains materials from novels (19.6%),
textbooks (50.3%), newspapers (9.8%) and politics (22.9%)
[1]. In corpus annotation, the 10-million word corpus has been
completed the part-of-speech tagging [2][3] and fixed phrase
tagging [4]. And some shallow parsing is carried out on the 1-
million word corpus, such as phrase tagging [5][6][7],
automatic sentence segmentation [8] and automatic predicate
segment recognition [9]. (IJACSA) International Journal of Advanced Computer Science and Applications,
Vol. 6, No. 10, 2015 (IJACSA) International Journal of Advanced Computer Science and Applications,
Vol. 6, No. 10, 2015 Sponsored by National Natural Science Foundation (60763033), National
Natural Science Foundation (61262046), and National Social Science
Foundation(10CYY022). II.
DESIGN AND REALIZATION OF MONGOLIAN SYNTACTIC
RETRIEVAL SYSTEM Dependency tree-based Mongolian syntactic retrieval
system is divided into two parts, syntactic tree display module
and retrieval statistics module. Realization of the two modules
is presented as follows. From 2008 to 2011, funded by National Social Science
Foundation and National Natural Science Foundation, using
the method of automatic parsing and manual proofreading,
Language Research Institute of Inner Mongolia University has
constructed a 500,000-word Mongolian dependency treebank
(MDTB) based on middle school Mongolian textbooks that
were extracted from the 1-million-word modern Mongolian
corpus [10]. MDTB has an annotation set of 17 dependency
relations under 5 categories [11]. The 5 categories are special
relation, dominant relation, conjunctional relation, auxiliary
relation and non-syntactical elements. The 17 dependency
relations include: key word in a sentence (HEAD),
independent element (INDE), subject (SUBJ), direct object
(DOBJ), indirect object (IOBJ), attribute (ATT), adverbial
(ADV), coordinate (COO), appositive (APP), summarization
(SUM), time-local words-auxiliary (TL-AUX), postposition-
auxiliary (PP-AUX), modal particles-auxiliary (MP-AUX),
modals-auxiliary (M-AUX), auxiliary verbs-auxiliary (AV- Keywords—Mongolian Language; Dependency Grammar;
Dependency Treebank; Syntactic Retrieval; Information Retrieval Keywords—Mongolian Language; Dependency Grammar;
Dependency Treebank; Syntactic Retrieval; Information Retrieval Design and Realization of Mongolian Syntactic
Retrieval System Based on Dependency Treebank S.Loglo
Language Research Institute
College of Mongolian Studies
Inner Mongolia University
Huhhot, Inner Mongolia Autonomous Region, China Sarula
Department of Journalism and publishing
College of Mongolian Studies
Inner Mongolia University
Huhhot, Inner Mongolia Autonomous Region, China AUX), contact verb-auxiliary (CV-AUX) and conjunction-
auxiliary (CJ-AUX). In the form of annotation, MDTB uses
two types of labeling, namely the brackets annotation and
graphical annotations. This treebank contains 461,240 words
in 31,722 sentences. The average sentence length is 14.54
words. Abstract—In the past seven years, Language Research
Institute of Inner Mongolia University has constructed a 500,000-
word scale Mongolian dependency treebank. The syntactic
treebank provides a favorable data platform for language
research and information processing. In order to effectively use
the treebank, we have designed and implemented a graphical
syntactic information retrieval system based on the Mongolian
dependency treebank. As an application system, this retrieval
system offers search and statistical analysis on word, phrase,
syntactic fragment and syntactic structure level. Mongolian dependency treebank contains rich syntactic
information, so the researchers can obtain all kinds of
information about syntax. On the dependency treebank,
researchers also can perform statistical analysis and example
sentence extraction. Therefore, it provides convenience for the
study and research of Mongolian traditional linguistics and
computational linguistics [12] [13]. However, at present,
treebank is usually used as a training and evaluation data for
syntactic parsing [14], but research about the syntactic
information retrieval is few and far between. This paper is
designed to expound a syntactic treebank retrieval system
based on the application system of the dependency treebank. The retrieval functions allow researchers to do enquiry and
statistical analysis on word, phrase, sentence constituents,
syntactic fragment and syntactic structure. 2) Contract or expand descendant nodes; and Treebank is an important resource for syntactic analysis
and evaluation, word sense disambiguation and semantic
analysis. MDTB provides a favorable data platform for
Mongolian language research and information processing. At
present, the use of treebank is mainly achieved through a
variety of retrieve technology-based statistical methods. As
such develop an efficient search algorithm is very necessary
for treebank-based systems [15] [16]. 3) Display the entire syntactic tree or only display the
search results. 3) Display the entire syntactic tree or only display the
search results. sea ch esults. Fig. 1. Node structure of a dependency tree
NextBrother ●
Word
● PrevBrother
LexicalInfo
Relation
xPos
Parent ●
Child ●
yPos
Angle
IsShown
IsMatched Fig. 1. Node structure of a dependency tree
NextBrother ●
Word
● PrevBrother
LexicalInfo
Relation
xPos
Parent ●
Child ●
yPos
Angle
IsShown
IsMatched The dependency treebank herein adopts two different
storage formats, text and graph. Text format is for treebank
that targets all users and can be opened and edited by any text
editing software. Graphical format, which MDTB adopts,
benefits both output and retrieval, although it does not perform
better than text format in terms of space utilization. Based on
graphical storage format, we have designed a treebank
retrieval algorithm with sub-tree query function. The query
conditions can be a sub-tree, a word or a syntactic fragment
with n nodes. Each node can have one or multiple
characteristic values such as vocabulary (can use wildcards
like ‗*‘ and ‗?‘), parts of speech, sub-categorization,
morphology, dependency relation type, father node and child
node. Fig. 1. Node structure of a dependency tree Fig. 1. Node structure of a dependency tree In Fig.1, PrevBrother stores a pointer that points to the
node's previous brother, NextBrother stores a pointer that
points to the node's next brother, Parent stores a pointer that
points to the node's parent node, Child stores a pointer that
points to the node's child node, Word stores the node's word,
LexicalInfo stores the node's lexical information, Relation
stores the node's dependency relation, xPos and yPos stores
the node's horizontal and vertical ordinate, Angle stores the
node's inclined angle of dependency arc, IsShown denotes
whether the node‘s descendent nodes are shown or not, and
IsMatched denotes that the node is among the research results. 2) Contract or expand descendant nodes; and The retrieval algorithm is as follows: 1) Traverse syntactic tree pre-orderly to find root node
(sRoot ) of query condition; 2) If a node (tNode) in the dependency tree satisfies the
requirement of the query condition’s root node (sRoot), then,
to find all the child nodes of sRoot in the child nodes of the
tNode; The output algorithm is as follows: The output algorithm is as follows:
Algorithm1. Dependency tree display algorithm
VOID ShowTree (CTree *T,int ShowMode)
//T denotes the dependency tree;
//ShowMode==‖0‖ indicates that the program will
//display all syntax trees; and "ShowMode==1" indicates
//that the program will display the results of the search. {If (ShowMode==1 &&
T->bSearchResult==FALSE) return;
//Visit RootNode on the dependency tree T;
If(!RootNode->IsShrunk())
// denotes that the root node is not shrunk;
ShowNode(RootNode);//draw the node (RootNode)
If(RootNode->bShowLexcicalInfo==TRUE)
ShowLexicalInfo(RootNode);
//shows the node‘s lexical information;
SortChildrens(RootNode);
//Sort the child nodes of RootNode;
//Traverse subtree forest of root nodes pre-orderly;
For(i=0;i<n;i++)a
//n is the number of child nodes of RootNode
ShowTree (CTi);
//CTi denotes a sub-tree whose root node is the ith
//child of T
Return;
} p
g
Algorithm1. Dependency tree display algorithm
VOID ShowTree (CTree *T,int ShowMode)
//T denotes the dependency tree;
//ShowMode==‖0‖ indicates that the program will
//display all syntax trees; and "ShowMode==1" indicates
//that the program will display the results of the search. {If (ShowMode==1 &&
T->bSearchResult==FALSE) return;
//Visit RootNode on the dependency tree T;
If(!RootNode->IsShrunk())
// denotes that the root node is not shrunk;
ShowNode(RootNode);//draw the node (RootNode)
If(RootNode->bShowLexcicalInfo==TRUE)
ShowLexicalInfo(RootNode);
//shows the node‘s lexical information;
SortChildrens(RootNode);
//Sort the child nodes of RootNode;
//Traverse subtree forest of root nodes pre-orderly;
For(i=0;i<n;i++)a
//n is the number of child nodes of RootNode
ShowTree (CTi);
//CTi denotes a sub-tree whose root node is the ith
//child of T
Return;
} 3) If all of sRoot’s child nodes are found in step (2),
recursively call step (2) until nodes that meet the requirements
can no longer be found or the rightmost descendent nodes of
query conditions are found; q
y
f
4) Continue to find sub-trees among the remaining nodes
of the current tree (excluding traversed nodes); and 5) Treebank search needs to call step (1) to step (4)
repeatedly. tNode represents one particular node on syntactic tree, and
sRoot denotes root node of one sub-tree under given query
conditions at given moment (including query condition itself). ShowLexicalInfo(RootNode); C. A Syntactic Retrieval Example //shows the node‘s lexical information; The rationale of retrieval algorithm is explained by finding
juxtaposed attributive in the following sentence. Fig.2 shows the dependency tree of the following sentence, A. Display Module of the Syntactic Tree Graphic display lies in the heart of treebank operation as
an essential technology. However formatted a treebank is, in
text or in graph, the output module can draw a complete tree
for each sentence, as shown in the Fig.5. The left window
displays corpus texts of which the current sentence is
displayed in selected model. The right window displays the
syntactic tree of the current sentence. Fig.1 shows the node structure of the syntactic tree. Each
node on the syntactic tree may have n child nodes which are
arranged from left to right according to the sequence in which
dependency relations were established. But this order dampens
the readability of treebank. To recover the original order of
brother nodes, we have added sorting function to the output
model. The output module also provides multiple optional
display modes which are presented as follows. 164 | P a g e 164 | P a g e www.ijacsa.thesai.org Algorithm1. Dependency tree display algorithm B. Design of Mongolian Syntactic Retrieval Algorithm )
p
f
p y f
2) Contract or expand descendant nodes; and )
p
f
p y f
2) Contract or expand descendant nodes; and JACSA) International Journal of Advanced Computer Science and Applications
Vol. 6, No. 10, 2015 (IJACSA) International Journal of Advanced Computer Science and Applications,
Vol. 6, No. 10, 2015 (IJACSA) International Journal of Advanced Computer Science and Applications,
Vol. 6, No. 10, 2015 (IJACSA) International Journal of Advanced Computer Science and Applications,
Vol. 6, No. 10, 2015 1) Open or close lexical information display function; Fig.2 shows the dependency tree of the following sentence, bi
uran=sibauxay-yin
xatagu=sirgagu
aǰilči=xödelműriči
ǰorig=sanag_a bolon uran=narin egűr sűlǰixű mergeǰil-i
űnen=sedxil-eče-ben bisiren_e. (I admire from the bottom of my
heart the brave and hard-working bird’s strong will and superb
nesting skill). The dotted lines represent syntactic fragments that meet
the requirements of query conditions. 165 | P a g e www.ijacsa.thesai.org (IJACSA) International Journal of Advanced Computer Science and Applications,
Vol. 6, No. 10, 2015 (IJACSA) International Journal of Advanced Computer Science and Applications,
Vol. 6, No. 10, 2015 Fig. 4. Node combinations that meet the query condition
uran=sibauxay-yin
ATT
aǰilči=xödelműriči
ATT
ǰorig=sanag_a
uran=sibauxay-yin
xatagu=sirgagu
ATT
ǰorig=sanag_a
ATT
xatagu=sirgagu
aǰilči=xödelműriči
ATT
ǰorig=sanag_a
ATT
(b)
(c)
(d)
uran=narin
sűlǰixű
ATT
mergeǰil-i
ATT
(a) uran=sibauxay-yin
xatagu=sirgagu
ATT
ǰorig=sanag_a
ATT
(b)
uran=narin
sűlǰixű
ATT
mergeǰil-i
ATT
(a) Fig. 2. Dependency Tree of the example sentence
bi
uran=narin
mergeǰil-i
bolon
uran=sibauxay-yin
<EOS>
xatagu=sirgagu
űnen=sedxil-eče-ben
bisiren_e. sűlǰixű
egűr
HEAD
DOBJ
SUBJ
ADV
ATT
COO
ATT
aǰilči=xödelműriči
ATT
ǰorig=sanag_a
ATT
CJ-AUX
ATT
DOBJ bisiren_e. (b) uran=sibauxay-yin
ATT
aǰilči=xödelműriči
ATT
ǰorig=sanag_a
(c) (c) xatagu=sirgagu
aǰilči=xödelműriči
ATT
ǰorig=sanag_a
ATT
(d) Fig. 2. Dependency Tree of the example sentence Query condition can be a word or phrase node, a
dependency arc or a syntactic fragment of any size. In the
query condition, node can have 16 attribute values including
the word or phrase itself, part of speech, syntactic relation and
affix. In the process of search, a query interface as shown in
the Fig.5 will pop up. A new node will be added by clicking
the white dot. A dependency relation can be established
between two nodes by dragging the mouse. Fig.3 shows the
query conditions of this example. Fig. 4. Node combinations that meet the query condition As the enquiry condition only contains two-layer nodes,
there is no need for recursive query. 2) Label with different colors syntactic fragments that
have been found. Continue to traverse the dependency tree to
find the next eligible syntactic fragment. Fig. 3. Query Condition
ATT
ATT
R
C2
C1 ATT
ATT
R
C2
C1 R 3) The combinations where node “ ǰorig=sanag_a” and
its child nodes meet the requirement are as shown in Fig.4
(b)—(d). 4) Search is done when the remaining nodes in the
dependency tree have been traversed and no eligible
fragments are found. Fig. 3. Query Condition Fig. 3. Query Condition Fig. 3. Query Condition It is worth noting that the program will restore the treebank
and clear the traces left from the previous query operation
before next query. If the search results need to be preserved, a
copy needs to be saved by using the pertinent functions in this
program. 1) Find nodes that match with R in dependency tree using
pre-order traversal. As R itself has no constraints, every node
in the dependency tree meets its requirement. The key is to
check whether the node has two ATT child nodes. In the
process of traversal, the node “mergeǰil-i” meets the
requirement, as shown in Fig.4 (a). Statistical analysis of syntactic fragment is done based on
query. Each search provides relevant statistical data, including
the number of times a syntactic fragment appears and the
number of sentences that contain the syntactic fragment. 166 | P a g e 166 | P a g e www.ijacsa.thesai.org (IJACSA) International Journal of Advanced Computer Science and Application
Vol. 6, No. 10, 201 (IJACSA) International Journal of Advanced Computer Science and Applications,
Vol. 6, No. 10, 2015 Fig. 5. The Editing, showing or query interface of Mongolian dependency Treebank [6] Hua Shabao, ―A tagging strategy of Mongolian phrases‖, Journal of the
Central University for Nationalities (Philosophy and Social Sciences
Edition), 2003, vol.30, N0.5, pp.98–100. III.
CONCLUSION The dependency tree that this Mongolian syntactic
retrieval system is based differs from binary phrase structure
tree in terms of node types and tree structures. Such difference
cannot be effectively handled at the current stage. Moving
forward, we will add to this system display, editing and
retrieval function for phrase structure tree and bidirectional
conversion between the two kind of tree structures. This
system has good universality, and has no particular
relationship with language per se. As such it can be applied to
other languages‘ treebank for editing and retrieval operations. [7] Wulan, Dabhurbayar, GUAN Xiaoda and ZHOU Qiang, ―Phrase
structure parsing of Mongolian‖, Journal of Chinese Information
Processing, 2014, vol.28, No.5, pp.162–169. [8] Wang
Serguleng,
―Rule-based
Mongolian
sentence
automatic
segmentation,‖ Journal of Inner Mongolia University (Philosophy &
Social Sciences (Mongolian Edition)), 2009, vol.38, No.3, pp.51–55. [9] Wang Serguleng, D.Sarana and Nasunurtu, ―Design and realization of
automatic annotation for modern Mongolian predicate segment‖,
Proceedings of 11th national symposium on minority languages,
Xishuangbanna, China, 2007, pp.420–427. [10] S.Loglo and Sarula, ―Construction of a Mongolian dependency
treebank‖, International Journal of Knowledge and Language
Processing, 2014, vol.5, No.2, pp.32–42. REFERENCE [1] Language Research Institute of Inner Mongolia University, ―About
modern Mongolian corpus‖, Journal of Inner Mongolia University
(Humanities & Social Sciences), 1992, vol.24, N0.1, pp. 1–5. [11] S.Loglo, ―Research on the modern Mongolian syntactic tagging system
based on the dependency grammar‖, Mongolian Studies of China, 2011,
vol.39, No.2, pp.116–119. [2] HUA Shabao, ―AYIMAG– A POS tagging system for Mongolian
corpus‖, Journal of Inner Mongolia University (Humanities & Social
Sciences), 1999, vol.31, N0.5, pp.31–35. [12] Liu Haitao, ―Dependency grammer (from Theory to practice)‖, Science
Press, Beijing, China, 2009, pp.1–15. [13] Jiří Mírovský, Netgraph, ―A tool for searching in prague dependency
treebank 2.0‖, Proceedings of the TLT 2006, pp.211–222. [3] Zhang Guanhong, S.Loglo and Odbal, ―Fusion of morphological
features for Mongolian part of speech based on maximum entropy
model‖, Journal of Computer Research and Development, 2011, vol.48,
N0.12, pp.2385–2390. [14] S.Loglo and Sarula, ―A rule-based Mongolian dependency parsing
model‖, International Journal of Knowledge and Language Processing,
2013, vol.4, No.3, pp.27–37. [4] S.Loglo and Sarula, ―Research on Mongolian lexical analyzer based on
NFA‖, Proceedings of 2010 IEEE International Conference on
Intelligent Computing and Intelligent Sytems, Xiamen, China, 2010,
vol.2, pp.240–245. [15] Jiří Mírovský, ―Searching in the prague dependency treebank‖,
Published by Institute of Formal and Applied Linguistics, Czech
Republic, 2009. [16] Laura Kallmeyer, ―On the complexity of queries for structurally
annotated linguistic data‖, Proceedings of ACIDCA, 2000, pp.1–6. [5] Hua Shabao and Dabhurbayar, ―A Phrase-tagging Research in
Mongolian Corpus‖, Journal of the Central University for Nationalities
(Philosophy and Social Sciences Edition), 2006, vol.33, No.5, pp.64–67. 167 | P a g e www.ijacsa.thesai.org
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English
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Absorptive Weak Plume Detection on Gaussian and Non-Gaussian Background Clutter
|
IEEE journal of selected topics in applied earth observations and remote sensing
| 2,021
|
cc-by
| 10,849
|
Absorptive Weak Plume Detection on Gaussian and
Non-Gaussian Background Clutter James Theiler Abstract—For additive signals on Gaussian clutter, the optimal
detectorisalinearmatchedfilterthatisadaptedtotheknownsignal
and the covariance of the background. This adaptive matched filter
is widely used for gas-phase plume detection, even though the effect
of the plume on the background is not strictly additive. Here, a
derivation of the matched filter for a strictly absorptive plume
produces, in the weak plume limit, a quadratic filter. This quadratic
matched filter is extended in two ways: an elliptically-contoured
multivariate t distribution is used to generalize the Gaussian back-
ground clutter, and a generalized likelihood ratio test detector is
derived to extend applicability to stronger plumes. In addition to
detectors whose purpose is to identify presence versus absence of a
plume, expressions are also derived for estimating plume strength. The performance of these various detectors is evaluated by im-
planting simulated plume into background images that are either
real hyperspectral images or simulated images based on different
(Gaussian, multivariate t, and lognormal) clutter distributions. The physics in this regime is simple—exponential Beer’s Law
absorption [11]—but it is not linear. Conventional lore holds that
for a sufficiently weak plume, the exponential is approximately
linear, and so a linear matched filter is appropriate. But even
for this simple model, the target is not truly additive even in the
weak plume limit. This problem also provides an exercise in composite hypoth-
esis testing. For such problems, a direct likelihood ratio test
cannot be employed, because there is a nuisance parameter (in
this case, the plume concentration) whose value is not a priori
known. The usual approach in this situation is to employ the
generalized likelihood ratio test (GLRT), but in the weak plume
limit the locally most powerful (LMP) test seems to make sense. It bears remarking that more general Bayesian methods [12] (of
which LMP is a special case) or Clairvoyant Fusion [13]–[15]
(for which GLRT is a special case) can also be considered. Index Terms—Adaptive signal detection, clutter, composite
hypothesis testing, elliptically-contoured distribution, gas-phase
plume detection, generalized likelihood ratio test, hyperspectral
imagery, lognormal distribution, matched filter. In the IGARSS conference paper [16] that this article follows,
the background was assumed to be Gaussian, and closed-form
solutions were derived for both the LMP and GLRT detec-
tors. IEEE JOURNAL OF SELECTED TOPICS IN APPLIED EARTH OBSERVATIONS AND REMOTE SENSING, VOL. 14, 2021 IEEE JOURNAL OF SELECTED TOPICS IN APPLIED EARTH OBSERVATIONS AND REMOTE SENSING, VOL. 14, 2021 6842 I. INTRODUCTION O O
NEofthetriumphsofmodernhyperspectralimagingisthe
remote detection of low-concentration gas-phase plumes
on cluttered backgrounds [1]–[10]. Such plumes are often very
nearly invisible, and the difference between an ON-plume pixel
spectrum and an OFF-plume spectrum may be very small. But
the gas absorption spectrum is known to high precision, and
because the observations are made at multiple (often hundreds
of) wavelengths, it is possible to detect the presence of these
weak plumes even in cluttered backgrounds. Absorptive Weak Plume Detection on Gaussian and
Non-Gaussian Background Clutter Here, the background is extended to the multivariate t-
distributed background (of which the Gaussian is a special case),
andnewclosed-formexpressionsarederived.Alognormalback-
ground model is also considered, based on the observation that
treating the data in log space reduces the exponential attenuation
to an additive target model. Finally, numerical experiments are
carried out in order to evaluate the relative performance of these
various detectors in different background scenarios. Manuscript received April 28, 2021; revised June 7, 2021; accepted June
27, 2021. Date of publication June 30, 2021; date of current version July 20,
2021. This work was supported by a NASA-sponsored project on “Compact
high-resolution trace-gas hyperspectral imagers, with agile on-board processing,
for trace gas monitoring” under Grant N7-INVEST17-0010, led by S. P. Love
at Los Alamos National Laboratory.
The author is with the Space Data Science, and Systems Group, Los Alamos
National Laboratory, Los Alamos, NM 87545 USA (e-mail: jt@lanl.gov).
Digital Object Identifier 10.1109/JSTARS.2021.3093820 der a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommon Manuscript received April 28, 2021; revised June 7, 2021; accepted June
27, 2021. Date of publication June 30, 2021; date of current version July 20,
2021. This work was supported by a NASA-sponsored project on “Compact
high-resolution trace-gas hyperspectral imagers, with agile on-board processing,
for trace gas monitoring” under Grant N7-INVEST17-0010, led by S. P. Love
at Los Alamos National Laboratory. II. ABSORPTIVE PLUME where τ = Trace(T). Thus, the likelihood ratio for a pixel x is
given by where τ = Trace(T). Thus, the likelihood ratio for a pixel x is
given by For an absorptive plume, we have from Beer’s Law that
the radiance observed at some wavelength λ is given by xλ =
zλ exp(−atλ), where zλ is the radiance that would observed in
theabsenceofplume,tλ istheabsorptioncoefficientoftheplume
gas, and a is the plume strength. For a sensor with d wavelengths,
we can express this in vector form, with d-dimensional vectors
x, z, and t, whose components correspond, respectively, to xλ,
zλ, and tλ: L(a, x) = Pplume(x)
Pbkg(x) = Pbkg(exp(aT)x) exp(aτ)
Pbkg(x)
. (5) (5) If we somehow did know the plume strength a (while at the same
time not knowing whether or not there even was a plume), then
this would be the optimal detector of that plume. It is known
as the clairvoyant detector [20] and is useful both conceptually
(as a starting point for detectors that do not require knowledge
of plume strength) and experimentally (as a best-case baseline
against which to compare other detectors). x = exp(−aT)z
(1) (1) where T = diag(t) is a diagonal matrix whose diagonal ele-
ments Tλλ are the absorption coefficients tλ. where T = diag(t) is a diagonal matrix whose diagonal ele-
ments Tλλ are the absorption coefficients tλ. THEILER: ABSORPTIVE WEAK PLUME DETECTION ON GAUSSIAN AND NON-GAUSSIAN BACKGROUND CLUTTER 6843 To first order in a (i.e., assuming a weak plume), (1) looks
like x ≈z −aTz, which suggests t∗= −Tz ≈−Tx, but this
expression for t∗is not constant, so the additive matched filter
is not applicable. A naive yet popular approximation (e.g.,
see [5]–[7]) takes t∗∝t; this is based on the argument that
the background varies much more slowly with wavelength than
the gas spectrum does, and so the approximation is that the
background is spectrally flat. A better approximation takes
t∗= −Tµ, where µ = ⟨x ⟩is the mean background over the
image. In this absorptive plume context, then, the AMF becomes TABLE I
BASIC NOTATION USED IN THIS EXPOSITION DTμ-AMF(x) = −(Tµ)′R−1(x −µ). (2) (2) TABLE II
DEFINITIONS OF SOME USEFUL INTERMEDIATE QUANTITIES Because (1) is not truly additive, the aim in this section is
to derive a detector that accounts for the multiplicative nature
of plume absorption. We begin by writing Pbkg(z) as the OFF-
plume background distribution and Pplume(x) as the distribution
of pixel values when plume is present. We can express the
relationship between these two using the usual formula for
change-of-variables in probability distributions Pplume(x) = Pbkg(z)
dz
dx
= Pbkg(exp(aT)x) |exp(aT)| (3) (3) on “characteristic” plume strength, and on why the LMP-based
detectors fare so poorly, are included in the Appendix. where | · | indicates the determinant. Note that the determinant
of the exponential can be expressed as the exponential of the
trace (since T is diagonal, this is readily verified) Table I provides a brief summary of the main symbols used
in the text. Because the search for closed-form solutions often
requires unwieldy algebra, various intermediate quantities were
introduced; they are defined, as they arise, in the text, and are
summarized in Table II. |exp(aT)| =
λ
exp(atλ) = exp
a
λ
tλ
= exp(a Trace(T)) = eaτ
(4) (4) A. Organization and Notation Plumes can be both absorptive and emissive, and although
emissive spectra in the thermal infrared can be extremely dis-
criminating, detection at visible wavelengths is much cheaper,
and requires less specialized hardware. The focus here is on
detection of plumes that are purely absorptive. SectionIIderivesthebasicequationsfortheabsorptiveplume,
including the “classic” matched filter and a clairvoyant detector
that is theoretically optimal when the background distribution
is specified. Section III begins with the assumption that the
background is Gaussian, and derives both a quadratic matched
filter based on the LMP and a GLRT detector, the latter of which
requires a maximum likelihood estimate of the plume strength,
which is of interest in its own right (though it is not the focus of
this article). In Section IV, the Gaussian results are generalized
to the case of a multivariate t-distributed background, and in
Section V a lognormal background is considered (and this is
generalized to a log-multivariate t-distributed background). Im-
plantedplumesonrealandsimulatedhyperspectralbackgrounds
are employed in Section VI to evaluate the various detectors that
have been derived in this article. Section VII concludes with a
summary of the main points of this article. Some further remarks A modification of the additive target model is used to de-
scribe the physics of absorptive plume detection in the visible
(and near-ultraviolet) wavelengths of hyperspectral imagery. A. Quadratic Matched Filter (QMF) Note that (13) is a transcendental equation, and an exact closed-
form solution is beyond the algebraic skills of this author. However, since we are interested in small a, we can approximate
the solution using a Taylor series expansion up to quadratic terms
in a. First, let us define Because we care about weak plumes, we will derive an LMP
detector [20] that is optimal for a →0. Note that a direct
substitution of a = 0 into (6) leads to log L(0, x) = 0, which
cannot be used as a detector. But we can always take a monotonic
transform of any detector without affecting its performance, and
as long as a > 0 (which is the case for absorptive plumes), the
transform log L(a, x)/a is monotonic. We, therefore, define A(x) = (x −µ)′R−1(x −µ)
(14) (14) which is the Mahalanobis distance from x to the centroid of the
distribution, and the basis of the RX anomaly detector [21]. Note
that DQAMF(x) = lim
a→0
log L(a, x)
a
= ∂
∂a log L(a, x)
a=0
(7) (7) A (exp(a T)x) = A
x + a Tx + 1
2a2T 2x + O(a3)
= (x −µ)′R−1(x −µ) + 2a(Tx)′R−1(x −µ)
+ a2(Tx)′R−1(Tx) + a2(T 2x)′R−1(x −µ) + O(a3)
= A(x) −2aQ(x) + a2E(x) + O(a3)
(15) with the expression on the right evaluated at a = 0. Define with the expression on the right evaluated at a = 0. Define Q(x) = lim
a→0
∂
∂a
−1
2(exp(aT)x −µ)′R−1(exp(aT)x −µ)
= lim
a→0
−∂
∂a(exp(aT)x −µ)′
R−1(exp(aT)x −µ)
= lim
a→0 [−exp(aT)Tx]′ R−1(exp(aT)x −µ)
= −(Tx)′R−1(x −µ)
(8) Q(x) = lim
a→0
∂
∂a
−1
2(exp(aT)x −µ)′R−1(exp(aT)x −µ)
= lim
a→0
−∂
∂a(exp(aT)x −µ)′
R−1(exp(aT)x −µ)
= lim [
exp(aT)Tx]′ R−1(exp(aT)x
µ) (15) where Q(x) is defined in (8) and where Q(x) is defined in (8) and = lim
a→0 [−exp(aT)Tx]′ R−1(exp(aT)x −µ) E(x) = (Tx)′R−1Tx + (T 2x)′R−1(x −µ)
(16) (16) = −(Tx)′R−1(x −µ) = −(Tx)′R−1(x −µ) (8) is defined here for convenience. By combining (13)–(15), we
have as a quadratic expression of interest, and observe that DQAMF(x) = lim
a→0
log L(a, x)
a
= Q(x) + τ
(9) DGLRT(x) = maxa [−A(exp(aT)x) + 2aτ + A(x)]
= maxa
2a (Q(x) + τ) −a2E(x) + O(a3)
. IEEE JOURNAL OF SELECTED TOPICS IN APPLIED EARTH OBSERVATIONS AND REMOTE SENSING, VOL. 14, 2021 IEEE JOURNAL OF SELECTED TOPICS IN APPLIED EARTH OBSERVATIONS AND REMOTE SENSING, VOL. 14, 2021 6844 = maxa [2 log Pbkg (exp(aT)x) + 2aτ −2 log Pbkg(x)]
(12)
= maxa
−(exp(aT)x −µ)′ R−1 (exp(aT)x −µ)
+2aτ + (x −µ)′R−1(x −µ)
. (13) = maxa [2 log Pbkg (exp(aT)x) + 2aτ −2 log Pbkg(x)]
(12) which provides a clairvoyant detector of absorptive plume on a
Gaussian background. And although our interest in this exposi-
tion is with weak plumes, note that (6) is optimal for all positive
values of a. = maxa
−(exp(aT)x −µ)′ R−1 (exp(aT)x −µ)
+2aτ + (x −µ)′R−1(x −µ)
. (13) +2aτ + (x −µ)′R−1(x −µ)
. (13) (13) A. Quadratic Matched Filter (QMF) (17) (9) is the “quadratic matched filter.” Note that the additive term τ
has no influence on the performance of the QMF as a detector,
and could safely be discarded. One advantage of keeping it in the
definition, however, is that the expected value of DQAMF(z) over
non-plume pixels is zero. As a further aside, comparison with
(2) shows that the quadratic Q(x) looks like the linear matched
filter, but the match is to Tx instead of Tµ. In this formulation, (17) is maximized at a = a, where In this formulation, (17) is maximized at a = a, where a = Q(x) + τ
E(x)
=
−(Tx)′R−1(x −µ) + τ
(Tx)′R−1Tx + (T 2x)′R−1(x −µ) (18) (18) though we should properly restrict a ≥0; i.e., reset negative
quantities to zero: a ←max(0, a). though we should properly restrict a ≥0; i.e., reset negative
quantities to zero: a ←max(0, a). A. Linear Adaptive Matched Filter (AMF) For a Gaussian background, we have Pbkg(x) ∝exp[−1
2(x −
µ)′R−1(x −µ)];incorporatingthisexpressionin(5),andtaking
logarithms, we have The classic AMF [17]–[19] was originally applied to radar
signal detection, but is widely used for plume detection. Its
derivation requires an additive model; i.e., a model of the form
x = z + at∗forsomeconstantsignaturet∗.Forthispurelyaddi-
tive model, assuming a Gaussian distribution for the OFF-plume
clutter z, the optimal (indeed, uniformly most powerful [12])
detector is given by DAMF(x) = t∗′R−1(x −µ), where x is the
measured spectrum at a pixel (which may or may not have plume
present), and the prime symbol (′) indicates transpose. log L(a, x) = log Pbkg(exp(aT)x) + aτ −log Pbkg(x)
= −1
2(exp(aT)x −µ)′R−1(exp(aT)x −µ)
+ aτ + 1
2(x −µ)′R−1(x −µ)
(6) + 1
2(x −µ)′R−1(x −µ) (6) THEILER: ABSORPTIVE WEAK PLUME DETECTION ON GAUSSIAN AND NON-GAUSSIAN BACKGROUND CLUTTER (27) L(a, x)−2/(ν+d) = (ν −2) + A(exp(a T)x)
(ν −2) + A(x)
× exp
−2aτ
ν + d
=
1 + A(exp(a T)x) −A(x)
(ν −2) + A(x)
× exp
−2aτ
ν + d
=
1 + Fν(x)2 [A(exp(a T)x) −A(x)]
ν −1
aAC = 1
r × aNAC = 1
r × −(Tµ)′R−1(x −µ)
(Tµ)′R−1(Tµ)
(21)
L(a, x)−2/(ν+d) = (ν −2) + A(exp(a T
(ν −2) + A(x) where the scalar factor r is given by r = x′µ/µ′µ. We observe that the a defined in (18) has a similar AC charac-
ter, although albedo correction was never explicitly incorporated
into it; rather, the form of the expression in (18) arose naturally
from the maximum likelihood estimate, based on exponential
absorption. (27) THEILER: ABSORPTIVE WEAK PLUME DETECTION ON GAUSSIAN AND NON-GAUSSIAN BACKGROUND CLUTTER 6845 can be thought of as a kind of “fattening” factor. Observe that
in the ν →∞limit, we have that Fν = 1, and (23) reverts to
the standard AMF in (2). At the other extreme, as ν →2, we
obtain the ACE detector (which is usually derived under other
assumptions [27]–[29]) anyway. And weirdly, the cheat is often better in the region of the
ROC curve we do not usually care about, getting better results
in the high false alarm rate regime. (One author has interpreted
this weirdness as a flaw in the GLRT itself [13].) 2) Some Remarks on Albedo-Corrected (AC) Estimates of
Plume Strength: In Foote et al. [22], an AC estimate of a
is suggested. To explain it, we begin with a non-AC (NAC)
estimate, based on the matched filter in (2). From the expres-
sion x = z −aTµ, where z is the plume-free pixel spectrum,
assumed to be distributed as a Gaussian with mean µ and
covariance R, we can obtain a best estimate of a at a pixel x DACE(x) = F2(x) DAMF(x) = DAMF(x)/
A(x). (25) (25) The extension of the quadratic matched filter to this fatter-
tailed background begins with the likelihood ratio The extension of the quadratic matched filter to this fatter-
tailed background begins with the likelihood ratio L(a, x) = Pplume(x)
Pbkg(x)
= Pbkg(exp(a T)x) exp(aτ)
Pbkg(x)
= [ν −2 + A(exp(a T)x)]−d+ν
2
[ν −2 + A(x)]−d+ν
2
× exp(aτ). (26) aNAC = −(Tµ)′R−1(x −µ)
(Tµ)′R−1(Tµ)
. (20) (20) (26) Based on the observation that this estimate is biased high for
high-albedo ground pixels, Foote et al. [22] proposed an AC
estimate Thus, Thus, aAC = 1
r × aNAC = 1
r × −(Tµ)′R−1(x −µ)
(Tµ)′R−1(Tµ)
(21)
L( (21)
arac-
rated
rally
ntial
L(a, x)−2/(ν+d) = (ν −2) + A(exp(a T)x)
(ν −2) + A(x)
× exp
−2aτ
ν + d
=
1 + A(exp(a T)x) −A(x)
(ν −2) + A(x)
× exp
−2aτ
ν + d
=
1 + Fν(x)2 [A(exp(a T)x) −A(x)]
ν −1
× exp
−2aτ
ν + d
. A. Clairvoyant Detector for EC Distribution The detectors above for GLRT in (19) and LMP in (9), as
well as the linear matched filter in (2), were all derived under
the assumption that the background distribution is Gaussian. In
this section, these expressions are generalized for elliptically-
contoured fat-tailed backgrounds given by the multivariate t
distribution, which is often more descriptive of actual hyper-
spectral clutter [23], [24]. The multivariate t is a fatter-tailed
distribution than the Gaussian, but like the Gaussian, depends
on x though the elliptical term A(x) = (x −µ)′R−1(x −µ). It is given by If a is known, then the optimal detector is given by a mono-
tonic rescaling of the likelihood ratio. Here, we can write D(a, x) = (ν −1)
1 −exp
2aτ
ν + d
L(a, x)−2/(ν+d)
(28
(28) (
)
as
the
monotonic
transform,
and
then
use
(27)
for
L(a, x)−2/(ν+d) to obtain the clairvoyant detector D(a, x) = F2
ν(x) [A(x) −A(exp(a T)x)] . (29) (29) Pbkg(x) = c [(ν −2) + A(x)]−d+ν
2 (22) (22) B. Generalized Likelihood Ratio Test q
( , )
Substituting this a into (17) leads to the GLRT detector DGLRT(x) = Q(x) + τ
E(x) The GLRT formulation recognizes the dependence of the
detector on plume strength a. The abovementioned LMP for-
mulation considered the small a limit; by contrast, the GLRT
formulation takes two steps: first an estimated plume strength a
is computed and then the likelihood is evaluated at that estimate. These steps can be combined into a single expression that defines
the GLRT for any background distribution Pbkg =
−(Tx)′R−1(x −µ) + τ
(Tx)′R−1Tx + (Tx)′TR−1(x −µ)
. (19) (19) These steps can be combined into a single expression that defines
the GLRT for any background distribution Pbkg 1) Brief Remark on Quadratic Detector Functions: In (17),
we see a monotonic rescaling of the likelihood ratio expressed
in terms of a Taylor series of the form [2aA −a2B + O(a3)]. This is a pattern will arise again in Section IV-D, and is
fairly common in general. Taking the derivative and setting
to zero produces a = A/B, but restricting to nonnegative a
makes that a = max(0, A)/B. Inserting this expression back
into the quadratic [2aA −a2B] gives a GLRT that looks like
[max(0, A)2/B]. We can further rescale with the monotonic
f(x) = sign(x)
|x|, and that leads to max(0, A)/
√
B. As a
detector, though, we can cheat and simply write A/
√
B since
negative values of the detector will not count as detections, D∗
GLRT(x) = maxaPbkg (exp(aT)x) exp(aτ)
Pbkg(x)
. (10) (10) The maximum likelihood estimator for the plume strength a is
the value of a that achieves the maximum in (10) The maximum likelihood estimator for the plume strength a is
the value of a that achieves the maximum in (10) a = argmaxaPbkg (exp(aT)x) exp(aτ)/Pbkg(x). (11) a = argmaxaPbkg (exp(aT)x) exp(aτ)/Pbkg(x). (11) (11) For the Gaussian distribution, the logarithm provides a conve-
nient monotonic transform For the Gaussian distribution, the logarithm provides a conve-
nient monotonic transform DGLRT(x) = 2 log D∗
GLRT(x) TABLE III
TAXONOMY OF EXPRESSIONS FOR ABSORPTIVE PLUME DETECTORS case, namely that the combination of a square root and the
imposition of a ≥0 yields the form A/
√
B, we have case, namely that the combination of a square root and the
imposition of a ≥0 yields the form A/
√
B, we have TABLE IV
HYPERSPECTRAL DATASETS USED FOR EVALUATION OF ALGORITHMS TABLE IV
HYPERSPECTRAL DATASETS USED FOR EVALUATION OF ALGORITHMS DGLRT-EC(x) =
F2
ν(x)Q(x) + Θν
F2ν(x)E(x) + 4Q(x)Θν+2Θ2ν
ν−1
. (37) (37) Observethatintheν →∞limit,Fν →1andΘν →τ,which
reduces to a = (Q(x) + τ)/E(x), in agreement with (18) for a
Gaussian background. In this limit, (37) similarly reduces to
(Q(x) + τ)/
E(x), which is the form in (19). +
a2
ν −1
F2
ν(x)E(x) + 4Q(x)Θν
ν −1
+ 2Θ2
ν
ν −1
+ O(a3)
(31)
The ν →2 limit is also of interest; this is the ACE-ification
limit, and here it leads to (31) with DGLRT-ACE(x) = lim
ν→2 DGLRT-EC(x) DGLRT-ACE(x) = lim
ν→2 DGLRT-EC(x)
=
Q(x)/A(x) + Θ2
E(x)/A(x) + 4Q(x)Θ2 + 2Θ2
2
(38) Θν = (ν −1)τ
ν + d . (32) (32) =
Q(x)/A(x) + Θ2
E(x)/A(x) + 4Q(x)Θ2 + 2Θ2
2
(38) (38) So now a monotonic rescaling of the likelihood can be expressed So now a monotonic rescaling of the likelihood can be expressed D(a, x) = (ν −1)(1 −L(a, x)−2/(ν+d))
= 2a
F2
ν(x)Q(x) + Θν
−a2
F2
ν(x)E(x) + 4Q(x)Θν + 2Θ2
ν
ν −1
+ O(a3). (33) with Θ2 = τ/(d + 2). with Θ2 = τ/(d + 2). C. LMP Detector for EC Distribution log x = log z −at. (39) (39) At this point, we can define the LMP detector in terms of the
limit In particular, Schaum [34] suggested the detector In particular, Schaum [34] suggested the detector Dlog-AMF(x) = t′ R−1(log x −µ)
(40) DQEC(x) = lim
a→0
D(a, x)
2a
−Θν = F2
ν(x) Q(x). (34) (40) (34) where µ and R are the mean and covariance, respectively, of the
background in log space. If the background is lognormal (that is:
ifthebackgroundisGaussianinlogspace),thenthislogmatched
filter is the uniformly most powerful (UMP) detector [34]. The ACE-ification of QMF is obtained by taking the ν →2 limit
of QEC The ACE-ification of QMF is obtained by taking the ν →2 limit
of QEC DQACE(x) = Q(x)/A(x)
(35) (35) Two natural extensions to this expression consider the case
that the log space is not Gaussian but multivariate t-distributed. These expressions do not share the UMP property of the log-
AMF detector, even in when the background is accurately mod-
eled by a multivariate t, but they are better fits to these more
general background distributions. Here, following (23), we can
write Two natural extensions to this expression consider the case
that the log space is not Gaussian but multivariate t-distributed. V. LOGNORMAL BACKGROUND A number of authors have recognized that exponential atten-
uation becomes linear when you are working in log space [30]–
[34]. That is to say, (1) becomes (33) B. Weak Plume Regime (Small a) To derive the LMP and GLRT solutions for weak plumes,
we need to express the likelihood ratio in terms of small a, and
this will be done with Taylor series formula that expresses these
quantities as low-order polynomials in a. Recall from (15) that
we can write A(exp(a T)x) −A(x) as a polynomial in a with
coefficients that depend on Q(x) and E(x). We can also expand
the exponential where d is the dimension of x and c is a constant prefactor. The scalar parameter ν characterizes how fat the tails are. The
limit ν →∞corresponds to the Gaussian, and smaller values
of ν describe distributions with fatter tails. As long as ν > 2, the
second moment exists (and so the covariance is well defined). We can estimate ν from the data using either an exceedance
plot [24] or a moment method [25]. The extension of AMF to elliptically-contoured multivariate
t-distributed backgrounds was developed in [26] exp
−2aτ
ν + d
= 1 −2aτ
ν + d +
2a2τ 2
(ν + d)2 + O(a3)
(30) (30) DEC(ν, x) = Fν(x) DAMF(x)
(23) DEC(ν, x) = Fν(x) DAMF(x) (23) so the product in (27) becomes where where L(a, x)−2/(ν+d) = Fν(x) =
(ν −1)
(ν −2) + A(x)
(24)
( , )
1 −
2a
ν −1
F2
ν(x)Q(x) + Θν
Fν(x) =
(ν −1)
(ν −2) + A(x)
(24)
( , )
1 − 1 −
2a
ν −1
F2
ν(x)Q(x) + Θν
(24) (24) IEEE JOURNAL OF SELECTED TOPICS IN APPLIED EARTH OBSERVATIONS AND REMOTE SENSING, VOL. 14, 2021 6846 TABLE III
TAXONOMY OF EXPRESSIONS FOR ABSORPTIVE PLUME DETECTORS TABLE III
TAXONOMY OF EXPRESSIONS FOR ABSORPTIVE PLUME DETECTORS THEILER: ABSORPTIVE WEAK PLUME DETECTION ON GAUSSIAN AND NON-GAUSSIAN BACKGROUND CLUTTER The mean µ and covariance R are estimated
from the OFF-plume data (in other words, we are neglecting
contamination effects, arguing that in an operational scenario,
the ON-plume pixels will be rare). This is a kind of matched-
pair approach [35], [36] that Rotman calls the two histogram
method [37]. The obtained detector is applied to both ON-plume
and OFF-plume pixels and from these a ROC curve is computed. Three statistics of interest to us are as follows: Here,
Fν and
A are essentially the same as Fν and A [defined,
respectively, in (24) and (14)], but using µ and R in place of µ
and R. A taxonomy of all of these detectors is shown in Table III. (Note that many of these detectors are expressed in terms of
intermediate quantities, which are summarized in Table II.) Three statistics of interest to us are as follows: 1) false alarm rate at threshold with detection rate of 80%
(FAR@DR80); THEILER: ABSORPTIVE WEAK PLUME DETECTION ON GAUSSIAN AND NON-GAUSSIAN BACKGROUND CLUTTER THEILER: ABSORPTIVE WEAK PLUME DETECTION ON GAUSSIAN AND NON-GAUSSIAN BACKGROUND CLUTTER 6847 TABLE V
PERFORMANCE OF VARIOUS DETECTORS OF PLUMES THAT ARE IMPLANTED ON REAL HYPERSPECTRAL DATA. BOLDFACE VALUES INDICATE THE BEST (O
NEARLY BEST) NONCLAIRVOYANT PERFORMANCE FOR EACH STATISTIC
re,
Fν and
A are essentially the same as Fν and A [defined,
pectively, in (24) and (14)], but using µ and R in place of µ
R. A taxonomy of all of these detectors is shown in Table III. ote that many of these detectors are expressed in terms of
ermediate quantities, which are summarized in Table II.)
VI. NUMERICAL EVALUATION
One way to measure the quality of a detection algorithm is
x = exp(−aT)z. The mean µ and covariance R are estim
from the OFF-plume data (in other words, we are negle
contamination effects, arguing that in an operational scen
the ON-plume pixels will be rare). This is a kind of matc
pair approach [35], [36] that Rotman calls the two histog
method [37]. The obtained detector is applied to both ON-p
and OFF-plume pixels and from these a ROC curve is comp
Three statistics of interest to us are as follows:
1) false alarm rate at threshold with detection rate of
(FAR@DR80);
2) detection rate at threshold with false alarm rate of
(DR@FAR05); TABLE V
PERFORMANCE OF VARIOUS DETECTORS OF PLUMES THAT ARE IMPLANTED ON REAL HYPERSPECTRAL DATA. BOLDFACE VALUES INDICATE THE BEST (OR
NEARLY BEST) NONCLAIRVOYANT PERFORMANCE FOR EACH STATISTIC TABLE V
ETECTORS OF PLUMES THAT ARE IMPLANTED ON REAL HYPERSPECTRAL DATA. BOLDFACE VALUES INDICATE THE BEST (OR
NEARLY BEST) NONCLAIRVOYANT PERFORMANCE FOR EACH STATISTIC TABLE V
ERFORMANCE OF VARIOUS DETECTORS OF PLUMES THAT ARE IMPLANTED ON REAL HYPERSPECTRAL DATA. BOLDFACE VAL
NEARLY BEST) NONCLAIRVOYANT PERFORMANCE FOR EACH STATISTIC x = exp(−aT)z. The mean µ and covariance R are estimated
from the OFF-plume data (in other words, we are neglecting
contamination effects, arguing that in an operational scenario,
the ON-plume pixels will be rare). This is a kind of matched-
pair approach [35], [36] that Rotman calls the two histogram
method [37]. The obtained detector is applied to both ON-plume
and OFF-plume pixels and from these a ROC curve is computed. Three statistics of interest to us are as follows: x = exp(−aT)z. D. GLRT Detector for EC Distribution These expressions do not share the UMP property of the log-
AMF detector, even in when the background is accurately mod-
eled by a multivariate t, but they are better fits to these more
general background distributions. Here, following (23), we can
write While the LMP detector required only terms linear in a, the
GLRT detector requires the quadratic terms as well. From the
formula for D(a, x) in (33), which can be expressed as 2aA −
a2B + O(a3), we can see that this expression is maximized at a = A
B =
F2
ν(x)Q(x) + Θν
F2ν(x)E(x) + 4Q(x)Θν+2Θ2ν
ν−1
. (36) Dlog-EC(x) =
Fν(log x) Dlog-AMF(x)
(41) (41) (36) and the ν →2 limit provides and the ν →2 limit provides Substituting a into D(a, x) gives an expression of the form
A2/B. Following the same argument we used for the Gaussian Dlog-ACE(x) = Dlog-AMF(x)/
A(log x). (42) (42) Dlog-ACE(x) = Dlog-AMF(x)/
A(log x). IEEE JOURNAL OF SELECTED TOPICS IN APPLIED EARTH OBSERVATIONS AND REMOTE SENSING, VOL. 14, 2021 6848 TABLE VI
IMPLANTED PLUMES IN GAUSSIAN BACKGROUNDS. BOLDFACE VALUES INDICATE THE BEST (OR NEARLY BEST) NONCLAIRVOYANT PERFORMANCE FOR EACH
STATISTIC. HERE, WE EXPECT (AND OBSERVE) THAT THE AMF AND EC VARIANTS OF THE VARIOUS DETECTORS PERFORM THE BEST, THOUGH OFTEN ONLY
MARGINALLY SO. FOR THESE DATASETS, THE GLRT-BASED DETECTORS ARE SUBSTANTIALLY BETTER THAN THE Tµ-BASED MATCHED FILTERS, WHILE THE
LOG-BASED DETECTORS ARE MUCH WORSE TABLE VI
IMPLANTED PLUMES IN GAUSSIAN BACKGROUNDS. BOLDFACE VALUES INDICATE THE BEST (OR NEARLY BEST) NONCLAIRVOYANT PERFORMANCE FOR EACH
STATISTIC. HERE, WE EXPECT (AND OBSERVE) THAT THE AMF AND EC VARIANTS OF THE VARIOUS DETECTORS PERFORM THE BEST, THOUGH OFTEN ONLY
MARGINALLY SO. FOR THESE DATASETS, THE GLRT-BASED DETECTORS ARE SUBSTANTIALLY BETTER THAN THE Tµ-BASED MATCHED FILTERS, WHILE THE
LOG-BASED DETECTORS ARE MUCH WORSE LOG BASED DETECTORS ARE MUCH WORSE
ovides a kind of compromise between the first two, except
t it places most of its weight on performance at high false
rm rates, so it is not usually a good choice in target detection
plications. All of these statistics are scalar values between 0
d 1; in the Tables, we have listed FAR, 1-AUC, and 1-DR so
t smaller values are always better. As described in Table IV, four hyperspectral datasets, and
ee different gas species (NO2, SO2, and CH4), were used to
aluate the algorithms. These datasets are as follows. a) An SO2 plume is implanted into an image obtained from
the Ozone Monitoring Instrument (OMI) [32], and then
b) An NO2 plume implanted into a different OMI scene [4
and again resampled to produce a 320 × 320 × 1444 pi
image. c) A plume of CH4 is implanted into a a 598 × 1200 c
of an AVIRIS-NG image of a landfill in India (sc
ang20160211t075004) [41]; although there are 4
total channels in the scene, we only use the 212 long
wavelength channels, that match the methane absorpt
spectrum. d) A CH4 plume is implanted into a 213 ×598 × 15
chip of an AVIRIS-NG image acquired over an oil a provides a kind of compromise between the first two, except
that it places most of its weight on performance at high false
alarm rates, so it is not usually a good choice in target detection
applications. VI. NUMERICAL EVALUATION 2) detection rate at threshold with false alarm rate of 0.05
(DR@FAR05); One way to measure the quality of a detection algorithm is
to implant a plume into a hyperspectral image, and see if the
algorithm can find it. We will do that here, but in a formalized
way that makes two copies of the hyperspectral data. The first
copyisuntouched,whilethesecondcopyhasplumeimplantedin
every pixel. Thus, if z is the pixel in the first (presumably plume-
free) copy, then the corresponding pixel in the ON-plume copy is 3) area under the ROC curve (AUC). 3) area under the ROC curve (AUC). The first statistic is more appropriate for most detection
scenarios (where low false alarm rates are crucial), but the
second statistic provides an alternative that might be relevant in
scenarios for which detections are crucial and some false alarms
can be tolerated. The AUC is widely employed, and arguably THEILER: ABSORPTIVE WEAK PLUME DETECTION ON GAUSSIAN AND NON-GAUSSIAN BACKGROUND CLUTTER THEILER: ABSORPTIVE WEAK PLUME DETECTION ON GAUSSIAN AND NON-GAUSSIAN BACKGROUND CLUTTER 6849 TABLE VII
IMPLANTED PLUMES IN MULTIVARIATE t-DISTRIBUTED BACKGROUNDS, WITH ν = 3.0. BOLDFACE VALUES INDICATE THE BEST (OR NEARLY BEST)
NONCLAIRVOYANT PERFORMANCE FOR EACH STATISTIC. HERE, WE EXPECT (AND OBSERVE) THAT THE EC AND ACE VARIANTS OF THE DETECTORS TO BE
BETTER, AND THEY ARE OFTEN SUBSTANTIALLY BETTER THAN THEIR AMF COUNTERPARTS. AS WITH THE GAUSSIAN BACKGROUND, THE LOG-BASED
DETECTORS PERFORM POORLY TABLE VII
IMPLANTED PLUMES IN MULTIVARIATE t-DISTRIBUTED BACKGROUNDS, WITH ν = 3.0. BOLDFACE VALUES INDICATE THE BEST (OR NEARLY BEST)
NONCLAIRVOYANT PERFORMANCE FOR EACH STATISTIC. HERE, WE EXPECT (AND OBSERVE) THAT THE EC AND ACE VARIANTS OF THE DETECTORS TO BE
BETTER, AND THEY ARE OFTEN SUBSTANTIALLY BETTER THAN THEIR AMF COUNTERPARTS. AS WITH THE GAUSSIAN BACKGROUND, THE LOG-BASED
DETECTORS PERFORM POORLY DETECTORS PERFORM POORLY
ect of the plumes on the spectra x, however, was quite small,
ically less than about half a percent. Table V shows the performance of the various algorithms in
ecting plume that has been implanted on the hyperspectral
a. In all four cases, we observe that Tµ is always better than
s a target vector. The QMF family of detectors, based on the
MP hypothesis, is in all cases worse than the matched filters
ed on the Tµ targets. Although LMP is optimal for weak
mes, that optimality does not hold up when the plumes are
ong enough to be detectable at reasonable false alarm rates
e Appendix A-B). The GLRT detectors are almost always
ter than the matched filter detectors (and the Clairvoyant
ectors are usually better still, but that detector requires a pri-
knowledge of plume strength, and in practice that is not
ilable). For these real datasets, the backgrounds were fatter
a better model, as is evidenced by the (often considera
better performance obtained by the ACE and EC variant
these detectors. That the ACE and EC detectors were ne
identical is a consequence of the ν ≪d that was observed
these datasets. The log-based detectors, especially log-ACE
log-EC, perform fairly well on this data, even outperformin
the OMI NO2 case) the clairvoyant detectors; that can hap
because the clairvoyant detectors are optimized for spe
background distributions (Gaussian for AMF, multivariate
EC) that may not correspond to the actual distribution of the
hyperspectral data. The log-transformed data provided interesting results. IEEE JOURNAL OF SELECTED TOPICS IN APPLIED EARTH OBSERVATIONS AND REMOTE SENSING, VOL. 14, 2021 All of these statistics are scalar values between 0
and 1; in the Tables, we have listed FAR, 1-AUC, and 1-DR so
that smaller values are always better. b) An NO2 plume implanted into a different OMI scene [40],
and again resampled to produce a 320 × 320 × 1444 pixel
image. c) A plume of CH4 is implanted into a a 598 × 1200 chip
of an AVIRIS-NG image of a landfill in India (scene
ang20160211t075004) [41]; although there are 425
total channels in the scene, we only use the 212 longer-
wavelength channels, that match the methane absorption
spectrum. As described in Table IV, four hyperspectral datasets, and
three different gas species (NO2, SO2, and CH4), were used to
evaluate the algorithms. These datasets are as follows. a) An SO2 plume is implanted into an image obtained from
the Ozone Monitoring Instrument (OMI) [32], and then
resampled to match the wavelengths available on the
Nanosat Atmospheric Chemistry Hyperspectral Observa-
tion System [38], [39] to produce a 320 band image with
320 × 1444 pixels. d) A CH4 plume is implanted into a 213 ×598 × 1500
chip of an AVIRIS-NG image acquired over an oil and
natural gas field near Carlsbad, New Mexico, USA (scene
ang20191023t151141) [41]. a) An SO2 plume is implanted into an image obtained from
the Ozone Monitoring Instrument (OMI) [32], and then
resampled to match the wavelengths available on the Nanosat Atmospheric Chemistry Hyperspectral Observa-
tion System [38], [39] to produce a 320 band image with
320 × 1444 pixels. In all four cases, the plume strength was chosen to be several
times the characteristic strength ao, defined in (46). The direct THEILER: ABSORPTIVE WEAK PLUME DETECTION ON GAUSSIAN AND NON-GAUSSIAN BACKGROUND CLUTTER log-AMF detector, which would be UMP if the background w
lognormal [34], was never as good as the GLRT-ACE or GL
EC detectors. But the log-ACE and log-EC detectors prov a better model, as is evidenced by the (often considerably)
better performance obtained by the ACE and EC variants of
these detectors. That the ACE and EC detectors were nearly
identical is a consequence of the ν ≪d that was observed for
these datasets. The log-based detectors, especially log-ACE and
log-EC, perform fairly well on this data, even outperforming (in
the OMI NO2 case) the clairvoyant detectors; that can happen
because the clairvoyant detectors are optimized for specific
background distributions (Gaussian for AMF, multivariate t for
EC) that may not correspond to the actual distribution of the real
hyperspectral data. effect of the plumes on the spectra x, however, was quite small,
typically less than about half a percent. Table V shows the performance of the various algorithms in
detecting plume that has been implanted on the hyperspectral
data. In all four cases, we observe that Tµ is always better than
t as a target vector. The QMF family of detectors, based on the
LMP hypothesis, is in all cases worse than the matched filters
based on the Tµ targets. Although LMP is optimal for weak
plumes, that optimality does not hold up when the plumes are
strong enough to be detectable at reasonable false alarm rates
(see Appendix A-B). The GLRT detectors are almost always
better than the matched filter detectors (and the Clairvoyant
detectors are usually better still, but that detector requires a pri-
ori knowledge of plume strength, and in practice that is not
available). For these real datasets, the backgrounds were fatter
tailed than Gaussian, and the multivariate t distribution provided The log-transformed data provided interesting results. The
log-AMF detector, which would be UMP if the background were
lognormal [34], was never as good as the GLRT-ACE or GLRT-
EC detectors. But the log-ACE and log-EC detectors provided
excellent performance, outperforming even the GLRT-ACE and IEEE JOURNAL OF SELECTED TOPICS IN APPLIED EARTH OBSERVATIONS AND REMOTE SENSING, VOL. 14, 2021 EEE JOURNAL OF SELECTED TOPICS IN APPLIED EARTH OBSERVATIONS AND REMOTE SENSING, VOL. 14, 2021 6850 TABLE VIII
LANTED PLUMES IN LOGNORMAL BACKGROUND. BOLDFACE VALUES INDICATE THE BEST (OR NEARLY BEST) NONCLAIRVOYANT PERFORMANCE FOR EA
TISTIC. AS PREDICTED, THE LOG-AMF IS OPTIMAL AGAINST THIS BACKGROUND. THE OTHER AMF-BASED DETECTORS, HOWEVER, ARE NOT AS GOOD
THE EC AND ACE VARIANTS TABLE VIII
IMPLANTED PLUMES IN LOGNORMAL BACKGROUND. BOLDFACE VALUES INDICATE THE BEST (OR NEARLY BEST) NONCLAIRVOYANT PERFORMANCE FOR EACH
STATISTIC. AS PREDICTED, THE LOG-AMF IS OPTIMAL AGAINST THIS BACKGROUND. THE OTHER AMF-BASED DETECTORS, HOWEVER, ARE NOT AS GOOD AS
THE EC AND ACE VARIANTS TABLE VIII
IMPLANTED PLUMES IN LOGNORMAL BACKGROUND. BOLDFACE VALUES INDICATE THE BEST (OR NEARLY BE
STATISTIC. AS PREDICTED, THE LOG-AMF IS OPTIMAL AGAINST THIS BACKGROUND. THE OTHER AMF-BASE
THE EC AND ACE VARIANTS GLRT-EC detectors in three of the four cases (and coming very
close in the fourth case). GLRT-based detectors are substantially better than the linear
Tµ-based matched filters. On the other hand, the log-based
detectors are much worse (the FAR@DR80 statistic is hundreds
of times larger for the log-based detectors, compared to the
GLRT detectors). In Tables VI–VIII, the background data are simulated from
known distributions, with mean and covariance adjusted to
match the mean and covariance of the original datasets. At a
minimum, this provides a sanity check that the algorithms that
areoptimizedforthosedistributionsindeedperformbestinthose
cases. Beyond that, however, these experiments also provide
a measure of stability of the various algorithms to different
background distributions. Table VII uses a multivariate t-distributed background with
verysmallparameter(ν = 3.0)correspondingtoaveryfat-tailed
elliptically-contoured distribution. Here, the ACE and EC de-
tectors are best, and the AMF-based detectors are often much
worse. The log-based detectors generally fare poorly here, as
well, though they are not as bad as they were against Gaussian
backgrounds (except for the OMI background with SO2 plume). Interestingly, and somewhat counter-intuitively, the lowest false
alarm rates for these fat-tailed backgrounds are lower than the Table VI uses a Gaussian background distribution, and in
this case we see—as expected—that the AMF variants of the
different detectors achieve the best statistics. But we also see
that the ACE variants are nearly as good. For these datasets, the THEILER: ABSORPTIVE WEAK PLUME DETECTION ON GAUSSIAN AND NON-GAUSSIAN BACKGROUND CLUTTER 6851 Fig. 1. IEEE JOURNAL OF SELECTED TOPICS IN APPLIED EARTH OBSERVATIONS AND REMOTE SENSING, VOL. 14, 2021 Comparison of performance (based on the false alarm rate when detection rate is 80%) for different detectors against implanted CH4 plumes on different
backgrounds. (a) Hyperspectral background is based on the 2019 AVIRIS-NG image over Carlsbad, New Mexico [data set (d) in the text, and in Tables V–VIII]. (b)
Simulated Gaussian background with same mean and covariance as the AVIRIS-NG data. (c) Simulated multivariate t-distributed background; note, the missing
bars for Clairvoyant-EC and Clairvoyant-ACE correspond to zero false alarms. (d) Simulated lognormal background. For a Gaussian background, we expect the
AMF to perform best, and although that is what we observe, we also observe that the EC and ACE variants exhibit nearly identical performance. Against the
multivariate t background, the EC variants are (as expected) best, and against the lognormal background, the log-AMF detector is (again, as predicted by theory)
best. Against the real hyperspectral background, we see the EC and ACE variants consistently and substantially outperforming their AMF counterparts. We also
see the simple Tµ-based matched filters nearly identical to the GLRT, which in turn is nearly as good as the clairvoyant detectors. And the log-EC and log-ACE
detectors are also seen to do fairly well. Those log-based detectors, however, perform very poorly against the simulated Gaussian and multivariate t backgrounds. In every case, we see that the t-based matched filters are outperformed by the Tµ-based detectors, and the LMP-based Quadratic detectors do not fare well at all. Fig. 1. Comparison of performance (based on the false alarm rate when detection rate is 80%) for different detectors against implanted CH4 plumes on different
backgrounds. (a) Hyperspectral background is based on the 2019 AVIRIS-NG image over Carlsbad, New Mexico [data set (d) in the text, and in Tables V–VIII]. (b)
Simulated Gaussian background with same mean and covariance as the AVIRIS-NG data. (c) Simulated multivariate t-distributed background; note, the missing
bars for Clairvoyant-EC and Clairvoyant-ACE correspond to zero false alarms. (d) Simulated lognormal background. For a Gaussian background, we expect the
AMF to perform best, and although that is what we observe, we also observe that the EC and ACE variants exhibit nearly identical performance. Against the
multivariate t background, the EC variants are (as expected) best, and against the lognormal background, the log-AMF detector is (again, as predicted by theory)
best. IEEE JOURNAL OF SELECTED TOPICS IN APPLIED EARTH OBSERVATIONS AND REMOTE SENSING, VOL. 14, 2021 Against the real hyperspectral background, we see the EC and ACE variants consistently and substantially outperforming their AMF counterparts. We also
see the simple Tµ-based matched filters nearly identical to the GLRT, which in turn is nearly as good as the clairvoyant detectors. And the log-EC and log-ACE
detectors are also seen to do fairly well. Those log-based detectors, however, perform very poorly against the simulated Gaussian and multivariate t backgrounds. In every case, we see that the t-based matched filters are outperformed by the Tµ-based detectors, and the LMP-based Quadratic detectors do not fare well at all. lowest false alarm rates on Gaussian backgrounds (as reported
in Table VI), even though the plume strength is the same in both
cases. background distribution. This explains, for instance, how
the clairvoyant-AMF (which assumes a Gaussian back-
ground) can fare so poorly in a lognormal background. Finally, Table VIII uses a lognormal background. The log of
this data will have the same mean and covariance as the original
data in log space. As predicted by theory, the log-based detectors
are best, and in particular the log-AMF lives up to its theoretical
optimality in this situation. In three of the four cases (b)–(d),
we see that the GLRT-EC detector, while not as good as the
log-AMF, was still competitive, with only a few times as many
false alarms at a fixed detection rate. 4) Although the AMF detectors are provably optimal when
the background is Gaussian, they are only slightly better
than the EC and ACE detectors in that scenario. And for
non-Gaussian backgrounds, the AMF detectors are often
substantially worse. 5) Although EC detectors are theoretically better than both
AMF and ACE detectors, adapting themselves to the de-
gree of fat-tailed-ness in the background distribution, they
were found in these experiments to be only marginally
better than the ACE detectors. 5) Although EC detectors are theoretically better than both
AMF and ACE detectors, adapting themselves to the de-
gree of fat-tailed-ness in the background distribution, they
were found in these experiments to be only marginally
better than the ACE detectors. Fig. 1 illustrates the main results in these four tables, restrict-
ing consideration to a single statistic (FAR@DR80) and to case
(d) abovementioned: a CH4 plume implanted in four different
backgrounds with the same mean and covariance as the 2019
AVIRIS-NG dataset. A. Remark on the Strength of Weak Plumes It bears remarking that although a plume whose strength is
on the order of the characteristic strength ao is just detectable,
it is still a weak plume. Consider a typical pixel, for which
x = µ, and notice that the magnitude of the difference between
OFF-plume and ON-plume is given by ∥exp(−aoT)µ −µ∥. A
natural comparison is the magnitude of the pixel itself, which is
∥µ∥, and the ratio is given by As computers become faster and cheaper, the need for closed-
form solutions has arguably diminished. Particularly with mod-
ern machine learning tools, it is possible to produce detectors
that are adapted for virtually any physical situation that can be
modeled [35], [36], [42]; there is, therefore, less need for linear
approximations or simplifying assumptions. But even in this en-
vironment, simple expressions do still have advantages. They are
useful when computation is limited (e.g., on small space-borne
platforms [38], [39]), or when intuitive interpretation is desired. ∥Δx∥
∥x∥
∼∥exp(−aoT)µ −µ∥
∥µ∥
≈∥aoTµ∥
∥µ∥
∼∥Tµ∥
∥µ∥×
1
∥R−1/2Tµ∥. (47) ∼∥Tµ∥
∥µ∥×
1
∥R−1/2Tµ∥. (47) (47) A slightly (but only slightly) more accurate derivation yields VII. CONCLUSION To obtain an “n sigma” detection requires To obtain an “n sigma” detection requires |Mean| = n
√
Variance
(45) (45) It is often, if informally, asserted that a linear filter is appro-
priate for gas-phase plume detection because the exponential
in Beer’s law becomes linear in the weak plume limit. But a
more careful derivation shows that the linear matched filter is
not strictly appropriate even in the limit as plume strength goes
to zero. which is to say that a(Tµ)′R−1(Tµ) = n
(Tµ)′R−1(Tµ). That is: a = n/
(Tµ)′R−1(Tµ). We will, therefore, take our
characteristic plume strength to be which is to say that a(Tµ)′R−1(Tµ) = n
(Tµ)′R−1(Tµ). That is: a = n/
(Tµ)′R−1(Tµ). We will, therefore, take our
characteristic plume strength to be which is to say that a(Tµ)′R−1(Tµ) = n
(Tµ)′R−1(Tµ). That is: a = n/
(Tµ)′R−1(Tµ). We will, therefore, take our
characteristic plume strength to be ao = 1/
(Tµ)′R−1(Tµ) = 1/∥R−1/2Tµ∥
(46) (46) Furthermore, this weak plume limit is itself not quite ap-
propriate if it implies that the plume is so weak that it is
undetectable. The LMP solution that corresponds to the weak
plume limit (a →0) and that leads to the quadratic matched filter
is empirically found to be less effective than the GLRT solution
(indeed, it is often less effective than the linear matched filter)
when the plume is strong enough to be detected with reasonably
small false alarm rate. and recognize that we will need plumes that are several times
stronger than this in order to get detections at low false alarm
rate. Perhaps the main use of ao is to inform our choice of a in
simulations assessing algorithms for detecting weak plumes. We may simulate multiple scenarios (e.g., different gases on
different backgrounds), but our baseline comparison is in terms
of the characteristic ao for the different scenarios. In deriving, the GLRT solutions, estimates of plume strength
a are made along the way, and it is observed in Section III-B2
that these estimates provide a built-in albedo correction, similar
in flavor to the albedo correction in [22]. A useful future study
would be to evaluate the relative performance of these different
estimators. IEEE JOURNAL OF SELECTED TOPICS IN APPLIED EARTH OBSERVATIONS AND REMOTE SENSING, VOL. 14, 2021 IEEE JOURNAL OF SELECTED TOPICS IN APPLIED EARTH OBSERVATIONS AND REMOTE SENSING, VOL. 14, 2021 6852 = (Tµ)′R−1(Tµ). (43) 8) The t-based matched filters were in virtually every case
outperformed by the Tmu-based equivalents. 8) The t-based matched filters were in virtually every case
outperformed by the Tmu-based equivalents. (43) In the presence of plume of magnitude a, we have a mean value
for the detector of 9) The LMP-based “quadratic matched filters” did not per-
form well at all, and are not recommended for practi-
cal use. Appendix A-B discusses this from a theoretical
perspective. Mean
(Tµ)′R−1(x −µ)
=
(Tµ)′R−1(z −aTµ −µ)
= −a(Tµ)′R−1(Tµ). (44) (44) (44) IEEE JOURNAL OF SELECTED TOPICS IN APPLIED EARTH OBSERVATIONS AND REMOTE SENSING, VOL. 14, 2021 6) The log-AMF detector, although it has nice theoretical
properties when the background distribution is lognor-
mal [34], does not perform well under other background
distributions. On the other hand, the log-EC and log-ACE
detectors, although they also fare poorly with Gaussian
and multivariate t-distributed backgrounds, did surpris-
ingly well in the four real datasets. 6) The log-AMF detector, although it has nice theoretical
properties when the background distribution is lognor-
mal [34], does not perform well under other background
distributions. On the other hand, the log-EC and log-ACE
detectors, although they also fare poorly with Gaussian
and multivariate t-distributed backgrounds, did surpris-
ingly well in the four real datasets. Numerical results are necessarily anecdotal and, therefore,
always somewhat tentative, but here are some general patterns
that can be discerned from these simulations: 1) There can be orders-of-magnitude differences in the per-
formance of different plume detectors. 7) Although the GLRT detectors are designed to be better
than the simple matched-filters, we find that the Tµ-ACE
detector is very often competitive with the best GLRT
detector for a given scenario. This bears some emphasis
because that detector is algebraically very simple, but
still different from the more widely employed Tµ-AMF
detector. 2) One of the greatest sources of this variability in perfor-
mance is differences in the background model, and how
well the model matches the actual background. 3) When including the clairvoyant detectors in these
comparisons, it is important to remember that what they
are clairvoyant about is the plume strength, not the B. Remark on the Inadequacy of the QMF (And of the LMP
Generally) pp
[11] A. Beer, “Bestimmung der absorption des rothen lichts in farbigen flus-
sigketiten,” Ann. Physik, vol. 86, pp. 78–88, 1852. [12] E. L. Lehmann and J. P. Romano, Testing Statistical Hypotheses. New
York, NY, USA: Springer, 2005. This notion of a characteristic plume strength also allows us
to explain why the QMF, which is “optimized” for weak plumes,
does such a poor job of detecting weak plumes. In the derivation
of QMF, we neglect the O(a2) terms on grounds that a is small
for weak plumes. But small compared to what? In particular,
one of the quadratic terms that we neglected, as seen in (17),
was a2(Tx)′R−1(Tx). But in terms of the characteristic plume
strength ao, we see that [13] A. Schaum, “Continuum fusion: A theory of inference, with applications
to hyperspectral detection,” Opt. Exp., vol. 18, pp. 8171–8181, 2010. [14] J. Theiler, “Confusion and clairvoyance: Some remarks on the compos-
ite hypothesis testing problem,” in Proc. SPIE, vol. 8390, 2012, Art. no. 839003. [15] A. Schaum, “Clairvoyant fusion: A new methodology for designing robust
detection algorithms,” in Proc. SPIE, vol. 10004, 2016, Art. no. 100040C. [16] J. Theiler and A. Schaum, “Some closed-form expressions for absorptive
plume detection,” in Proc. IEEE Int. Geosci. Remote Sens. Symp., 2020,
pp. 1786–1789. pp
[17] E. J. Kelly, “Performance of an adaptive detection algorithm: Rejection
of unwanted signals,” IEEE Trans. Aerosp. Electron. Syst., vol. 25, no. 2,
pp. 122–133, Mar. 1989.
a2(Tx)′R−1(Tx)
> a2(Tµ)′R−1(Tµ) = a2/a2
o. (50) [18] I. S. Reed, J. D. Mallett, and L. E. Brennan, “Rapid convergence rate in
adaptive arrays,” IEEE Trans. Aerosp. Electron. Syst., vol. AES-10, no. 6,
pp. 853–863, Nov. 1974. If our “weak” plume is at least strong enough to be detected,
then this term is greater than 1, and can hardly be assumed to
approach zero. Put another way, the QMF detector is optimized
for plumes that are too weak (i.e., for a ≪ao) to be effectively
detected. If our “weak” plume is at least strong enough to be detected,
then this term is greater than 1, and can hardly be assumed to
approach zero. Put another way, the QMF detector is optimized
for plumes that are too weak (i.e., for a ≪ao) to be effectively
detected. [19] F. C. Robey, D. R. Fuhrmann, E. J. Kelly, and R. Nitzberg, “A CFAR
adaptive matched filter detector,” IEEE Trans. Aerosp. Electron. THEILER: ABSORPTIVE WEAK PLUME DETECTION ON GAUSSIAN AND NON-GAUSSIAN BACKGROUND CLUTTER THEILER: ABSORPTIVE WEAK PLUME DETECTION ON GAUSSIAN AND NON-GAUSSIAN BACKGROUND CLUTTER 6853 (Tµ)′R−1Tµ/∥Tµ∥2 ≫1/∥µ∥2 from which it follows: [4] S. J. Young, “Detection and quantification of gases in industrial-stack
plumes using thermal-infrared hyperspectral imaging,” the aerospace cor-
poration, Tech. Rep. ATR-2002(8407)-1, 2002. ∥Tµ∥
∥µ∥×
1
∥R−1/2Tµ∥≪1. (49) (49) p
p
[5] B. R. Foy, R. R. Petrin, C. R. Quick, T. Shimada, and J. J. Tiee, “Com-
parisons between hyperspectral passive and multispectral active sensor
measurements.” in Proc. SPIE, vol. 4722, pp. 98–109, 2002. That is: ∥Δx∥≪∥x∥, which means that the effect of the a = ao
plume is small. Numerically, we find with the datasets used
here, that this ratio is less than one in a thousand. (Specifically,
∥Δx∥/∥x∥= 0.00087 for the for OMI data with the NO2
plume, 0.00063 for OMI data with the SO2 plume, 0.00047
for the 2016 AVIRIS-NG data with CH4 plume, and 0.00054
for the 2019 CH4 data. The values in Table IV are somewhat
larger than this because a > ao for those plumes.) Thus, the
multiplyingeffectofmatchedfiltersisconsiderablehere.Plumes
that are virtually invisible in the raw data can be readily evident
in matched filter projections. That is: ∥Δx∥≪∥x∥, which means that the effect of the a = ao
plume is small. Numerically, we find with the datasets used
here, that this ratio is less than one in a thousand. (Specifically, pp
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Speech, Signal Process., vol. 38, no. 10, pp. 1760–1770, Oct. 1990. ACKNOWLEDGMENT The author is grateful to Alan Schaum for valuable insights
and useful discussions and, in particular, for emphasizing the
importance of the |dz/dx| factor in (3). He also thanks Bernard
Foy, with whom he had many insightful discussions over the
years, for help acquiring and interpreting the OMI data and
Philip Dennison for help understanding the AVIRIS-NG data at
the NASA-JPL website [41]. The author also thanks both these
organizations for making their data freely available. p
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Fink, Eds., vol. 116. Washington, DC, USA: Amer. Geophysical Union,
2000, pp. 117–138. APPENDIX A
CHARACTERISTIC PLUME STRENGTH ao ∥Δx∥
∥x∥
∼∥exp(−aoT)x −x∥
∥x∥
≈∥aoTx∥
∥x∥
∼
∥Tµ∥2 + t′Rt
∥µ∥2 + Trace(R) ×
1
∥R−1/2Tµ∥. (48) The detectability of a plume certainly depends on its strength
a, but it also depends on the absorption spectrum t, and the mean
μ and covariance R of the clutter. For a given t, μ, and R, we
seek a characteristic plume strength ao that corresponds roughly
to a minimum detectable quantity. The actual MDQ will depend
on x as well (or, more properly, on z, the pixel spectrum in the
absence of plume), and of course on the level of false alarm rate
that you can tolerate with your detection. (48) We find in practice this ratio is often much–much less than one,
even when the plume to clutter ratio in the matched filter space
is of order one. Observe that the covariance R typically has its maximum
eigen-direction along a vector that is roughly parallel to µ;
but since the data values are positive, we can say that the
largest eigenvalue is roughly bounded by ∥µ∥2, and that most
eigenvalues tend to be quite a bit smaller than that. Thus,
the smallest eigenvalue of R−1 is of order 1/∥µ∥2 with most
eigenvalues quite a bit larger than that. Thus, for most vectors u,
we expect u′R−1u/u′u ≫1/∥µ∥2. Taking u = Tµ, we obtain: We will go back to the approximation x = z −aTµ. The de-
tector (Tµ)′R−1(x −µ) will, in the absence of plume (x = z),
be a scalar random variable with mean zero and variance given
by Variance
(Tµ)′R−1(z −µ)
=
(Tµ)′R−1(z −µ)(z −µ)′R−1(Tµ)
Variance
(Tµ)′R−1(z −µ) =
(Tµ)′R−1(z −µ)(z −µ)′R−1(Tµ)
IEEE JOURNAL OF SELECTED TOPICS IN APPLIED EARTH OBSERVATIONS AND REMOTE SENSING, VOL. 14, 2021 IEEE JOURNAL OF SELECTED TOPICS IN APPLIED EARTH OBSERVATIONS AND REMOTE SENSING, VOL. 14, 2021 6854 [30] J. Li, C. C. Schmidt, J. P. Nelson III, T. J. Schmit, and W. Menzel,
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Use of sonic anemometry for the study of confined swirling flows in large industrial units
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Flow measurement and instrumentation
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Use of sonic anemometry for the study of confined
swirling flows in large industrial units
Francia Garcia, Victor; Martín, Luis; Bayly, Andrew E.; Simmons, Mark J.h. DOI:
10.1016/j.flowmeasinst.2016.06.017
License:
Creative Commons: Attribution (CC BY) Document Version
Publisher's PDF, also known as Version of record Citation for published version (Harvard):
Francia Garcia, V, Martín, L, Bayly, AE & Simmons, MJH 2016, 'Use of sonic anemometry for the study of
confined swirling flows in large industrial units', Flow Measurement and Instrumentation, vol. 50, pp. 216-228. https://doi.org/10.1016/j.flowmeasinst.2016.06.017 Citation for published version (Harvard):
Francia Garcia, V, Martín, L, Bayly, AE & Simmons, MJH 2016, 'Use of sonic anemometry for the study of
confined swirling flows in large industrial units', Flow Measurement and Instrumentation, vol. 50, pp. 216-228. https://doi.org/10.1016/j.flowmeasinst.2016.06.017 Link to publication on Research at Birmingham portal General rights
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•Users may not further distribute the material nor use it for the purposes of commercial gain. Where a licence is displayed above, please note the terms and conditions of the licence govern your use of this document. Where a licence is displayed above, please note the terms and conditions of the licence govern your use of th When citing, please reference the published version. a r t i c l e i n f o Article history:
Received 29 October 2015
Received in revised form
24 May 2016
Accepted 22 June 2016
Available online 23 June 2016
Keywords:
Full scale measurement
Vortex flow
Spray dryer
Sonic anemometry
Swirl
Scale up Article history:
Received 29 October 2015
Received in revised form
24 May 2016
Accepted 22 June 2016
Available online 23 June 2016
Keywords:
Full scale measurement
Vortex flow
Spray dryer
Sonic anemometry
Swirl
Scale up This work explores the methodology and errors involved in using a commercial sonic anemometer to
study confined industrial swirling air flows, such as those in large cyclones or dryers in the order of
hundreds of m3. Common sources of uncertainty in time-of-flight techniques and multiple-path ane-
mometry are evaluated and corrections and methodology guidelines are proposed to deal with issues
typical of full scale measurement. In particular, this paper focuses on quantifying the error associated
with the disruption of the local flow caused by a
−
HS
50 horizontal sonic anemometer under a range of
turbulence characteristic of industrial swirl towers. Under the guidelines proposed and the conditions
studied here, the presence of the instrument originates a measurement error <
−
1
4% in velocity, <
−
°
1
3
in direction and <
−
7
31% in turbulent kinetic energy for an isothermal flow in the absence of solids. These ranges are above traditional uses of sonic anemometry in meteorology due to the limitations
inherent to industrial units, but remain within reasonable margins for engineering applications. Laser
diagnostic methods are widely used in laboratory and pilot scale cyclones or dryers but are rarely ap-
plicable to large production scales. In this context, the data collected with sonic anemometers render
much lower resolution but appear in agreement with historical Particle Image Velocimetry. Methods
such as the one proposed here can be a useful alternative to improve the level of detail of fluid dynamic
studies in industrial units, which are often qualitative or with a limited validation. Article history:
Received 29 October 2015
Received in revised form
24 May 2016
Accepted 22 June 2016
Available online 23 June 2016 & 2016 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/). Use of sonic anemometry for the study of confined swirling flows in
large industrial units Víctor Francia a,b,n, Luis Martín b, Andrew E. Bayly b,1, Mark J.H. Simmons a School of Chemical Engineering, University of Birmingham, Birmingham, UK
b Procter & Gamble R & D, Newcastle Innovation Centre, Newcastle upon Tyne, UK Take down policy down policy
the University of Birmingham exercises care and attention in making items available there are rare occasions when an it
ded in error or has been deemed to be commercially or otherwise sensitive. this is the case for this document, please contact UBIRA@lists.bham.ac.uk providing details and we will remove access
ely and investigate. If you believe that this is the case for this document, please contact UBIRA@lists.bham.ac.uk providing details and we
the work immediately and investigate. Download date: 24. Oct. 2024 Flow Measurement and Instrumentation 50 (2016) 216–228 Contents lists available at ScienceDirect n Corresponding author at: School of Chemical Engineering, University of Bir-
mingham, Birmingham, UK
E-mail address: v.francia.chemeng@gmail.com (V. Francia).
1 Present address: School Chemical and Process Engineering, University of
Leeds, Leeds, UK http://dx.doi.org/10.1016/j.flowmeasinst.2016.06.017
0955-5986/& 2016 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). nst.2016.06.017
ished by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). http://dx.doi.org/10.1016/j.flowmeasinst.2016.06.017
0955-5986/& 2016 The Authors. Published by Elsevier Ltd. This is an open access art
n Corresponding author at: School of Chemical Engineering, University of Bir-
mingham, Birmingham, UK
E-mail address: v.francia.chemeng@gmail.com (V. Francia).
1 Present address: School Chemical and Process Engineering, University of
Leeds, Leeds, UK http://dx.doi.org/10.1016/j.flowmeasinst.2016.06.017
0955-5986/& 2016 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). 1. Introduction Fluid dynamics models de-
velop new designs and study stability [23,24], collection efficiency
[25] or heat and mass transfer [26–28], but are hardly ever vali-
dated in full scale, which implies that some characteristics in-
herent to production are neglected, namely: (a) specific designs in
industry, in particular inlet nozzles and exhaust lines (b) range of
Reynolds, Re, and hence swirl stability and (c) comparable friction,
materials or deposits. Counter-current swirl drying towers are
good examples of the issues that may arise. Extensive efforts have
been made to numerically describe the swirling flow [15,29,30]
and compare models to data collected in laboratory [31] and pilot
scale towers [12]. The attenuation of the swirl was found negli-
gible in simplified scenarios, but PIV data [13] collected in pro-
duction units, and later measurements with the method described
here indicate that friction [32] is key to explaining how the flow
and turbulence structure are generated in units with rough walls
and deposits [33]. The effects of friction are beginning to be
brought into modelling [34] but could only be identified after the
experiments moved into full scale. Fig. 1b describes the velocity and turbulence profiles observed
in the chamber [32]. Within the cylinder the vortex exhibits a
“concentrated” shape [35] whereby the tangential velocity
̅θ
U
shows a forced inner core and an outer free vortex that changes in
extension as the flow approaches the top exit. At the central re-
gion, an axial jet (Fig. 1b2) coveys the flow towards the top exit
duct. In this area, the core of the vortex core precesses around the
cylinder axis. The displacement of the core results in an area of
higher variability (Fig. 1b3), which indicates a periodical change in
the core position rather than any real turbulent kinetic energy
[32]. A more thorough description of the structure of the flow is
out of the scope of this paper ; the reader is referred to other
works to find more a detailed analysis of stability, structure and
turbulence in a cleaned dryer [32], and how the scale and the
deposits affect the structure [33]. The following sections report
data sets in two spray drying towers, denoted Scale I and II in
Table 1. Section 4 reports data at three radial positions in the
tower Scale I. 1. Introduction important drawbacks: (a) disruption due to optics, (b) time and
lack of flexibility due to set up and safety protocols, (c) reliability
in industrial environments, (d) limited measurement range and
importantly, costs associated to (e) engineering modifications,
(f) time production units need to be shut down and (e) seeding. In
full scale cyclones or dryers, vane anemometers [14] and flow vi-
sualization [15,16] are commonly used, but they provide a poor
level of detail and cannot address many of the questions arising
from models, optimization or scale up. In addition to new acoustic
sensors, thermal anemometers represent a good compromise be-
tween quality and robustness [17]. Typical arrays of hot-wires are
an excellent alternative for studies in a pilot scale [10,18] thanks to
a high response frequency, but the set up is time consuming and
too delicate for a quick and reliable use in industrial cases. Laser diagnostics methods such as Particle Image Velocimetry,
PIV [1], or Laser Doppler Anemometry, LDA [2–4], and the use of
hot-wires [5,6] are widely applied to study swirling flows in small
devices (e.g. pipes, swirlers, concentric cylinders or combustors)
and larger process units such as cyclones [7–9] and dryers [10,11]
in laboratory or pilot devices. On a large scale, access to these units
is more complicated. The space and time available are restricted
and the cost to collect data increases significantly, which makes
experimentation less frequent. Lasers can provide a high spatial
and time resolution but are difficult to apply in an industrial en-
vironment. Most studies are limited to scaled down devices de-
signed adhoc. The application to pilot [12] or even large geometries
[13] is possible but it is typically uneconomical because it carries As a result of the lack of alternatives, to this date the data in
large scale swirl towers or cyclones are mostly qualitative, com-
prised of unidirectional velocities, with no turbulence information
or restricted to small sections. Consequently, scaling up the par-
ticle dynamics into the largest devices remains a challenge [19–
21]. Scale-up rules arise from the experience of manufacturers,
dimensional analysis of response times and the tendency of the V. Francia et al. 1. Introduction ′
′
′
W
U
V
,
,
Spar measurement axis of the HS50 Solent Anem-
ometer in Fig. 3. C O
,
With and without the use of the internal calibration. E
Reference estimated value. t b
,
Associated with top or bottom transducers. *
For the door-anemometer ensemble. −
Indicates time average. Absolute error, ∆=
̅
−
̅
X
X
C
E for variable X. current swirl dryers property of Procter & Gamble. An outline of
the typical unit is given in Fig. 1a; Table 1 summarizes the design. The air enters the bottom of the unit with angular momentum due
to the alignment of the inlet ports. It forms a vortex entering the
cylindrical chamber and exiting through the top duct. The method
proposed here allows the study of the flow in the cylindrical
section where the drying droplets spend most of their residence
time. The experiments were conducted under a target air mass
flow rate and exit pressure, under ambient conditions and without
particle production. Control loops are disabled to reduce noise. powder to migrate to the walls [22]. Fluid dynamics models de-
velop new designs and study stability [23,24], collection efficiency
[25] or heat and mass transfer [26–28], but are hardly ever vali-
dated in full scale, which implies that some characteristics in-
herent to production are neglected, namely: (a) specific designs in
industry, in particular inlet nozzles and exhaust lines (b) range of
Reynolds, Re, and hence swirl stability and (c) comparable friction,
materials or deposits. Counter-current swirl drying towers are
good examples of the issues that may arise. Extensive efforts have
been made to numerically describe the swirling flow [15,29,30]
and compare models to data collected in laboratory [31] and pilot
scale towers [12]. The attenuation of the swirl was found negli-
gible in simplified scenarios, but PIV data [13] collected in pro-
duction units, and later measurements with the method described
here indicate that friction [32] is key to explaining how the flow
and turbulence structure are generated in units with rough walls
and deposits [33]. The effects of friction are beginning to be
brought into modelling [34] but could only be identified after the
experiments moved into full scale. powder to migrate to the walls [22]. 1. Introduction Section 5 discusses particularities of the method
using data from both towers and Section 6 provides a comparison
of sonic anemometry and laser based measurements. In the interest of exploring measurements in an industrial en-
vironment, this work discusses the application of a sonic anem-
ometer to characterise the flow in large cyclones or dryers. The
paper develops an alternative to characterise swirl flows in an
array of industrial devices where lasers cannot be generally ap-
plied [17]; it provides engineers with insight to use sonic anem-
ometers in this context and with a reasonable evaluation of some
of the errors that must be expected. Guidelines to collect and
correct velocity and turbulence data with a HS-50 solent sonic
anemometer are given, along with a range for the measurement
error under turbulence levels characteristic of industrial swirl
towers. 2. Application to a large confined swirling flow Common techniques to study swirling flows cannot deal with
the largest scales in the process industry, where some units lack
fluid dynamic information to validate models and draw design and
scale up criteria. As discussed before, some of the limitations arise
from the use of lasers and the cost of a delicate and time 1. Introduction / Flow Measurement and Instrumentation 50 (2016) 216–228 217 Nomenclature
D
Cylinder diameter, m
Gi
The i-th gyration matrix
H
Distance from air inlets to vortex finder, m
R
Cylinder radius, m
Re
Reynolds number
ρ
μ
=
̅
̅
Re
D U
. . /
av
, -
̅MA
Inlet mass rate of air, kg s1
Ps
Combined path shadow
= ∑=
=
P
s
s
i
i
i
1
3
̅Ui
Air velocity / or component in i-th-axis , m s1
̅Uav
Bulk or superficial air velocity
πρ
̅
=
̅
U
M
R
/
av
A
2, m s1
d
Diameter of the vortex finder, m
g
Gyration angle, °
r
Radial position, m
ra
Rotation angle, °
si
Shadow of the i-th path x 1/3
ui
Air velocity fluctuation in the i-axis, m s1
x
Distance from the inner wall along D, m
y
Cartesian axis in Fig. 12,
=
y
0 for
=
r
0 , m
z
Coordinate in the axial direction, m
Greek letters
∆
Absolute error, ∆=
̅
−
̅
X
X
C
E for variable X. α
Misalignment, gyration over a2, °
β
Misalignment, gyration over a3, °
δ
Angle of attack to the horizontal plane, °
ε
Relative error,
(
)
ε =
−
X
X
100. /
1
C
E
for variable X. γ
Misalignment, gyration over a1, °
λ
Angle of attack to the frame axis a2, °
κ
Specific turbulent kinetic energy, m2 s2
ρ
Density, kg m3
σij
Variance/Covariance u u
i
j, m2 s2
Subscripts , superscripts and caps
1,2,3
Auxiliary axis in Fig. 5 or sonic path numbers. a
a
a
,
,
1
2
3 Frame of reference of the anemometer in Fig. 3. θ
r z
,
,
Polar coordinates, along radial, vertical and tangential
direction. ′
′
′
W
U
V
,
,
Spar measurement axis of the HS50 Solent Anem-
ometer in Fig. 3. C O
,
With and without the use of the internal calibration. E
Reference estimated value. t b
,
Associated with top or bottom transducers. *
For the door-anemometer ensemble. −
Indicates time average. 1,2,3
Auxiliary axis in Fig. 5 or sonic path numbers. a
a
a
,
,
1
2
3 Frame of reference of the anemometer in Fig. 3. θ
r z
,
,
Polar coordinates, along radial, vertical and tangential
direction. 2.3. Instrumentation and methodology A commercial research instrument HS50 (Horizontal Symmetry
50 Sonic Solent Anemometer, Gill Instruments Ltd.) was selected
because it is designed to minimise the drag caused by the head
and transducers. The lay-out is shown in Fig. 2 along the no-
menclature and frames of reference used in this paper and the
definition of the angles of attack between the air and the anem-
ometer, denoted δ and λ. Three independent pairs of transducers
are mounted in a single frame with a fork-like head. The anem-
ometer is inserted through a door that allows the movement of the
instrument across the diameter of the dryer as depicted in Fig. 3a. Measurements are gathered at an acquisition frequency of
Hz
50
during
s
60
and can be taken with and without the use of an in-
ternal function that corrects the signal for the drag associated with
the anemometer according to a wind tunnel calibration. Section 4
focuses in detail in quantifying the error due to the local distortion
to the flow in a relevant range of turbulence for this application. Fig. 1. (a) Outline of a tall swirl drying tower, not to scale. (b) Characteristic velocity
profiles, Scale I tower, levels
D
3. 4
and
D
7. 7
[32] (b1) Tangential velocity
̅θ
U (b2)
Axial velocity
̅Uz (b3), turbulent kinetic energy κ. consuming experimental plan. The application of sonic anem-
ometers can address some of the issues. Being designed for me-
teorological research, commercial devices are robust, handle
changes in temperature and even the presence of a small amount
of droplets/dust (i.e rain, snow). The instruments are easy to apply
to a large confinement with affordable changes and no need for
seeding. Sonic anemometers estimate the shift in the velocity of
the sound when it propagates in a moving medium by monitoring consuming experimental plan. The application of sonic anem-
ometers can address some of the issues. Being designed for me-
teorological research, commercial devices are robust, handle
changes in temperature and even the presence of a small amount
of droplets/dust (i.e rain, snow). The instruments are easy to apply
to a large confinement with affordable changes and no need for
seeding. 2.1. Operation of swirl tall-form dryers Their application into industrial devices has many benefits but
there are some important drawbacks that need to be noticed:
(a)
limited
spatial
resolution
and
acquisition
frequency,
(b) anemometers cannot handle the process conditions in most
industrial units and thus, they are limited to study the carrier
phase alone in an isothermal field (c) measurement error depends
on the local disruption caused by the probe and needs to be
quantified in an adequate range of turbulence, and (d) care must
be taken to ensure that the instrument does not disrupt the overall
pressure field. Fig. 1. (a) Outline of a tall swirl drying tower, not to scale. (b) Characteristic velocity
profiles, Scale I tower, levels
D
3. 4
and
D
7. 7
[32] (b1) Tangential velocity
̅θ
U (b2)
Axial velocity
̅Uz (b3), turbulent kinetic energy κ. 2.1. Operation of swirl tall-form dryers This work discusses data collected in two industrial counter- V. Francia et al. / Flow Measurement and Instrumentation 50 (2016) 216–228 218 Table 1
Tower design and volume V ranges. Design
Scale I
Scale II
V V
/ ScaleI
1.0
16.1
H D
/
10.58
2.97
d D
/
0.29
0.28
−
Re.10 5
1.0–2.0
6.0–7.0 Fig. 1. (a) Outline of a tall swirl drying tower, not to scale. (b) Characteristic velocity
profiles, Scale I tower, levels
D
3. 4
and
D
7. 7
[32] (b1) Tangential velocity
̅θ
U (b2)
Axial velocity
̅Uz (b3), turbulent kinetic energy κ. the time-of-flight of consecutive acoustic pulses between two or
three independent pairs of transducers. Commercial research in-
struments provide velocity measurements in a finite sampling
volume with a temporal resolution of 50 Hz. Of course, the size of
the instrument limits spatial resolution but considering the scale
of the industrial units (e.g. >>
m
100
3) when an anemometer is
applied to sufficiently large devices, the measurement volume
becomes comparable to characteristic length scales of the turbu-
lence such as in the use of finite size sensors like hot wires in
smaller devices [5]. Nevertheless, accuracy is a function of the
scale treated and it must be checked on a case-per-case basis
contrasting sonic data with punctual measurements at high re-
solution. The technique itself is long established in wind en-
gineering
[36,37]
to
validate
fluid
dynamic
geomorphologic
models [38,39], study meteorological phenomena [40] and atmo-
spheric turbulence [41–43], particularly the closure of the energy
balance and the boundary layer [44,45]. Often, 2D devices are used
to measure wind velocity, direction and fluctuation simulta-
neously in a set of fixed locations, but 3D instruments can also
render a full spatial resolution [46]. Multi-path anemometers are
common in aeronautics, for eddy covariance analysis [47], for in-
stance the estimation of axial fluxes of greenhouse gases [48–50]. 3.1. Velocity gradients in the mean flow Commercial anemometers emit alternate acoustic pulses of a
frequency
>
Hz
4.104
to ensure that the wave length is much
smaller than the measurement path and so apply a geometrical
acoustic approach to derive the air velocity [60]. The time-of-flight
principle used is only strictly applicable when the air velocity
across the path of the pulse is constant. In three dimensional
flows, gradients and the curvature of the flow field can make the
pulse deviate from a linear path and so there is a need to integrate
the propagation velocity in a non-linear trajectory. The error as-
sociated to commercial instruments (i.e. assuming a linear path)
has been studied for ideal flow fields and related to the velocity
gradients and the field symmetry and curvature [60,61]. In general
terms, errors are in the order of the second power of the Mach
number. As opposed to wind engineering or aeronautics where
wind speeds are very high, they pose no major concern for in-
dustrial applications (i.e. Mach o o0.1). In a long vortex (Fig. 1b
[32]) the tangential flow is the dominant motion. In order to
minimise gradients and curvature, the optimum position results more complex set up). Once the door is positioned, the frame axis
a2 is aligned perpendicular to the door. The head is positioned at a
target distance from the wall x, sliding the frame in and twisting
the orientation to align the axis a1 to the cylinder vertical axis z. Fig. 3a shows the alignment left / right, denoted α, (i.e. rotation of
the plane
−
a
a
1
3 over a2) and up / down, denoted β (i.e. angle
between the frame and the horizontal plane) and the alignment
between the radial and the entry direction, denoted γ. The fol-
lowing sections detail the reasons why the target alignment is set
as
α
β
γ
=
=
= 0 to minimise the sources of error. During the
measurement however, one must expect small deviations from
this target, among other reasons because of (a) space and time
restrictions (b) bending of
the extension at long distances
(c)
alignment
of
the
entry
points
and
the
door,
and
(d) misalignment of the door and the inner wall. Section 5 de-
scribes the transformations required to correct the raw signal and
discuses some of the restrictions to be accounted for. more complex set up). 3. Issues with time-of-flight techniques Sources of error in sonic anemometry have been widely studied
in single [52] and multiple-path devices [53]. The following sec-
tions briefly discuss common issues with time-of-flight techni-
ques, such as velocity gradients in the measurement volume or the
transducer lay-out. The main source of error in sonic anemometry
arises from the local disruption that the instrument itself causes in
the measurement region. Errors have been related to specific de-
signs [54] and the methodology and corrections applied to the
signal depending on the orientation of the anemometer [55,56]. In
general terms, the distortion is minimized by an adequate selec-
tion of the instrument and the set up [57], and later corrected for
by a wind tunnel calibration or other post processing algorithms
[58,59]. Fig. 2. Anemometer. Spar measurement axis (
′
′
′)
W
U V
,
,
and frame of reference
(
)
a
a
a
,
,
1
2
3 for an anticlockwise air vortex (flow left to right) [51]. Angles of attack,δ,
λ. (a) perspective view. (b) top and (c) front views. Not to scale. 2.3. Instrumentation and methodology Spar measurement axis (
′
′
′)
W
U V
,
,
and frame of reference
(
)
a
a
a
,
,
1
2
3 for an anticlockwise air vortex (flow left to right) [51]. Angles of attack,δ,
λ. (a) perspective view. (b) top and (c) front views. Not to scale. 2.3. Instrumentation and methodology Sonic anemometers estimate the shift in the velocity of
the sound when it propagates in a moving medium by monitoring The ensemble is dismantled and moved between access points
in dryers or cyclones for a period of days, weeks or months to
adapt to the availability of production units (i.e. impossible with a V. Francia et al. / Flow Measurement and Instrumentation 50 (2016) 216–228 219 3. Issues with time-of-flight te
Sources of error in sonic anem
in single [52] and multiple-path
tions briefly discuss common i
ques, such as velocity gradients i
transducer lay-out. The main sou
arises from the local disruption t
the measurement region. Errors
signs [54] and the methodolog
signal depending on the orientat
general terms, the distortion is
tion of the instrument and the s
by a wind tunnel calibration or
[58,59]. 3.1. Velocity gradients in the mea
Fig. 2. Anemometer. Spar measurement axis (
′
′
′)
W
U V
,
,
and frame of reference
(
)
a
a
a
,
,
1
2
3 for an anticlockwise air vortex (flow left to right) [51]. Angles of attack,δ,
λ. (a) perspective view. (b) top and (c) front views. Not to scale. Fig. 3. (a) Ensemble, electronic unit EU
PCI [51]. Polar frame of reference (
θ
rz ),
alignments,α β γ
,
,
[32]. (b) and (c) use o
V. Francia et al. / Flow Measurement and Instrumentation 50 (2016) 216–228 Fig. 2. Anemometer. Spar measurement axis (
′
′
′)
W
U V
,
,
and frame of reference
(
)
a
a
a
,
,
1
2
3 for an anticlockwise air vortex (flow left to right) [51]. Angles of attack,δ,
λ. (a) perspective view. (b) top and (c) front views. Not to scale. / Fig. 3. (a) Ensemble, electronic unit EU [51] and power communication interface,
PCI [51]. Polar frame of reference (
θ
rz ), anemometer axes (a1, a2, a3) (a a a
1 2 3) and
alignments,α β γ
,
,
[32]. (b) and (c) use of extensions. Not to scale. Fig. 3. (a) Ensemble, electronic unit EU [51] and power communication interface,
PCI [51]. Polar frame of reference (
θ
rz ), anemometer axes (a1, a2, a3) (a a a
1 2 3) and
alignments,α β γ
,
,
[32]. (b) and (c) use of extensions. Not to scale. Fig. 2. Anemometer. 3.1. Velocity gradients in the mean flow A wind tunnel calibration is used to
correct the raw signal for the effects of the frame and the trans-
ducers as a function of the measured values of the angles of attack
δ and λ. In a HS-50, the calibration reports an accuracy <
RMS
1%
in
velocity and
°
1 in direction as long as (a) λ>
°
30 , which ensures the
frame and the head do not block the flow (see Fig. 2a) and (b)
δ<
°
50 , which ensures that the transducers themselves do not block
the measurement region (see Fig. 2a). The level of turbulence in wind/air flows also has important
effects on the error [63,64]. As fluctuations increase, the air attacks
the instrument across a wider range of direction (δ and λ), which
cannot be described properly by using average values to calibrate
the instrument or to set the limit angles of attack to consider
measurements acceptable. In a traditional meteorological appli-
cation the instrument would be realigned to the mean wind di-
rection in order to minimise δ close to
°
0 and maximise λ close to
°
180 . In this manner, most of the fluctuations from the average
direction remain within the limit angles and the disruption is
minimal. Measurements in a confined unit are necessarily differ-
ent. The level of turbulence is higher and so the error must be
studied independently. Besides, it is impossible to realign the an-
emometer to an optimum orientation every time because it would
imply enormous costs in terms of time and engineering entry
points. Instead, this work proposes to keep the anemometer at a
constant orientation versus the cylinder (i.e. a1 aligned with z thus
α = 0, and a2 aligned with r thus β
γ
=
= 0), which optimises the
values of the angles of attack ( δ, λ) when the flow is tangential
(δ= °
0 , λ=
°
90 ). Indeed, cyclones or dryers exhibit a strong swirling
flow where the tangential motion is dominant, but in general, also
present smaller axial and radial velocities. In this way, when the
anemometer is placed at different locations in the flow field
(moved from one to another place in the dryer), the angles of at-
tack ( δ, λ) become a function of the air direction in that specific
point. 3.1. Velocity gradients in the mean flow Some re-
gions of the chamber are excluded from this study because gra-
dients increase significantly and the errors have not been quanti-
fied: (a) force inner vortex
<
r
R
0.10 , (b) boundary layer
>
r
R
0.98
[13] and (c) vicinity of the exit
>
z
H
0.95 . from aligning the anemometer head, a1 with the cylinder axis z, so
that axial and radial velocity gradients in the paths are minimum
(i.e. order of <
−
s
0.1
1 and <
−
s
10
1 respectively). The exact error in
this position could only be quantified if the field was known a
priori with much higher spatial resolution (e.g. PIV); in this case
significant errors can be discarded on the following basis: (a) the
analysis in Section 4 shows that errors are limited and have an
aerodynamic origin even when the gradients increase by a factor
of a hundred, which should have made the error rise by several
orders of magnitude if curvature was relevant [61], (b) integration
of
̅Uz across the cross-section of the dryer is in agreement with
measurement of the volume rate, and (c) measurements were
found in agreement with PIV [13] (see
̅θ
U in Section 6). Some re-
gions of the chamber are excluded from this study because gra-
dients increase significantly and the errors have not been quanti-
fied: (a) force inner vortex
<
r
R
0.10 , (b) boundary layer
>
r
R
0.98
[13] and (c) vicinity of the exit
>
z
H
0.95 . ⎛
⎝
⎜
⎜⎜
⎞
⎠
⎟
⎟⎟
λ =
̅
̅
+
̅
−
cos
U
U
U
a
a
a
1
2
2
2
2
2
3 ⎛
⎝
⎜
⎜⎜
⎞
⎠
⎟
⎟⎟
λ =
̅
̅
+
̅
−
cos
U
U
U
a
a
a
1
2
2
2
2
2
3 ⎛
⎝
⎜
⎜⎜
⎞
⎠
⎟
⎟⎟
λ =
̅
̅
+
̅
( )
−
cos
U
U
U
2
a
a
a
1
2
2
2
2
2
3 ( )
2 Each anemometer is designed to operate in a range of δ and λ
where errors are minimum. 3.3. Shadow of transducers and supports The presence of the anemometer causes a local disruption to
the flow that depends on the relative orientation between the
instrument and the mean air direction, known as the angle of
attack. The orientation is defined by the angle of attack to the
horizontal plane, δ, and the angle of attack to the frame axis, λ,
shown in Fig. 2a and defined below: ⎛
⎝
⎜
⎜
⎜
⎜
⎞
⎠
⎟
⎟
⎟
⎟
δ =
̅
+
̅
̅
+
̅
+
̅
( )
−
cos
U
U
U
U
U
1
a
a
a
a
a
1
2
2
2
2
2
2
3
1
2
3 ⎛
⎝
⎜
⎜
⎜
⎜
⎞
⎠
⎟
⎟
⎟
⎟
δ =
̅
+
̅
̅
+
̅
+
̅
( )
−
cos
U
U
U
U
U
1
a
a
a
a
a
1
2
2
2
2
2
2
3
1
2
3 ( )
1 ( )
1 Fig. 4. Examples of singular orientations in the angle of attack δ. 3.2. Small angles between the sonic pulses Relative small angles between the pulses reduce the ability to
characterise three dimensional flows and lead to errors in the
correction procedure of anemometers. A mathematical singularity
arises at certain orientations that reduce the determinant of the
Jacobian in the calibration to a null value. In essence, some di-
rections become undefined after applying the correction to the
signal, which distorts the measurement [62]. Fig. 4 illustrates this
phenomenon when using a HS50. The population of the angle of
attack
δ defined below is shown for raw and calibrated mea-
surements in two instances that exhibit the singularity. The un-
defined orientation occurs only when using the calibration and in
the proximity of the limit angle of attack δ <
°
45
given later for
aerodynamic considerations, which ensures that it originates no
further error in any of the measurements considered acceptable in
following sections. 3.1. Velocity gradients in the mean flow Using the orientation proposed here to study a large long
vortex results in angles of attack below the limits for the most part
of the chamber, and therefore the measurements are adequate. Only in sections where the axial flow (
=
)
U
U
a
z
1
dominates the
motion δ rises >
°
50 and the measurements must be discarded (e.g. core in Fig. 1b). The horizontal angle of attack λ is less restrictive
because with the exception of the core and the exit duct, the radial
velocity is very low and λ~
°
90 . Of course, lower spatial resolution,
stronger turbulence and angles of attack that are in general above
the optimum make errors higher than in typical uses of sonic
anemometer such as the research of atmospheric turbulence. However, considering the lack of alternatives in this type of units,
the data can easily accept higher errors and remain valuable. 3.1. Velocity gradients in the mean flow Once the door is positioned, the frame axis
a2 is aligned perpendicular to the door. The head is positioned at a
target distance from the wall x, sliding the frame in and twisting
the orientation to align the axis a1 to the cylinder vertical axis z. Fig. 3a shows the alignment left / right, denoted α, (i.e. rotation of
the plane
−
a
a
1
3 over a2) and up / down, denoted β (i.e. angle
between the frame and the horizontal plane) and the alignment
between the radial and the entry direction, denoted γ. The fol-
lowing sections detail the reasons why the target alignment is set
as
α
β
γ
=
=
= 0 to minimise the sources of error. During the
measurement however, one must expect small deviations from
this target, among other reasons because of (a) space and time
restrictions (b) bending of
the extension at long distances
(c)
alignment
of
the
entry
points
and
the
door,
and
(d) misalignment of the door and the inner wall. Section 5 de-
scribes the transformations required to correct the raw signal and
discuses some of the restrictions to be accounted for. V. Francia et al. / Flow Measurement and Instrumentation 50 (2016) 216–228 220 from aligning the anemometer head, a1 with the cylinder axis z, so
that axial and radial velocity gradients in the paths are minimum
(i.e. order of <
−
s
0.1
1 and <
−
s
10
1 respectively). The exact error in
this position could only be quantified if the field was known a
priori with much higher spatial resolution (e.g. PIV); in this case
significant errors can be discarded on the following basis: (a) the
analysis in Section 4 shows that errors are limited and have an
aerodynamic origin even when the gradients increase by a factor
of a hundred, which should have made the error rise by several
orders of magnitude if curvature was relevant [61], (b) integration
of
̅Uz across the cross-section of the dryer is in agreement with
measurement of the volume rate, and (c) measurements were
found in agreement with PIV [13] (see
̅θ
U in Section 6). Table 2 rotation angle ra in Set A, where
> °
g 0 (moving from left to right
with
the
bottom
transducers
upstream)
and
Set
B,
where
< °
g 0 (moving
from
right
to
left
with
the
top
transducers
upstream). Fig. 5. Revolution of the anemometer facing a tangential flow. was rotated (see Fig. 5) in such a way that the transducers are
progressively aligned against the flow (e.g. Ua1 and δ increase while
the velocity and turbulence remain constant). The analysis has
been repeated at positions that cover the range of turbulence ki-
netic energy from κ
̅U
/
E
2 =0.005 to 0.026 (data available in Sup-
plementary material). p
)
The response of the measurement when the head revolves is
described in Fig. 7. The real values are given by the best estimate of
velocity, direction and turbulence statistics, denoted by an
E
subscript and obtained from the average of measurements where
δ <
°
5 . The variation expected for each variable during the re-
volution is denoted by a solid line and computed according to the
change in coordinates. The actual measurements are recorded
with or without the calibration and denoted with open and solid
markers and O or C subscripts respectively. Fig. 7 shows how the
measurements start to deviate significantly from the expected
values when the angles of attack increase above a certain limit. The revolution commences at
=
°
g
0
where the disruption is
negligible (see δ~ °
0 in Fig. 7c, where δ
th
90
denotes the 90th per-
centile of δ). As the head revolves, the frame and the transducers
start to disrupt the flow and the angle of attack increases. As a
result of the drag the measurement of the velocity starts to exhibit
a deficit error between the estimated and measured values,
̅ −
̅
U
U
C
E (i.e. Fig. 7a). The change in coordinates makes the velocity
in the axial and tangential direction
̅Ua1 and
̅Ua3 to be swapped
during the revolution (Fig. 7e and f). The axial flow (i.e. ̅Ua1) is the
most susceptible to drag in this anemometer and thus it shows the
highest measurement bias, −20% at the largest value of the angle
of attack, δ
th
90 , when the transducers block the flow. In addition, Fig. 6 illustrates the revolution. 4. Quantification of the local flow disruption in a large tur-
bulent vortex Wind tunnel calibrations are carried out under near laminar
conditions and cannot be a priori considered representative of
such industrial applications where turbulence must be accounted
for [63,64]. This section quantifies the measurement error in this
application as function of the angle of attack δ and under a range
of turbulence characteristic of a swirl dryer. It studies how the
velocity signal changes when the anemometer is revolved at a fix
point. The instrument was placed at three radial positions in the
tower Scale I where the flow is mainly tangential, and the head Fig. 4. Examples of singular orientations in the angle of attack δ. V. Francia et al. / Flow Measurement and Instrumentation 50 (2016) 216–228 221 Fig. 5. Revolution of the anemometer facing a tangential flow. Table 2
Equivalent positions in the revolution. Design
Set A
Set B
Gyration angle, g
<
<
g
0
180
−
<
<
g
180
0
Rotation angle, ra
=
ra
g
=
+
ra
g
180
Upstream transducers
bottom
top Table 2
Equivalent positions in the revolution. Table 2 / Flow Measurement and Instrumentation 50 (2016) 216–228 222 Fig. 7. Evolution of the error in the measurement of velocity and turbulence during the revolution in the anemometer frame of reference (a a a
1 2 3). Data for Scale I tower,
=
r
R
0. 35 , κ
̅U
/
E
2 =0. 005. (a)
̅U (b) λ (c) δ (d)
̅Ua2 (e)
̅Ua3 (f)
̅Ua1 (g) σa a
1 1 (h) σa a
2 2 (i) σa a
3 3 (j) σa a
1 2 (k) σa a
1 3(l)σa a
2 3. E, C and O denote respectively estimate, calibrated and non-
calibrated measurements. Fig. 7. Evolution of the error in the measurement of velocity and turbulence during the revolution in the anemometer frame of reference (a a a
1 2 3). Data for Scale I tower,
=
r
R
0. 35 , κ
̅U
/
E
2 =0. 005. (a)
̅U (b) λ (c) δ (d)
̅Ua2 (e)
̅Ua3 (f)
̅Ua1 (g) σa a
1 1 (h) σa a
2 2 (i) σa a
3 3 (j) σa a
1 2 (k) σa a
1 3(l)σa a
2 3. E, C and O denote respectively estimate, calibrated and non-
calibrated measurements Fig. 7. Evolution of the error in the measurement of velocity and turbulence during the revolution in the anemometer frame of reference (a a a
1 2 3). Data for Scale I tower,
=
r
R
0. 35 , κ
̅U
/
E
2 =0. 005. (a)
̅U (b) λ (c) δ (d)
̅Ua2 (e)
̅Ua3 (f)
̅Ua1 (g) σa a
1 1 (h) σa a
2 2 (i) σa a
3 3 (j) σa a
1 2 (k) σa a
1 3(l)σa a
2 3. E, C and O denote respectively estimate, calibrated and non-
lib
t d
t Fig. 7. Evolution of the error in the measurement of velocity and turbulence during the revolution in the anemometer frame of reference (a a a
1 2 3). Data for Scale I tower,
=
r
R
0. 35 , κ
̅U
/
E
2 =0. 005. (a)
̅U (b) λ (c) δ (d)
̅Ua2 (e)
̅Ua3 (f)
̅Ua1 (g) σa a
1 1 (h) σa a
2 2 (i) σa a
3 3 (j) σa a
1 2 (k) σa a
1 3(l)σa a
2 3. Table 2 Initially, the head frame a1a1 is
aligned with the vertical axis of the tower, z z, and the velocity
components in polar coordinates (
θ
U U U
r
z
) are equivalent to those in
the anemometer frame of reference ( U U U
a
a
a
1
2
3) defined in Fig-
s. 2 and 3. The head then rotates over a2a2 and measurements are
taken in calibrated and un-calibrated mode at 50 gyration angles,
denoted g g (
> °
g 0
clockwise and
< °
g 0
anticlockwise; note that
α
=
g
, this notation is used here to distinguish the error analysis
from the standard methodology). As the head revolves a2 remains
aligned with the radial direction r and only a1 and a3 change. It is
important noticing that due the way the transducers are layout in
a HS50, the position obtained for g in clockwise or anticlockwise
sense differs (e.g. compare
=
°
g 60
and
= −
°
g
60
in Fig. 6). The
same geometry is obtained for two sets of rotation angles ra such
that
=
ra
g in anti-clockwise direction and
=
+
°
ra
g 180 in clock-
wise direction (e.g. compare
=
°
g 60 and
= −
°
g
120 in Fig. 6). Ta-
ble 2 defines two equivalent sets of positions resulting from one
revolution, Sets A and B. Fig. 6 illustrates the position for each Fig. 6. Orientation of the head and the transducers during the revolution. Position and anemometer frame of reference in a clockwise rotation (moving from left to right Set
A, Table 2) and an anticlockwise rotation (moving from right to left Set B , Table 2). The set of transducers upstream is responsible of the drag and given in bold: it
corresponds to the bottom in Set A or the top in Set B. Fig. 6. Orientation of the head and the transducers during the revolution. Position and anemometer frame of reference in a clockwise rotation (moving from left to right Set
A, Table 2) and an anticlockwise rotation (moving from right to left Set B , Table 2). The set of transducers upstream is responsible of the drag and given in bold: it
corresponds to the bottom in Set A or the top in Set B. V. Francia et al. 2
2
In Fig. 7, the profiles are not symmetrical about
~ °
g 0 , but Fig. 8. Disrupted volume. Projection of along the streamline (the flow moves into the page) as function of δ for λ=
°
105 . Grey areas delimit the shadow cast by the surface
comprised of the transducers upstream. Fig. 9. Correlation of error to path shadow. Calibrated mode, Scale I tower,
=
r
R
0. 35 , κ
̅ =
U
/
0. 005
E
2
, filled and open symbols denote Set A and B in Table 2. Velocities
̅Ur and
̅Uz (left axes) and normal stresses σr r,
σz z,
(right axes). The path
shadow Ps (central axis) gives the ratio of the paths included in the volume dis-
rupted (Fig. 8). PsU gives the shadow cast by the mean air direction and
Ps the
time-averaged shadow. si denotes the contribution of each path to
PsU (see
nomenclature). set of positions that share the same geometry in Fig. 6 and Table 2
show overlapping trends in Fig. 9. The regions at either side of
=
°
ra 90 correspond to the positions where the transducer 1 blocks
the sonic path
tb
1
or the transducers
−
2
3 block the paths
tb
2
and
tb
3
(see δ=
°
75
in Fig. 8 and the corresponding individual path
shadows in Fig. 9). Interestingly, both sides of
=
°
ra 90 show clearly
different profiles and it is precisely the transition between them
which originates the change in sense of the radial flow (Fig. 9a)
and the large perturbation observed in the variability (Fig. 9b). Data extended to the other positions and normal and Reynolds
stresses are given in Supplementary material. The fact that this
asymmetry exists and that the error pattern can be linked to the
shadows, exclusive function of geometry, is relevant. It shows the
aerodynamic origin of the error and confirms that it is not purely
determined by δ but also by the sense of Ua3, in an indication that δ
may be better defined from
−
°
0
180 . Fig. 10 shows the resulting distribution of error in the velocity
magnitude, direction, and turbulence as a function of δ
th
90
for the
measurements during the revolution. Table 3 presents the max-
imum error range for the measurements that comply with the
limits δ
<
°
45
th
90
and λ
>
°
30
th
90
. 5.1. Time averaging maximum errors are reached at
~−
°
g
75 and
~
°
g 105 . This is a 3D
effect that cannot be described with the usual definition of δ given
in Eq. (1) between 0 and
°
90 but can be explained by considering
the shadow cast by the transducers. Each transducer disrupts the
field downstream, but it is the orientation what determines
whether the local perturbation affects the measurement volume. Fig. 8 shows the projection of the transducers head along the air
streamline as function of g and δ in a purely tangential flow. As the
head revolves the shadow cast by the transducers is projected into
the path of the sonic pulses, which causes the increase in error. Notice that positions with the same δ can in fact produce different
drag areas (e.g. see how δ=
°
75 may result from two layouts where
either one transducer, `1´, or two , `2´ & `3´, are responsible for the
drag depending on whether the bottom or top transducers are
upstream in Sets A or B). Fig. 9 presents the correlation between
the shadow cast by the transducers and the error pattern. The two The measurement time needs to be sufficiently high to ensure
the convergence of the statistics with sufficient accuracy. It is
particularly true in cyclonic flows that tend to exhibit variability in
large time scales as well as oscillations and periodical flows
[23,24,65]. It is equally true that the costs of working with a
production unit make impossible to collect data for very long
periods of time. Indeed, costs explain the lack of data and one
must try reducing the measurement time as much as possible
while keeping the data meaningful. Of course, any limitation
makes studying the largest time scales in the flow impossible and
introduces an error, but in practise, it is essential to access many
production units. In an oscillating swirling flow the minimum time
would depend on the design and scale of the unit and thus it
needs to be checked on a case-per-case basis. In this work, the
error arising from reducing the averaging time was checked in
several positions. Fig. 11 illustrates how a certain large time scale maximum errors are reached at
~−
°
g
75 and
~
°
g 105 . These ranges are characteristic of
the turbulence in an industrial tower and cover the full range of
possible angles of attack when one moves the instrument from
one to another location. Thus, they may be consider a conservative
estimate of the error when one collects data with a
−
HS 50 at any
position as long as the flow exhibits a similar structure and the
same limit angles and guidelines are considered. Fig. 9. Correlation of error to path shadow. Calibrated mode, Scale I tower,
=
r
R
0. 35 , κ
̅ =
U
/
0. 005
E
2
, filled and open symbols denote Set A and B in Table 2. Velocities
̅Ur and
̅Uz (left axes) and normal stresses σr r,
σz z,
(right axes). The path
shadow Ps (central axis) gives the ratio of the paths included in the volume dis-
rupted (Fig. 8). PsU gives the shadow cast by the mean air direction and
Ps the
time-averaged shadow. si denotes the contribution of each path to
PsU (see
nomenclature). Table 2 E, C and O denote respectively estimate, calibrated and non-
calibrated measurements. flow in Fig. 7b and d translates here into artificial measurement of
turbulence at the positions associated to the strongest drag (i.e. δ
>
°
45
th
90
), which becomes particularly visible in the radial normal
stress σa a
,
2
2 (Fig. 7h). the mean direction,
λ (i.e. Fig. 7b), becomes strongly distorted
when the angle of attack δ
th
90
moves >
°
45 . Above this point, the
drag originates a clear perturbation in the radial flow,
̅
−
̅
U
U
a C
a E
,
,
2
2
that changes sense (i.e. Fig. 7d). Fig. 7g to l include the deviation of
the turbulence statistics. The perturbation observed in the radial V. Francia et al. / Flow Measurement and Instrumentation 50 (2016) 216–228 V. Francia et al. / Flow Measurement and Instrumentation 50 (2016) 216–228
22 223 Fig. 8. Disrupted volume. Projection of along the streamline (the flow moves into the page) as function of δ for λ=
°
105 . Grey areas delimit the shadow cast by the surface
comprised of the transducers upstream. 5.1. Time averaging Variable X
XE
∆or ε
average
range
min
max
=
r
R
0.35
δ
2.4°
2.4°
0.6°
1.8°
λ
99.5°
7.2°
5.4°
1.8°
̅U
̅UE
5.3%
4.3%
1.0%
κ
̅ ∙
U
/
10
E
2
3
5.3
22.3%
6.7%
15.6%
̅
̅
u u
U
/
.10
z
z
E
2
3
4.2
50.6%
18.7%
32.4%
̅
̅
θ
θ
u u
U
/
.10
E
2
3
3.6
32.6%
13.4%
19.3%
̅
̅
u u U
/
.10
r
r
E
2
3
2.7
39.5%
22.4%
16.5%
=
r
R
0.60
δ
12.3°
3.0°
1.4°
1.6°
λ
95.8°
3.9°
2.8°
1.1°
̅U
̅UE
3.3%
3.0%
0.4%
κ
̅ ∙
U
/
10
E
2
3
7.8
37.5%
6.2%
31.3%
̅
̅
u u
U
/
.10
z
z
E
2
3
5.9
57.5%
16.5%
41.0%
̅
̅
θ
θ
u u
U
/
.10
E
2
3
4.2
42.6%
19.4%
23.1%
̅
̅
u u U
/
.10
r
r
E
2
3
5.5
42.1%
6.7%
35.3%
=
r
R
0.10
δ
25.9°
8.1°
1.5°
6.6°
λ
108.0°
7.5°
3.0°
4.5°
̅U
̅UE
19.1%
14.5%
4.6%
κ
̅ ∙
U
/
10
E
2
3
26.5
31.3%
17.7%
13.6%
̅
̅
u u
U
/
.10
z
z
E
2
3
21.9
35.0%
18.9%
16.1%
̅
̅
θ
θ
u u
U
/
.10
E
2
3
17.1
71.0 %
31.5%
39.4%
̅
̅
u u U
/
.10
r
r
E
2
3
13.9
34.7%
15.5%
19.3%
Fig. 11. Average air velocity for decreasing averaging times. Table 3
Range for the measurement error in the revolution when the direction complies
with the limit angle of attack δ
<
°
45
th
90
, λ
>
°
30
th
90
under calibrated mode. Absolute
∆and relative error ε (see nomenclature). Fig. 11. Average air velocity for decreasing averaging times. Fig. 10. Errors as a function of the 90th percentile of the angle of attack δ
th
90 . Scale I
tower, =
r
R
0. 35 , κ
̅ =
U
/
0. 005
E
2
. Calibrated mode. 5.1. Time averaging The solid symbols indicate the data
complying with λ
>
°
30
th
90
, δ
<
°
45
th
90
, Absolute ∆or relative error ε for (a)
̅U (b) δ
(c) λ (d) κ (e) σθ θ,
, σ θ
r,
(f) σz z,
, σ θ
z,
(g) σr r, ,σr z, . Fig. 11. Average air velocity for decreasing averaging times. variability appears for averaging times <
s
360 . In this particular
case study, a measurement time of
s
60
was considered a good
compromise to limit error and preserve the flexibility of the
method. It allows very large geometries to be covered and it was
sufficient to reduce the variability in the time-averaged velocity
(<1% and <2% in Fig. 11a and b for κ
̅ =
U
/
0.008
E
2
and 0.035) and the
turbulence statistics below the errors expected from the local
disruption. β are measured by an in-built inclinometer. In a large scale unit, it
is vital to account for deviations from the target orientation. In
particular, one must consider the adjustment of the mechanical
fittings, see Fig. 3a, because the doors are often misaligned with
the walls. Any deviation between a2 and the radial direction, r,
denoted γ, can lead to very large errors in
θ
U and Ur. The trans-
formations outlined below express the raw signal in polar co-
ordinates. The position of measurement is related in Eq. (6) to the
position and alignment of the ensemble shown in Fig. 3. The latter
can be estimated by manually measuring the offset between the
radial direction r and the projection of the orthogonal to the door 5.2. Correction for the actual anemometer orientation 5.1. Time averaging This is a 3D
effect that cannot be described with the usual definition of δ given
in Eq. (1) between 0 and
°
90 but can be explained by considering
the shadow cast by the transducers. Each transducer disrupts the
field downstream, but it is the orientation what determines
whether the local perturbation affects the measurement volume. Fig. 8 shows the projection of the transducers head along the air
streamline as function of g and δ in a purely tangential flow. As the
head revolves the shadow cast by the transducers is projected into
the path of the sonic pulses, which causes the increase in error. Notice that positions with the same δ can in fact produce different
drag areas (e.g. see how δ=
°
75 may result from two layouts where
either one transducer, `1´, or two , `2´ & `3´, are responsible for the
drag depending on whether the bottom or top transducers are
upstream in Sets A or B). Fig. 9 presents the correlation between
the shadow cast by the transducers and the error pattern. The two V. Francia et al. / Flow Measurement and Instrumentation 50 (2016) 216–228 224 variability appears for averaging times <
s
360
In this particular
Fig. 10. Errors as a function of the 90th percentile of the angle of attack δ
th
90 . Scale I
tower, =
r
R
0. 35 , κ
̅ =
U
/
0. 005
E
2
. Calibrated mode. The solid symbols indicate the data
complying with λ
>
°
30
th
90
, δ
<
°
45
th
90
, Absolute ∆or relative error ε for (a)
̅U (b) δ
(c) λ (d) κ (e) σθ θ,
, σ θ
r,
(f) σz z,
, σ θ
z,
(g) σr r, ,σr z, . / variability appears for averaging times <
s
360 . In this particular
case study, a measurement time of
s
60
was considered a good
compromise to limit error and preserve the flexibility of the
method. It allows very large geometries to be covered and it was
sufficient to reduce the variability in the time-averaged velocity
(<1% and <2% in Fig. 11a and b for κ
̅ =
U
/
0.008
E
2
and 0.035) and the
turbulence statistics below the errors expected from the local
disruption. β are measured by an in-built inclinometer. 5.1. Time averaging In a large scale unit, it
is vital to account for deviations from the target orientation. In
particular, one must consider the adjustment of the mechanical
fittings, see Fig. 3a, because the doors are often misaligned with
the walls. Any deviation between a2 and the radial direction, r,
denoted γ, can lead to very large errors in
θ
U and Ur. The trans-
formations outlined below express the raw signal in polar co-
ordinates. The position of measurement is related in Eq. (6) to the
Fig. 10. Errors as a function of the 90th percentile of the angle of attack δ
th
90 . Scale I
tower, =
r
R
0. 35 , κ
̅ =
U
/
0. 005
E
2
. Calibrated mode. The solid symbols indicate the data
complying with λ
>
°
30
th
90
, δ
<
°
45
th
90
, Absolute ∆or relative error ε for (a)
̅U (b) δ
(c) λ (d) κ (e) σθ θ,
, σ θ
r,
(f) σz z,
, σ θ
z,
(g) σr r, ,σr z, . Table 3
Range for the measurement error in the revolution when the direction complies
with the limit angle of attack δ
<
°
45
th
90
, λ
>
°
30
th
90
under calibrated mode. Absolute
∆and relative error ε (see nomenclature). 5.1. Time averaging Variable X
XE
∆or ε
average
range
min
max
=
r
R
0.35
δ
2.4°
2.4°
0.6°
1.8°
λ
99.5°
7.2°
5.4°
1.8°
̅U
̅UE
5.3%
4.3%
1.0%
κ
̅ ∙
U
/
10
E
2
3
5.3
22.3%
6.7%
15.6%
̅
̅
u u
U
/
.10
z
z
E
2
3
4.2
50.6%
18.7%
32.4%
̅
̅
θ
θ
u u
U
/
.10
E
2
3
3.6
32.6%
13.4%
19.3%
̅
̅
u u U
/
.10
r
r
E
2
3
2.7
39.5%
22.4%
16.5%
=
r
R
0.60
δ
12.3°
3.0°
1.4°
1.6°
λ
95.8°
3.9°
2.8°
1.1°
̅U
̅UE
3.3%
3.0%
0.4%
κ
̅ ∙
U
/
10
E
2
3
7.8
37.5%
6.2%
31.3%
̅
̅
u u
U
/
.10
z
z
E
2
3
5.9
57.5%
16.5%
41.0%
̅
̅
θ
θ
u u
U
/
.10
E
2
3
4.2
42.6%
19.4%
23.1%
̅
̅
u u U
/
.10
r
r
E
2
3
5.5
42.1%
6.7%
35.3%
=
r
R
0.10
δ
25.9°
8.1°
1.5°
6.6°
λ
108.0°
7.5°
3.0°
4.5°
̅U
̅UE
19.1%
14.5%
4.6%
κ
̅ ∙
U
/
10
E
2
3
26.5
31.3%
17.7%
13.6%
̅
̅
u u
U
/
.10
z
z
E
2
3
21.9
35.0%
18.9%
16.1%
̅
̅
θ
θ
u u
U
/
.10
E
2
3
17.1
71.0 %
31.5%
39.4%
̅
̅
u u U
/
.10
r
r
E
2
3
13.9
34.7%
15.5%
19.3%
Fig. 11. Average air velocity for decreasing averaging times. /
(
) variability appears for averaging times <
s
360 . In this particular
case study, a measurement time of
s
60
was considered a good
compromise to limit error and preserve the flexibility of the
method. It allows very large geometries to be covered and it was
sufficient to reduce the variability in the time-averaged velocity
(<1% and <2% in Fig. 11a and b for κ
̅ =
U
/
0.008
E
2
and 0.035) and the
turbulence statistics below the errors expected from the local
disruption. 5 2
C
ti
f
th
t
l
t
i
t ti
β are measured by an in-built inclinometer. In a large scale unit, it
is vital to account for deviations from the target orientation. In
particular, one must consider the adjustment of the mechanical
fittings, see Fig. 5.1. Time averaging 3a, because the doors are often misaligned with
the walls. Any deviation between a2 and the radial direction, r,
denoted γ, can lead to very large errors in
θ
U and Ur. The trans-
formations outlined below express the raw signal in polar co-
ordinates. The position of measurement is related in Eq. (6) to the
iti
d
li
t
f th
bl
h
i
Fi
3 Th
l tt
Fig. 10. Errors as a function of the 90th percentile of the angle of attack δ
th
90 . Scale I
tower, =
r
R
0. 35 , κ
̅ =
U
/
0. 005
E
2
. Calibrated mode. The solid symbols indicate the data
complying with λ
>
°
30
th
90
, δ
<
°
45
th
90
, Absolute ∆or relative error ε for (a)
̅U (b) δ
(c) λ (d) κ (e) σθ θ,
, σ θ
r,
(f) σz z,
, σ θ
z,
(g) σr r, ,σr z, . Table 3
Range for the measurement error in the revolution when the direction complies
with the limit angle of attack δ
<
°
45
th
90
, λ
>
°
30
th
90
under calibrated mode. Absolute
∆and relative error ε (see nomenclature). 5.1. Time averaging Variable X
XE
∆or ε
average
range
min
max
=
r
R
0.35
δ
2.4°
2.4°
0.6°
1.8°
λ
99.5°
7.2°
5.4°
1.8°
̅U
̅UE
5.3%
4.3%
1.0%
κ
̅ ∙
U
/
10
E
2
3
5.3
22.3%
6.7%
15.6%
̅
̅
u u
U
/
.10
z
z
E
2
3
4.2
50.6%
18.7%
32.4%
̅
̅
θ
θ
u u
U
/
.10
E
2
3
3.6
32.6%
13.4%
19.3%
̅
̅
u u U
/
.10
r
r
E
2
3
2.7
39.5%
22.4%
16.5%
=
r
R
0.60
δ
12.3°
3.0°
1.4°
1.6°
λ
95.8°
3.9°
2.8°
1.1°
̅U
̅UE
3.3%
3.0%
0.4%
κ
̅ ∙
U
/
10
E
2
3
7.8
37.5%
6.2%
31.3%
̅
̅
u u
U
/
.10
z
z
E
2
3
5.9
57.5%
16.5%
41.0%
̅
̅
θ
θ
u u
U
/
.10
E
2
3
4.2
42.6%
19.4%
23.1%
̅
̅
u u U
/
.10
r
r
E
2
3
5.5
42.1%
6.7%
35.3%
=
r
R
0.10
δ
25.9°
8.1°
1.5°
6.6°
λ
108.0°
7.5°
3.0°
4.5°
̅U
̅UE
19.1%
14.5%
4.6%
κ
̅ ∙
U
/
10
E
2
3
26.5
31.3%
17.7%
13.6%
̅
̅
u u
U
/
.10
z
z
E
2
3
21.9
35.0%
18.9%
16.1%
̅
̅
θ
θ
u u
U
/
.10
E
2
3
17.1
71.0 %
31.5%
39.4%
̅
̅
u u U
/
.10
r
r
E
2
3
13.9
34.7%
15.5%
19.3%
Fig. 11. Average air velocity for decreasing averaging times. β
d b
i
b ilt i
li
t
I
l
l
it it
Table 3
Range for the measurement error in the revolution when the direction complies
with the limit angle of attack δ
<
°
45
th
90
, λ
>
°
30
th
90
under calibrated mode. Absolute
∆and relative error ε (see nomenclature). 5.2. Correction for the actual anemometer orientation (7) and sets the
positive direction of a3 with the sense of rotation of the flow,
where S reads 1 or 1 for an anticlockwise or clockwise vortex. The first and second terms in Eq. (8) account for the alignment of
the head with a first gyration over the frame axis a2 in G1, and a
second in G2 over the perpendicular to the anemometer plane
(axis 3 in Fig. 12). In this manner, one axis aligns with z while the
remaining (2 and 3) are contained in the horizontal plane
θ
−
r
. The third term G3 accounts for the door. A gyration over z aligns
both axes in the horizontal plane with r and θ, setting a positive
sense in the radial direction outwards. G0 sets the reference axes in the raw signal in Eq. (7) and sets the
positive direction of a3 with the sense of rotation of the flow,
where S reads 1 or 1 for an anticlockwise or clockwise vortex. The first and second terms in Eq. (8) account for the alignment of
the head with a first gyration over the frame axis a2 in G1, and a
second in G2 over the perpendicular to the anemometer plane
(axis 3 in Fig. 12). In this manner, one axis aligns with z while the
remaining (2 and 3) are contained in the horizontal plane
θ
−
r
. The third term G3 accounts for the door. A gyration over z aligns
both axes in the horizontal plane with r and θ, setting a positive
sense in the radial direction outwards. Fig. 12. Position and misalignment of the ensemble in the horizontal plane. in the plane
θ
−
r
, denoted Δy and depicted in Fig. 12. The sub-
sequent offsets in x and θ are denoted Δx and Δθ and computed in
Eqs. (3) and (4). Δ
Δ
β
=
−
−
*·
( )
R
x cos
3
x
y
2
2 Δ
Δ
β
=
−
−
*·
R
x cos
x
y
2
2 ( )
3 ⎛
⎝⎜
⎞
⎠⎟
Δ
Δ
=
( )
θ
−
sin
r
4
y
1 ⎛
⎝⎜
⎞
⎠⎟
Δ
Δ
=
θ
−
sin
r
y
1 5.2. Correction for the actual anemometer orientation ⎡
⎣
⎢
⎢
⎢
⎤
⎦
⎥
⎥
⎥
⎡⎣
⎤⎦
=
′
′
′ ·
( )
u
u
u
W
U
V
G
7
a
a
a
0
1
2
3
⎡
⎣
⎢
⎢
⎤
⎦
⎥
⎥
⎡⎣
⎤⎦
=
·
·
·
( )
θ
u
u
u
u
u
u
G G G
8
z
r
a
a
a
1
2
3
1
2
3
h
f
i
f h
i
li ( )
7 ⎡
⎣
⎢
⎢
⎤
⎦
⎥
⎥
⎡⎣
⎤⎦
=
·
·
·
( )
θ
u
u
u
u
u
u
G G G
8
z
r
a
a
a
1
2
3
1
2
3 ( )
8 where Gi are functions of the instrument alignment: where Gi are functions of the instrument alignment: ⎡
⎣
⎢
⎢
⎤
⎦
⎥
⎥
=
( )
G
S
1 0 0
0 1 0
0 0
9
0
⎡
⎣
⎢
⎢
⎤
⎦
⎥
⎥
α
α
α
α
=
−
(
)
G
cos
sin
sin
cos
0
0
1
0
0
10
1
⎡
⎣
⎢
⎢
⎢
⎤
⎦
⎥
⎥
⎥
β
β
β
β
=
−
(
)
G
cos
sin
sin
cos
0
0
0
0
1
11
2
⎡
⎣
⎢
⎢
⎢
⎤
⎦
⎥
⎥
⎥
γ
γ
γ
γ
=
−
(
)
G
cos
sin
sin
cos
1
0
0
0
0
12
3 ⎡
⎣
⎢
⎢
⎤
⎦
⎥
⎥
=
( )
G
S
1 0 0
0 1 0
0 0
9
0
⎡
⎣
⎢
⎢
⎤
⎦
⎥
⎥
α
α
α
α
=
−
(
)
G
cos
sin
sin
cos
0
0
1
0
0
10
1
⎡
⎣
⎢
⎢
⎢
⎤
⎦
⎥
⎥
⎥
β
β
β
β
=
−
(
)
G
cos
sin
sin
cos
0
0
0
0
1
11
2
⎡
⎣
⎢
⎢
⎢
⎤
⎦
⎥
⎥
⎥
γ
γ
γ
γ
=
−
(
)
G
cos
sin
sin
cos
1
0
0
0
0
12
3 ( )
9 (
)
10 (
)
11 (
)
12 G0 sets the reference axes in the raw signal in Eq. 5.2. Correction for the actual anemometer orientation ⎡
⎣
⎢
⎢
⎢
⎤
⎦
⎥
⎥
⎥
⎡⎣
⎤⎦
=
′
′
′ ·
( )
u
u
u
W
U
V
G
7
a
a
a
0
1
2
3
⎡
⎣
⎢
⎢
⎤
⎦
⎥
⎥
⎡⎣
⎤⎦
=
·
·
·
( )
θ
u
u
u
u
u
u
G G G
8
z
r
a
a
a
1
2
3
1
2
3
where Gi are functions of the instrument alignment:
⎡
⎣
⎢
⎢
⎤
⎦
⎥
⎥
=
( )
G
S
1 0 0
0 1 0
0 0
9
0
⎡
⎣
⎢
⎢
⎤
⎦
⎥
⎥
α
α
α
α
=
−
(
)
G
cos
sin
sin
cos
0
0
1
0
0
10
1
⎡
⎢
⎢
⎤
⎥
⎥
β
β
β
β
=
−
G
cos
sin
sin
cos
0
0
2 ref
not
⎡
⎣
⎢
⎢
⎢
u
u
u
⎡
⎣
⎢
⎢
θ
u
u
u
z
wh
G0
G1
G2 reference axes a
a
a
,
,
1
2
3 into the polar frame of the cylinder, de-
noted
θ
r z
,
,
. reference axes a
a
a
,
,
1
2
3 into the polar frame of the cylinder, de
noted
θ
r z
,
,
. 5.2. Correction for the actual anemometer orientation After positioning the anemometer, the actual alignmentsα and After positioning the anemometer, the actual alignmentsα and V. Francia et al. / Flow Measurement and Instrumentation 50 (2016) 216–228
225 225 Fig. 12. Position and misalignment of the ensemble in the horizontal plane. V. Francia et al. / Flow Measuremen reference axes a
a
a
,
,
1
2
3 into the polar fram
noted
θ
r z
,
,
. ⎡
⎣
⎢
⎢
⎢
⎤
⎦
⎥
⎥
⎥
⎡⎣
⎤⎦
=
′
′
′ ·
u
u
u
W
U
V
G
a
a
a
0
1
2
3
⎡
⎣
⎢
⎢
⎤
⎦
⎥
⎥
⎡⎣
⎤⎦
=
·
·
·
θ
u
u
u
u
u
u
G G G
z
r
a
a
a
1
2
3
1
2
3
where Gi are functions of the instrument al
⎡
⎣
⎢
⎢
⎤
⎦
⎥
⎥
=
G
S
1 0 0
0 1 0
0 0
0
⎡
⎣
⎢
⎢
⎤
⎦
⎥
⎥
α
α
α
α
=
−
G
cos
sin
sin
cos
0
0
1
0
0
1
⎡
⎣
⎢
⎢
⎢
⎤
⎦
⎥
⎥
⎥
β
β
β
β
=
−
G
cos
sin
sin
cos
0
0
0
0
1
2
⎡
⎣
⎢
⎢
⎢
⎤
⎦
⎥
⎥
⎥
γ
γ
γ
γ
=
−
G
cos
sin
sin
cos
1
0
0
0
0
3
G0 sets the reference axes in the raw signal
positive direction of a3 with the sense of
where S reads 1 or 1 for an anticlockwis
Fig. 12. Position and misalignment of the ensemble in the horizontal plane. V. Francia et al. / Flow Measurement and Instrumentation 50 (2016) 216–228 reference axes a
a
a
,
,
1
2
3 into the polar frame of the cylinder, de-
noted
θ
r z
,
,
. 7. Conclusions This work introduces the use of sonic anemometry to study
industrial confined swirling flows. It does not develop a new
technique, but from an applied perspective looks into methods
typical of meteorological, geomorphologic and aeronautics re-
search to discuss the particularities of using these instruments in
industrial devices. Further research would be required to extend
the use of these instruments to other flow structures, particularly
when significant velocity gradients develop in the measurement
volume. In this paper, a useful guide has been given for engineers
studying large cyclones or dryers, along with restrictions, appro-
priate signal corrections and an estimation of conservative range
for the error. The main source is associated to the disruption
caused by the instrument and has been linked to its geometry and
orientation. The maximum range of measurement error when
using a HS50 under a turbulence level characteristic of swirl dryers
and within recommended angles of attack, δ
<
°
45
th
90
and λ
>
°
30
th
90
,
was found <
−
1
4% in
̅U, <
−
°
1
3 in direction and <
−
7
31% in κ,
which is considered sufficient for this type of engineering appli-
cations where alternatives are very limited. Certain regions are
excluded from the analysis (a) the core, <
r
R
0.10 , (b) the boundary
layer,
>
r
R
0.98
and (c) regions nearby the contraction,
>
z
H
0.95 . Other limitations arising from time averaging, calibration or the
use of extensions have also been discussed. 5.3. Disruption of the development of the vortex In this
way, measurements collected when the arrangement crosses the
centre provide a rough indication of velocity but should not be
used to study variability. 5.3. Disruption of the development of the vortex ( )
4 Whenever
Δ ≠0
y
, a gyration angle
γ is required to align the
auxiliary axes 2 and 3 in Fig. 12 with the true r and θ. Depending
upon the relative position of the anemometer versus the centre-
line, γ reads Swirling flows oscillate and can be easily distorted. Given this
method is intrusive in nature, one must ensure that the presence
of the instrument does not affect the overall development of the
pressure field, which is an obvious assumption in meteorology or
natural swirling flows, but not in a confinement. It has been no-
ticed that the presence of the anemometer in the central region
(i.e. jet-like region in Fig. 1) distorts the turbulence field. Fig. 13
shows examples where an extension was used to collect data (see
Fig. 3b). Measurements taken when the anemometer reaches a
position without crossing the centre are denoted with solid sym-
bols (the anemometer penetrates a length
<
x
R at a given θ) and
compared to measurements where the same position is reached
from the opposite side of the walls and so the arrangement crosses
the centre, denoted with open symbols (i.e. the extension and
frame penetrate a length
−
>
D
x
R at
θ+
°
180 ). A clear radial
profile is obtained in velocity and turbulence regardless of the use
or not of an extension as long as the instrument does not cross the
centre; otherwise a bias appears on the turbulence statistics, see ⎪
⎪⎧
⎨
⎩
γ
Δ
Δ
π
Δ
Δ
=
>
−
<
( )
θ
θ
for
for
0
0
5
x
x ( )
5 The position in cylindrical coordinates
θ
r z
,
,
is expressed in Eq. (6) as function of the position of the door θ* and
*
z and the dis-
tance of the head into the cylinder
*
x . ⎡
⎣
⎢
⎢
⎤
⎦
⎥
⎥
⎡
⎣
⎢
⎢
⎢
⎢
⎤
⎦
⎥
⎥
⎥
⎥
θ
Δ
Δ
β
θ
γ
=
+
* +
*·
* +
( )
r
z
z
x sin
6
x
y
2
2 ( )
6 The following transformations can be used to express the ve-
locity data measured along the spar axes
′
W ,
′
′
U V
,
and the V. Francia et al. 5.3. Disruption of the development of the vortex / Flow Measurement and Instrumentation 50 (2016) 216–228 226 Fig. 13. Disruption to the vortex when using extensions. Open and solid symbols
show respectively positions where the arrangement crosses or not the centre of the
chamber. (a)
̅U (b) κ, σθθ, σrr and σ θ
r . Scale I tower [32], Scale II tower [33]. Fig. 14. Comparison against laser diagnostics methods, PIV [13]. uption to the vortex when using extensions. Open and solid symbols i
1
C
i
i
l
di
i
h d
[13] Fig. 14. Comparison against laser diagnostics methods, PIV [13]. Fig. 13. Disruption to the vortex when using extensions. Open and solid symbols
show respectively positions where the arrangement crosses or not the centre of the
chamber. (a)
̅U (b) κ, σθθ, σrr and σ θ
r . Scale I tower [32], Scale II tower [33]. Fig. 13. Disruption to the vortex when using extensions. Open and solid symbols
show respectively positions where the arrangement crosses or not the centre of the
chamber. (a)
̅U (b) κ, σθθ, σrr and σ θ
r . Scale I tower [32], Scale II tower [33]. Fig. 14. Comparison against laser diagnostics methods, PIV [13]. the potential of the method and demonstrate the importance of
having alternatives for full scale measurement. In addition, despite
the limitations discussed, the measurement provided sufficient
resolution to assess the impact of friction on the turbulence [33]
and the frequency at which the core precesses [32]. bottom Fig. 13b. This phenomenon may be related to the preces-
sion of the vortex core, PVC, that is common in cyclones [65] and
dryers [32,33]. It seems reasonable to believe that when the frame
is allowed to destabilise the core it breaks the oscillation and
disrupts the transport of turbulence in the horizontal plane. In this
way, measurements collected when the arrangement crosses the
centre provide a rough indication of velocity but should not be
used to study variability. bottom Fig. 13b. This phenomenon may be related to the preces-
sion of the vortex core, PVC, that is common in cyclones [65] and
dryers [32,33]. It seems reasonable to believe that when the frame
is allowed to destabilise the core it breaks the oscillation and
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13 show some examples of the velocity fields in the towers named
Scale I and II (Table 1). The vortex structure divided between the
core and annular regions was found characteristic of a Rankine
pattern in good agreement with literature [12,15,29]. Fig 14 in-
cludes a direct comparison between the tangential velocity
θ
U
collected at two axial levels with PIV and the corresponding sonic
anemometry data [32] in comparable conditions. The agreement
with PIV is evident and has important consequences. The work of
Hassall [13] was the first to indicate that
θ
U in a production unit
was substantially lower than expected from LDA data collected in
the same type of dryers at laboratory scale and with smooth walls
[31]. Hassall could not describe the full structure of the flow nor
analyse the reasons behind this because PIV could only reach a
very limited area of the chamber. Access to the full geometry is
now possible with anemometry and confirms the attenuation of
the swirl. It also demonstrates that it is dependent on the rough-
ness of the walls [32] and the type and coverage of the deposits
formed in manufacture [33]. Velocity and turbulence data col-
lected with sonic anemometers in a cleaned tower were success-
fully replicated with numerical models including roughness and
friction [34]. This example and the agreement with PIV confirm In this way one could map very large geometries, far beyond
the possibilities of optical techniques. Data are collected with
simple modifications and in affordable way ( ~ min
5
of shutdown
time per location) but still retain a great level of detail. Consider-
ing that it should be compared with alternative techniques under V. Francia et al. / Flow Measurement and Instrumentation 50 (2016) 216–228 227 the same operational challenges and conditions (i.e. volume, op-
eration regime and time), the method proposed represents a
useful asset to improve the frequency of validation for large scale
computational models in the particle technology industry. It is
worth stressing that in the case of swirl dryers, it provided data in
agreement with the only source of data available with PIV and
allowed identification of features exclusively linked to production
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(2001) 1585–1608. [61] S. Franchini, A. Sanchez-Andres, A. Cuerva, Measurement of velocity in rota-
tional flows using ultrasonic anemometry: the flowmeter, Exp. Fluids 42
(2007) 903–911. [55] Gash JHC, A.J. Dolman, Sonic anemometer (co)sine response and flux mea-
surement I. The potential for (co)sine error to affect sonic anemometer-based
flux measurements, Agric. For. Meteorol. 119 (2003) 195–207. [62] A. Cuerva, A. Sanchez-Andres, O. Lopez, Singularities and undefinitions in the
calibration functions of sonic anemometers, J. Atmos. Ocean. Technol. 21
(2004) 1868–1875. [56] M.K. Van der Molen, J.H.C. Gash, J.A. Elbers, Sonic anemometer (co)sine re-
sponse and flux measurement II. The effect of introducing an angle of attack
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tunnel calibrations compared to atmospheric in situ calibrations against a
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[64] T. Nakai, K. Shimoyama, Ultrasonic anemometer angle of attack errors under
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English
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Multi-walled carbon nanotube-based composite materials as catalyst support for water–gas shift and hydroformylation reactions
|
RSC advances
| 2,019
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cc-by
| 8,379
|
Patrick Wolf,‡a Morten Logemann,‡b Markus Sch¨orner,a Laura Keller,bc
Marco Haumann
*a and Matthias Wessling
bc Patrick Wolf,‡a Morten Logemann,‡b Markus Sch¨orner,a Laura Keller,bc
Marco Haumann
*a and Matthias Wessling
bc In times of depleting fossil fuel reserves, optimizing industrial catalytic reactions has become increasingly
important. One possibility for optimization is the use of homogenous catalysts, which are advantageous
over heterogeneous catalysts because of mild reaction conditions as well as higher selectivity and
activity. A new emerging technology, supported ionic liquid phase (SILP), was developed to permanently
immobilize homogeneous catalyst complexes for continuous processes. However, these SILP catalysts
are unable to form freestanding supports by themselves. This study presents a new method to introduce
the SILP system into a support made from multi-walled carbon nanotubes (MWCNT). In a first step, SILP
catalysts were prepared for hydroformylation as well as low-temperature water–gas shift (WGS)
reactions. These catalysts were integrated into freestanding microtubes formed from MWCNTs, with
silica (for hydroformylation) or alumina particles (for WGS) incorporated. In hydroformylation, the activity
increased significantly by around 400% when the pure MWCNT material was used as SILP support. An
opposite trend was observed for WGS, where pure alumina particles exhibited the highest activity. A
significant advantage of the MWCNT composite materials is the possibility to coat them with separation
layers, which allows their application in membrane reactors for more efficient processes. Received 26th June 2019
Accepted 26th August 2019
DOI: 10.1039/c9ra04830h
rsc.li/rsc-advances Thus, the dissolved homogeneous catalyst does not evaporate
into the gaseous reactant stream and is retained inside the xed
SILP catalyst bed. The concept has been developed within the
past 15 years in academia and industry, including the success-
ful proof-of-concept for ultra-low temperature water–gas shi
reaction6 (in cooperation with Clariant and Linde) and gas-
phase hydroformylation7 (in cooperation with Evonik) by
some of the authors. Both reactions are of high industrial
importance. The water–gas shi(WGS) reaction, meaning the
exothermic conversion of carbon monoxide and water to carbon
dioxide and hydrogen, still plays an essential role in the tech-
nical hydrogen production nowadays. aLehrstuhl f¨ur Chemische Reaktionstechnik (CRT), Friedrich-Alexander-Universit¨at
Erlangen-N¨urnberg (FAU), Egerlandstr. 3, 91058 Erlangen, Germany. E-mail: marco.
haumann@fau.de
bChemical Process Engineering, RWTH Aachen University, Forckenbeckstr. 51, 52074
Aachen, Germany
cDWI – Leibniz Institute for Interactive Materials, Forckenbeckstr. 50, 52074 Aachen,
Germany
† Electronic
supplementary
information
(ESI)
available.
See
DOI:
10.1039/c9ra04830h
‡ Both authors contributed equally to the paper. Patrick Wolf,‡a Morten Logemann,‡b Markus Sch¨orner,a Laura Keller,bc
Marco Haumann
*a and Matthias Wessling
bc Carbon monoxide, which
is formed during hydrocarbon steam reforming and can act as
severe catalyst poison, e.g., for ammonia synthesis or fuel cell
(FC) catalysts, can be reduced to low levels below 0.5 vol%.8 Due
to the exothermic nature of the WGS equilibrium reaction, low
temperatures around 150 C and below are desirable to achieve
CO levels below 10 ppm.9 Commercial heterogeneous catalysts
require reaction temperatures around 200 C and yield a CO
level of 0.3 vol%. Such concentrations are still too high for e.g. the Pt catalyst in FC systems or the Fe catalyst in the Haber–
Bosch process.10,11 In 2010, a Ru-SILP catalyst was developed by
Werner et al. that allows operation between 120 C to 160 C,
thereby outperforming state of the art commercial catalysts.6,12
The SILP catalyst is made of a rutheniumchlorocarbonyl dimer
dissolved in the ionic liquid 1-butyl-2,3-dimethylimidazolium This journal is © The Royal Society of Chemistry 2019 RSC Advances RSC Advances Cite this: RSC Adv., 2019, 9, 27732
Received 26th June 2019
Accepted 26th August 2019
DOI: 10.1039/c9ra04830h
rsc.li/rsc-advances
PAPER
ublished on 03 September 2019. Downloaded by evonikindustries on 10/16/2019 5:15:40 AM. s article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Open Access Article. Published on 03 September 2019. Downloaded by evonikindustries on 10/16/2019 5:15:40 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Open Access Article. Published on 03 September 2019. Downloaded by evonikindustries on 10/16/2019 5:15:40 AM.
This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Promising SILP catalysts have also been developed that allow
hydroformylation of highly diluted or mixed feeds with excep-
tionally high selectivity, adding value to industrial waste
streams.13,14 For the rst time, the applicability of sophisticated
but labile phosphite ligands (see bpp in Fig. 1) in hydro-
formylation has been reported with catalyst stabilities exceeding
800
h
time-on-stream
with
cumulative
turn-over-numbers
exceeding 400.000 and high space-time-yields of 850 kgproduct
mSILP
1 h1.13 These values are high enough to make new
hydroformylation processes based on SILP materials economi-
cally viable. A possible drawback is the undesired aldol conden-
sation, in which two aldehyde molecules can react to form side
products with high boiling points that accumulate in the ionic
liquid lm.15,16 This side reaction has to be suppressed in order to
maintain high catalyst activity. One option is to remove the
formed aldehydes immediately from the SILP catalyst in
a membrane type reactor. Therefore, we aim at developing SILP
based membrane reactors for both WGS and hydroformylation
reactions. While the WGS will benet from product removal due
to the shiin equilibrium conversion, the yield of the desired
aldehyde in hydroformylation will benet from in situ removal
due to less aldol condensation (see Scheme 1). In this work, the fabrication procedure is altered to include
small-scale particles within the MWCNT network, which
enhance catalytic activity in a SILP reaction system. The ability
to tune microtubes via the addition of small particles was rst
reported by Keller et al.20 This particle inclusion offers possi-
bilities to adjust porosity and pore volume for a proper catalyst
distribution in the microtube. Previous experiments indicated
that silica (SiO2) is the support material of choice for the
hydroformylation reaction, while alumina (Al2O3) particles work
well in the water gas shireaction.12,21 Such small-scale particles are unable to act as a freestanding
membrane support material on their own. However, integrated
into a MWCNT network, the MWCNTs combined with small
particles build a freestanding catalytic support, which is
tunable to different reaction systems, depending on various
catalysts and reaction kinetics. In order to evaluate the appli-
cability of these microtubes as support for SILP catalyzed
reactions, we analyzed the performance of short MWCNT tube
fragments as a structured packing for both reactions mentioned
above. We included these particles into the microtube structure
with various particle ratios for a better understanding of the
reaction performances. This journal is © The Royal Society of Chemistry 2019 Introduction Homogeneous catalysts offer several advantages over their
heterogeneous counterparts: especially mild reaction condi-
tions, higher activity and better selectivity.1 In light of depleting
fossil fuel reserves, the use of such catalysts is highly recom-
mended.2 However, recycling of homogeneous catalysts from
the product stream constitutes a signicant drawback for
technical implementation.3 To simplify the recycling of liquid
catalysts, the supported ionic liquid phase (SILP) technology
was developed.4 SILP technology is a fundamental, new
approach to achieve the ambitious goal of effectively and
permanently immobilizing homogeneous catalyst complexes
for continuous processes.5 SILP technology involves surface
modication of a porous solid by dispersing a thin lm of non-
volatile ionic liquid on the inner surface of the support material. This journal is © The Royal Society of Chemistry 2019 27732 | RSC Adv., 2019, 9, 27732–27742 RSC Advances
View Article Online RSC Advances
View Article Online View Article Online Paper chloride [C4C1C1Im]Cl inside porous g-alumina (see Fig. 1). Further improvement of this promising catalyst system could be
better heat management to avoid hot spots and in situ removal
of one of the formed products (CO2 or H2) via membranes in
order to simplify downstream separation. polymer-based spherical activated carbon (PBSAC) can serve as
support for SILP hydroformylation catalysts.17,18 Proper surface
modication can yield SILP catalysts with TOF of 700 molpentanal
molRh h1 in gas-phase 1-butene hydroformylation.18 Using
another carbonaceous material, in 2014 Gendel et al. demon-
strated the fabrication of freestanding hollow bers made only
from multi-walled carbon nanotubes (MWCNT).19 Experimental microtubes with integrated particles in the MWCNT wall
structure. For the hydroformylation case study, 500 mg of
various particle combinations of MWCNT and SiO2 were sus-
pended in 500 mL deionized water. The tested fractions were 0,
30, 50, and 70 wt% SiO2 and are listed in Table S1.† For the case
study of the WGS reaction, alumina oxide particles from either
Sasol or Pengda were mixed with MWCNT and suspended in
500 mL deionized water. The combination of Al2O3 particles
and MWCNT added up to 500 mg. The tested fractions were 0,
10, 30 and 50 wt% Al2O3 and are listed in Table S2.† Further-
more, all three materials, MWCNT, SiO2 and Al2O3 were tested
in their powder form for reference values. All
gases
were
purchased
from
Linde
Gas. For
hydro-
formylation, 1-butene (99.5 vol%), CO (99.95 vol%) and H2
(99.9990 vol%) were used with He (99.9990 vol%) as balance. Catalytic WGS tests were conducted using a mixture of CO
(99.95 vol%) and H2O diluted with N2 (99.9990 vol%). Multiwalled carbon nanotubes were purchased from Sigma-
Aldrich. The MWCNT's have an outside diameter of 10 nm
1 nm, an inside diameter of 4.5 nm 0.5 nm and are 3–6 mm
long. Their carbon content is $98%. Silica particles SYLOID® C
803 with an average diameter of 3.4–4 mm were provided from
Grace. Two different alumina oxide particles were used in this
study. Puralox TM 100/150 UF with an average diameter of 2.5
mm were kindly donated from Sasol Germany GmbH. Gamma-
alumina (g-Al2O3) powder with a particle size of approx. 5 mm
was purchased from Pengda Munich GmbH. Polypropylene (PP)
S6/2 hollow ber membranes for ltration of MWCNT/MWCNT
+ particle suspension were ordered from Accurel (1.8 0.15 mm
inside diameter). Further chemicals used were Triton-X 100
(Carl Roth), iso-propanol (Applichem, 98% purity) and deion-
ized water (2.2 mS cm1). The fabrication process of the freestanding tubes was the
same for both SiO2 and Al2O3 particles integration. To ensure
well-dispersed particles within the suspensions, 5 mL of Triton-X
100 was added as a surfactant. Each suspension was sonicated for
three hours (Hielscher UP200S) and subsequently stirred with
a magnetic stirrer for an hour. Experimental In consecutive steps, each
suspension was ltered in an inside out ltration through the
polypropylene (PP) S6/2 hollow ber membrane, which had been
cut to a length of 250 mm and glued shut on one end. Filtration
occurred with a constant pressure of 5 bar until 3.4 mLsuspension
cmPP-membrane
1 were transported through the membrane. A
scheme of the experimental set-up is given in Fig. 2. Open Access Article. Published on 03 September 2019. Downloaded by evonikindustries on 10/16/2019 5:15:40 AM.
This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Scheme 1
Reaction schemes of SILP catalyzed water–gas shift (WGS) and hydroformylation (HyFo) with attempted in situ removal of one
product. R ¼ alkyl chain. This journal is © The Royal Society of Chemistry 2019 Open Access Article. Published on 03 September 2019. Downloaded by evonikindustries on 10/16/2019 5:15:40 AM.
This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. A material suitable for both membrane formation and SILP
impregnation has to be identied. Recently, we have shown that Fig. 1
Building blocks for water gas shift (WGS) and hydroformylation (HyFo) catalysis using SILP materials. Hydroformylation ligand biphephos
bpp (6,60-((3,30-di-tert-butyl-5,50-dimethoxy-[1,10-biphenyl]-2,20-diyl)bis(oxy))didibenzo[d,f][1,3,2]dioxaphosphepine). Fig. 1
Building blocks for water gas shift (WGS) and hydroformylation (HyFo) catalysis using SILP materials. Hydroformylation ligand biphephos
bpp (6,60-((3,30-di-tert-butyl-5,50-dimethoxy-[1,10-biphenyl]-2,20-diyl)bis(oxy))didibenzo[d,f][1,3,2]dioxaphosphepine). This journal is © The Royal Society of Chemistry 2019 RSC Adv., 2019, 9, 27732–27742 | 27733 View Article Online RSC Advances Scheme 1
Reaction schemes of SILP catalyzed water–gas shift (WGS) and hydroformylation (HyFo) with attempted in situ removal of one
product. R ¼ alkyl chain. tember 2019. Downloaded by evonikindustries on 10/16/2019 5:15:40 AM. under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. SILP material characterization High-resolution images were taken using two different scan-
ning electron microscopes (S-3000 and S-4800, Hitachi). The
samples were sputtered with an approximately 3 nm thick gold
layer in a high vacuum sputter coater (EM ACE600, Leica). For
sample preparation, MWCNT microtubes were placed in liquid
nitrogen for 2 minutes and broken immediately aer exiting the
liquid nitrogen bath. The Barrett, Joyner and Halenda (BJH)
method was used to estimate pore size and pore volume from
nitrogen adsorption isotherms.22 The nitrogen adsorption was
conducted at 196 C (ASAP 2020, Micromeritics). Prior to
analysis, all samples were dried in a vacuum oven overnight at
30 mbar and 50 C. Aerward, the samples were degassed at
250 C for four hours at 20 mbar. Thermogravimetric analysis
(TGA STA6000, PerkinElmer) was used to determine the thermal
stability of MWCNT microtubes. wRu ¼
mRu
msupport
100%
(1) (1) The pore lling degree with ionic liquid, dened as ratio
between ionic liquid volume and total pore volume, was set to
aIL ¼ 34 vol% (see eqn (2)). aIL ¼
VIL
Vsupport
100%
(2) (2) Open Access Article. Published on 03 September 2019. Downloaded by evonikindustries on 10/16/2019 5:15:40 AM.
This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Fig. 2
Schematic representation of the microtube production via filtration process. Alfa Aesar) was added, followed by another 15 min of stirring. Subsequently, 0.4 g of the MWCNT-Al2O3 support material
(crushed to approx. 1 mm length) was added and stirred for an
additional 5 min. The stirrer bar was taken out of the ask
before the solvent was removed, to reduce any further crushing
of the particles due to mechanical stress. The solvent with-
drawal was carried out in two steps in a rotary evaporator (step
1: 2 h, 40 C, 900 mbar; step 2: 1 h, 40 C, <3 mbar). Aer solvent
removal, free-owing, shiny black SILP catalyst particles were
obtained when using MWCNT supports, while pure alumina
yielded a yellow SILP powder. The resulting catalyst had
a nominal metal loading of wRu ¼ 2 wt% (see eqn (1)). This cleaning step is necessary because otherwise, the
MWCNT based microtubes exhibit defects.19 These defects
destabilize the microtubes, which would hinder their use as
a support material. Aer removing the surfactant, a drying step
was performed overnight in a vacuum oven at 30 C and 30
mbar. In a nal step, one end of the PP membrane was carefully
cut offwith a scalpel to pull out the formed MWCNT microtube
from the shell. Preparation of MWCNT-based SILP hydroformylation
catalysts Hydroformylation SILP catalysts were handled and prepared
under inert conditions inside a glove box, using standard
Schlenk techniques. For the impregnation of the MWCNT and
MWCNT-SiO2 support material, the ionic liquid tetradecyl-
trihexylphosphonium bis(triuoromethylsulfonyl)imide [P14666]
[NTf2] in dichloromethane was added to the support material
and stirred for 10 min. Next, a mixture of the precursor
Rh(acac)(CO)2 and the biphephos ligand bpp dissolved in DCM
was added and stirred for additional 20 min. The solvent was This journal is © The Royal Society of Chemistry 2019 Preparation of MWCNT, MWCNT-SiO2 and MWCNT-Al2O3
materials The MWCNT/particle suspension forms a lter cake on the
inside of the PP membrane during ltration. Aer the ltration,
a cleaning step was added, to remove any remaining Triton-X
100 impurities in the lter cake. 50 mL of isopropanol were
ushed through the PP membrane with 0.7 mL min1 using
a syringe pump (Harvard Apparatus, PHD Ultra). A detailed description of the production of MWCNT based
microtubes and possibilities for geometric variations, e.g., wall
thickness and length, can be found elsewhere.19 Within this
work, a focus was placed on the production of MWCNT This journal is © The Royal Society of Chemistry 2019 27734 | RSC Adv., 2019, 9, 27732–27742 RSC Advances
View Article Online RSC Advances
View Article Online Paper Paper Fig. 2
Schematic representation of the microtube production via filtration process. p
nloaded by evonikindustries on 10/16/2019 5:15:40 AM. Commons Attribution-NonCommercial 3.0 Unported Licence. Catalytic tests in hydroformylation The continuous gas-phase hydroformylation experiments were
conducted at 100 C and 11 bar pressure using a xed bed
reactor set-up (for details see Fig. S5†).24 The syngas ows (33H2
mLN min1, 33 CO mLN min1) were regulated by a mass ow
controller (Bronkhorst). 1-Butene (0.067 mL min1) was dosed
by a minipump (Smartline Pump 100 by Knauer) equipped with
a 10 mL pump head. An additional inert gas ow of helium was
applied by a mass ow controller (Bronkhorst) during the start
of the reaction (decreased in steps of 25 mLN min1 from 100 to
0 mLN min1). The gaseous substrate stream was contacted with
the SILP catalyst xed bed (0.124 g SILP + 0.9 g inert Silica 60,
63–200 mm) in a tubular reactor with a residence time of 7
seconds. An on-line gas chromatograph (Bruker 450 GC)
monitored the product feed stream with two FID detectors (CP-
Wax 52CB, 25 m length, 0.53 mm inner diameter, 2 mm coating
for C5–C10 separation; CP-Porabond Q, 25 m length, 0.53 mm
inner diameter for C4 separation) and one TCD detector
(permanent gases). Field emission scanning electron microscopy (FESEM)
allows to evaluate the particle distribution within the microtube
wall. The thickness of the microtube wall depends on the
ltration time and ltration ux, being in the range of 150–350
microns. Fig. 3d shows the catalyst-free microtube surface
structure of pure MWCNTs. Even though there are no particles
embedded into the network of MWCNTs, it seems as if some
small clusters form beneath the surface. These clusters consist
of agglomerated MWCNTs, and they can form dense structures,
which also affect the homogeneity in porosity of the MWCNT
network as explained in the next section. The number of clus-
ters depends much on the preparation and the corresponding
homogeneity of the solution to be ltered. In comparison to the pure MWCNT microtube from Fig. 3(d),
the integration of small particles considerably changes the
structure of the microtube. Fig. 4 is an example of a surface and
a cross-section view of a 50 wt% MWCNT/50 wt% SiO2 micro-
tube. The small SiO2 particles are evenly distributed within the
MWCNT network. The same effect is observed for Al2O3 parti-
cles as well, as can be seen in Fig. S1.† The SILP hydroformylation activity is expressed by means of
the turnover frequency TOF (in h1) which is calculated
according to eqn (3). ed on 03 September 2019. Downloaded by evonikindustries on 10/16/2019 5:15:40 AM.
le is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. The molar ow of substrate is known from the calibrated
MFC and the degree of conversion is calculated based on the CO
signal from the gas analyzer. Catalytic tests in WGS A continuously operated tubular reactor was used that has been
reported previously.23 The ow scheme of the test rig is given in
the ESI (Fig. S4†). For a typical experiment, 0.4 g of the prepared
SILP catalyst was placed in the tubular reactor on a glass wool
covered frit to keep the xed bed in place. Following a pressure
test, the reactor was heated to the reference temperature of
120 C under a nitrogen atmosphere. Once the temperature was
reached, the rig was switched into bypass mode, and the
streams of CO, N2 and H2O were adjusted with a partial pressure
ratio of pN2 : pCO : pH2O ¼ 7 : 1 : 2 and a total pressure of ptot ¼ 1
bar. When the IR-Analyzer (X-STREAM Enhanced XEGP, Emer-
son) showed stable values, the data logger was started to take
reference values for 15 minutes (one data point per 30 seconds)
before switching into reactor mode. The temperature of the
tubular reactor was varied at constant volume ows between
120 C and 140 C in steps of 5 C. Every set point is kept for 5
hours in which steady-state conditions were reached. MWCNT microtube fabrication Producing freestanding MWCNT based microtubes is possible
with the production procedure developed by Gendel et al.19 As
can be seen in Fig. 3(a–c), the microtubes produced in this work
are self-supporting, long and thin hollow bers. Measurements
show that all bers are above 200 mm in length and have an
outside diameter of around 1.7 mm. The outside diameter of
the microtube is slightly smaller than the inside diameter of the
PP ltration membrane because the microtube shrinks during
the drying step. Open Access Article. Published on 03 September 2019. Downloaded by evonikindustries on 10
This article is licensed under a Creative Commons Attribution-NonComme First, we present the results from the MWCNT microtube
fabrication and analysis, as well as the preparation of composite
MWCNT-SiO2 and MWCNT-Al2O3 materials. These materials
were tested in WGS and hydroformylation, which constitutes
the second part of this chapter. Preparation of MWCNT-based SILP WGS catalysts Water gas shiSILP catalyst were prepared using standard
Schlenk techniques. In a rst step, the ionic liquid 1-butyl-2,3-
dimethylimidazolium chloride [C4C1C1Im]Cl was dissolved in
dichloromethane (typically 30 mL DCM) in a round-bottom
Schlenk ask under argon atmosphere. Aer stirring the solu-
tion till complete dissolution of the ionic liquid (10–15 min at
25 C), the precursor complex [Ru(CO)3Cl2]2 (purchased from This journal is © The Royal Society of Chemistry 2019 RSC Adv., 2019, 9, 27732–27742 | 27735 View Article Online View Article Online RSC Advances Paper Paper Paper the online IR-analyzer. Here, the TOF has to be modied
according to eqn (4). the online IR-analyzer. Here, the TOF has to be modied
according to eqn (4). then slowly removed under reduced pressure (step 1: 2 h, 40 C,
900 mbar; step 2: 1 h, 40 C, <3 mbar) to facilitate the homo-
geneous coating of the support material, resulting in the
average, adjusted degree of pore lling. The resulting SILP
materials contained a rhodium loading of wRh ¼ 0.2 wt%,
a ligand/Rh ratio of 4 and an ionic liquid loading of aIL ¼
10 vol%. TOF ¼ n
substrate X
ncatalyst
(4) (4) This journal is © The Royal Society of Chemistry 2019 Catalytic tests in hydroformylation Particle loading has a strong inuence on microtube
stability. Mechanically
stable,
freestanding
MWCNT-SiO2
microtubes can be formed up to a particle loading of 70 wt% of
SiO2 particles. For MWCNT-Al2O3 microtubes, it was not
possible to increase the particle loading above 50 wt% without
breaking the bers during production. For particle loadings
larger than 70 wt% in SiO2 rich microtubes or 50 wt% in Al2O3
rich microtubes, the structures became unstable and some TOF ¼ n
product
ncatalyst
(3) TOF ¼ n
product
ncatalyst
(3) (3) For SILP WGS the molar ow of product cannot be deter-
mined in high accuracy due to a change of the total ow caused
by the condensation of water before the dry gas ows through This journal is © The Royal Society of Chemistry 2019 27736 | RSC Adv., 2019, 9, 27732–27742 RSC Advances
View Article Online RSC Advances
View Article Online Fig. 3
(a) Photo of freestanding microtubes, (b) photo of three microtubes next to a coin for size reference, (c) cross-section SEM picture of
a MWCNT microtube made from 50 wt% MWCNT and 50 wt% SiO2, (d) FESEM image of the outer surface of a pure MWCNT microtube. Lighter
grey spots show clustered MWCNTs. FESEM images were adjusted in contrast and brightness. Open Access Article. Published on 03 September 2019. Downloaded by evonikindustries on 10/16/2019 5:15:40 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Fig. 3
(a) Photo of freestanding microtubes, (b) photo of three microtubes next to a coin for size reference, (c) cross-section SEM picture of
a MWCNT microtube made from 50 wt% MWCNT and 50 wt% SiO2, (d) FESEM image of the outer surface of a pure MWCNT microtube. Lighter
grey spots show clustered MWCNTs. FESEM images were adjusted in contrast and brightness. samples broke upon removing them from the PP ltrations
membranes. Therefore, all results presented in the following
were obtained with microtube particle loadings between 0–
70 wt% in case of SiO2 rich microtubes and 0–50 wt% for Al2O3
rich microtubes. of the MWCNT interconnection, and thus, the stability of the
microtube can be achieved through crosslinking of amine-
functionalized MWCNT's.25 The crosslinking increased the
mechanical stability of buckypaper by more than two-fold,
according to Zhang et al.,26 and could be tested in follow up
work for MWCNT microtubes. This journal is © The Royal Society of Chemistry 2019 Open Access Article. Published on 03 September 2019. Downloaded by evonikindustries on 10/16/2019 5:15:40 AM.
This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Fig. 5
(a) BJH adsorption pore volume for MWCNT-SiO2 combinations, (b) BJH adsorption pore volume for MWCNT-Al2O3 combinations from
different suppliers. Black filled symbols are powders and white symbols are microtubes. temperature stage. By doing so, Fig. 7 is obtained, which shows
the same experimental run (Pengda, 5 mm particles) as a TOF-
temperature plot. In addition, results from powdered Sasol
support (Puralox TM 100/150 UF, 2.5 mm particles) and granu-
lated alumina (Puralox NWa 155, 200–500 mm particles) are
shown. From these data it becomes evident that the three
systems had a similar activity at each reaction temperature. amount of pore volume with SILP solution. Fig. 5 shows the
Barrett, Joyner and Halenda (BJH) adsorption pore volumes of
different amounts of all three kinds of particles integrated into
MWCNT microtubes, as well as the powder forms. For the SiO2 particles, the pore volume decreases from a pure
MWCNT microtube towards a 50% MWCNT-SiO2 combination. A further increase of SiO2 particles increases the pore volume
again, towards the SiO2 powder pore volume. A possible expla-
nation for this behavior is the entanglement of MWCNTs with
SiO2 particles and, resulting from this interaction, the forming
of new pores. Contrary to the MWCNT-SiO2 combination, the
pore volume increases slightly for both MWCNT-Al2O3 combi-
nations at 10 wt% Al2O3, before dropping continuously with
higher particle loadings. Again, the entanglement of MWCNT's
and particles seems to be a possible reason, which leads to the
forming of new pores. A more detailed explanation can be found
in the ESI including Fig. S2† of the differential analysis of pore
volume depending on particle loading. Next, we investigated the performance of MWCNT-alumina
composite support materials. Different support materials
made from varying fractions of MWCNTs and alumina-types
(purchased from Pengda and Sasol) were examined. Aer
preparation, the MWCNT material was reduced in length into
fragments of approx. 1 mm length each, which were placed into Fig. 6
Catalytic activity (as TOF) as a function of temperature and time
for a SILP catalyst based on pure alumina (Pengda) as support material. Reaction conditions: T ¼ 120–140
C, p ¼ 1 bar, precursor ¼
[Ru(CO)3(Cl)2]2, IL ¼ [C4C1C1Im]Cl, mSILP ¼ 0.4 g, wRu ¼ 2 wt%, aIL ¼
34 vol%, total flow rate ¼ 130.8 mLN min1, H2O : CO ¼ 2 : 1. 27738 | RSC Adv., 2019, 9, 27732–27742 Catalytic tests in hydroformylation The observations regarding MWCNT-microtube stability
match well with the work from Keller et al., who discovered that
bending strength of MWCNT microtubes reduced signicantly
with an increase in particle loading.20 A possible improvement For the SILP impregnation into the porous microtubes, it is
necessary to know the pore volume. As mentioned in the cata-
lyst preparation section, it is sufficient to ll only a certain Fig. 4
(a) FESEM image of the outer surface of a mixed MWCNT-SiO2 microtube. Particle loading of 50 wt% MWCNT/50 wt% SiO2, (b) FESEM
image of the cross-section of a mixed MWCNT-SiO2 microtube. Particle loading of 50 wt% MWCNT/50 wt% SiO2. FESEM images were adjusted in
contrast and brightness. Fig. 4
(a) FESEM image of the outer surface of a mixed MWCNT-SiO2 microtube. Particle loading of 50 wt% MWCNT/50 wt% SiO2, (b) FESEM
image of the cross-section of a mixed MWCNT-SiO2 microtube. Particle loading of 50 wt% MWCNT/50 wt% SiO2. FESEM images were adjusted in
contrast and brightness. RSC Adv., 2019, 9, 27732–27742 | 27737 This journal is © The Royal Society of Chemistry 2019 View Article Online Fig. 5
(a) BJH adsorption pore volume for MWCNT-SiO2 combinations, (b) BJH adsorption pore volume for MWCNT-Al2O3 combinations from
different suppliers. Black filled symbols are powders and white symbols are microtubes. RSC Advances
Paper
nloaded by evonikindustries on 10/16/2019 5:15:40 AM. Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online dvances RSC Advances Continuous hydroformylation In a similar manner as for WGS, we tested the MWCNT-SiO2
composite supports for gas-phase hydroformylation of 1-butene
in a continuous reactor setup. Fig. 9 shows the activity and
selectivity over time on stream for a composite support con-
taining 70 wt% MWCNT and 30 wt% SiO2. For a smoother start-
up, especially to avoid high aldol formation, the reaction
mixture was diluted with helium. The helium ow was reduced
every 80 minutes from 100 to 0 mLN min1 in four steps. Next to
the TOF as a critical characteristic for catalytic activity, the
selectivities for the primary and side products are shown as well
as the n/iso-selectivity within the aldehyde fraction. This selec-
tivity represents the amount of the desired n-pentanal in
comparison to all pentanals formed. Side reactions are the
hydrogenation to butane, the isomerization to cis-2-butene and
trans-2-butene (combined as isomerization selectivity) as well as
the
subsequent
reaction
of
n-pentanal
to
3-hydroxy-2-
propylheptanal which can undergo a condensation reaction to
form 2-propylhept-2-enal (combined as aldol selectivity). the reactor. The prepared systems had alumina contents of
0 (pure MWCNT), 10, 30, 50 and 100 wt%. In Fig. 8 the results
for the composite material based on alumina Pengda are
shown; the behavior for Puralox TM 100/150 UF can be found in
the ESI (Fig. S6†), which is almost identical. All systems showed
a higher activity with higher temperatures following Arrhenius
law without undergoing any deactivation over time. This
temperature dependency leads to the conclusion that the Fig. 8
Catalytic activity (as TOF) as a function of the alumina (Pengda)
content in MWCNT-alumina composite support materials. Reaction
conditions: T ¼ 120 C (filled squares), 130 C (open circles) and 140 C
(filled triangles), p ¼ 1 bar, precursor ¼ [Ru(CO)3(Cl)2]2, IL ¼ [C4C1C1Im]
Cl, mSILP ¼ 0.4 g, wRu ¼ 2 wt%, aIL ¼ 34 vol%, total flow rate ¼ 130.8
mLN min1, H2O : CO ¼ 2 : 1. The SILP catalyst required a long induction period of more
than 13 h to reach a stable performance (the rst two hours were
bypass measurements). The purely silica based SILP catalyst
also reached steady-state performance only aer 15 h (see
Fig. S7†). Interestingly, even aer the reactor temperature had
stabilized, which took approximately 10 hours, the system was
not stable, and the activity kept increasing. Continuous WGS catalysis This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. the re
0 (pur
for th
shown
the ES
a high
law w
tempe
Fig. 7
SILP c
Puralo
155 (C
1 bar,
wRu ¼
H2O :
Open Access Article. Published on 03 September 2019. Downloaded by evonikindustries on 10/16/2019 5:15:40 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. support materials themselves are stable at elevated tempera-
tures under reaction conditions. While all systems showed
a similar increase in activity with rising temperature, the system
with 50 wt% alumina deactivated to a minor degree from 120 to
130 C. Furthermore, a higher alumina content in the prepared
support materials for the catalysts had a positive inuence on
activity. The higher the alumina content, the higher the activity
of the investigated SILP catalyst system. We observe this trend
throughout all three plotted temperature stages. The trend can
be considered as quasi-linear, which is most prominent at
120 C. All discussed trends can be observed in the catalyst
systems using alumina from Sasol as well (see Fig. S6†). While the WGS catalytic activity did not benet from incor-
porating alumina particles in MWCNTs, the tubular geometries
that might be accessible by these new composite materials (e.g.,
hollow bers, tube bundles) can compensate for the effect of
lower activity. On the way to a membrane reactor that would
consist of several hundreds of MWCNT hollow bers, the lower
activity would be compensated by the more effective handling of
SILP catalysts in combination with separation. Fig. 7
Catalytic activity (as TOF) as a function of temperature for three
SILP catalysts based on powdered alumina Pengda (>), powdered
Puralox TM 100/150 UF (B) and granulated 200–500 m Puralox NWa
155 (C) as support material. Reaction conditions: T ¼ 120–140 C, p ¼
1 bar, precursor ¼ [Ru(CO)3(Cl)2]2, IL ¼ [C4C1C1Im]Cl, mSILP ¼ 0.4 g,
wRu ¼ 2 wt%, aIL ¼ 34 vol%, total flow rate ¼ 130.8 mLN min1,
H2O : CO ¼ 2 : 1. This journal is © The Royal Society of Chemistry 2019 Continuous WGS catalysis The basis for the evaluation of the MWCNT-Al2O3 composite
materials in WGS SILP was a temperature variation at constant
feed ows and compositions. A typical plot of results can be
seen in Fig. 6, which summarizes the obtained catalytic values
for a SILP catalyst based on pure alumina (Pengda) support
material. Aer every temperature variation, the temperature was reset
to the reference state of 120 C, to investigate whether the
system undergoes deactivation phenomena. The results show
that the catalyst promotes stable values at all temperature
stages aer a start-up period in the rst 120 C-stage. For the
sake of clarity and better comparability, every temperature
variation experiment is turned into a turnover frequency (TOF)
over temperature plot by averaging the measured data of
a stable operation regime over a time of 30 minutes within every Fig. 6
Catalytic activity (as TOF) as a function of temperature and time
for a SILP catalyst based on pure alumina (Pengda) as support material. Reaction conditions: T ¼ 120–140
C, p ¼ 1 bar, precursor ¼
[Ru(CO)3(Cl)2]2, IL ¼ [C4C1C1Im]Cl, mSILP ¼ 0.4 g, wRu ¼ 2 wt%, aIL ¼
34 vol%, total flow rate ¼ 130.8 mLN min1, H2O : CO ¼ 2 : 1. This journal is © The Royal Society of Chemistry 2019 27738 | RSC Adv., 2019, 9, 27732–27742 RSC Advances
View Article Online Fig. 7
Catalytic activity (as TOF) as a function of temperature for three
SILP catalysts based on powdered alumina Pengda (>), powdered
Puralox TM 100/150 UF (B) and granulated 200–500 m Puralox NWa
155 (C) as support material. Reaction conditions: T ¼ 120–140 C, p ¼
1 bar, precursor ¼ [Ru(CO)3(Cl)2]2, IL ¼ [C4C1C1Im]Cl, mSILP ¼ 0.4 g,
wRu ¼ 2 wt%, aIL ¼ 34 vol%, total flow rate ¼ 130.8 mLN min1,
H2O : CO ¼ 2 : 1. Paper
er 2019. Downloaded by evonikindustries on 10/16/2019 5:15:40 AM. er a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. RSC Advances
View Article Online View Article Online Paper Paper Open Access Article. Published on 03 September 2019. Downloaded by evonikindustries on 10/16/2019 5:15:40 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. t
0
f
s
t
a
l
t
F
S
P
1
1
w
H
Open Access Article. Published on 03 September 2019. Downloaded by evonikindustries on 10/16/2019 5:15:40 AM. Continuous hydroformylation Aer 15 hours, the
MWCNT-SiO2 based SILP catalyst reached a steady state of
approximately
1800
h1
activity,
corresponding
to
4.8%
conversion. The n-pentanal selectivity was very high at 99.2%,
the total selectivity toward aldehydes stabilized at 64.2% and
the isomerization tendency was around 19.4%. Aldol formation
as the most problematic side reaction in gas-phase hydro-
formylation was low during the induction period but increased
to a stable plateau of 13.5%. Such behavior hints at an ongoing Fig. 8
Catalytic activity (as TOF) as a function of the alumina (Pengda)
content in MWCNT-alumina composite support materials. Reaction
conditions: T ¼ 120 C (filled squares), 130 C (open circles) and 140 C
(filled triangles), p ¼ 1 bar, precursor ¼ [Ru(CO)3(Cl)2]2, IL ¼ [C4C1C1Im]
Cl, mSILP ¼ 0.4 g, wRu ¼ 2 wt%, aIL ¼ 34 vol%, total flow rate ¼ 130.8
mLN min1, H2O : CO ¼ 2 : 1. This journal is © The Royal Society of Chemistry 2019 RSC Adv., 2019, 9, 27732–27742 | 27739 View Article Online RSC Advances Fig. 9
Gas-phase 1-butene hydroformylation using MWCNT-SiO2 support (30 wt% SiO2). Rh-SILP activity (top) is given in turnover frequency
TOF (-). Selectivities Si (bottom) indicate amount of formed n-pentanal (>), 1-butene isomerization (O), aldols (B) and n/iso selectivity within
pentanals (,). Reaction conditions: temperature ¼ 100 C, total molar flow prior to reactor 3.75 mmol min1, partial pressures pH2 ¼ 4.75 bar,
pCO ¼ 4.62 bar, p1-butene ¼ 1.95 bar, amount of active metal mRh ¼ 0.45 mg, wRh ¼ 0.2 wt%, amount of SILP-catalyst mSILP ¼ 0.12 mg, amount of
inert silica 60, 70–230 mesh minert ¼ 0.90 mg, aIL ¼ 10 vol%, IL ¼ [P14666][NTf2], dotted line indicates stable reactor temperature. RSC Advances
Paper
Open Access Article. Published on 03 September 2019. Downloaded by evonikindustries on 10/16/2019 5:15:40 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Fig. 9
Gas-phase 1-butene hydroformylation using MWCNT-SiO2 support (30 wt% SiO2). Rh-SILP activity (top) is given in turnover frequency
TOF (-). Selectivities Si (bottom) indicate amount of formed n-pentanal (>), 1-butene isomerization (O), aldols (B) and n/iso selectivity within
pentanals (,). Continuous hydroformylation Reaction conditions: temperature ¼ 100 C, total molar flow prior to reactor 3.75 mmol min1, partial pressures pH2 ¼ 4.75 bar,
CO ¼ 4.62 bar, p1-butene ¼ 1.95 bar, amount of active metal mRh ¼ 0.45 mg, wRh ¼ 0.2 wt%, amount of SILP-catalyst mSILP ¼ 0.12 mg, amount of
nert silica 60, 70–230 mesh minert ¼ 0.90 mg, aIL ¼ 10 vol%, IL ¼ [P14666][NTf2], dotted line indicates stable reactor temperature. g. 9
Gas-phase 1-butene hydroformylation using MWCNT-SiO2 support (30 wt% SiO2). Rh-SILP activity (top) is given in turnover frequency
OF (-). Selectivities Si (bottom) indicate amount of formed n-pentanal (>), 1-butene isomerization (O), aldols (B) and n/iso selectivity within
entanals (,). Reaction conditions: temperature ¼ 100 C, total molar flow prior to reactor 3.75 mmol min1, partial pressures pH2 ¼ 4.75 bar,
CO ¼ 4.62 bar, p1-butene ¼ 1.95 bar, amount of active metal mRh ¼ 0.45 mg, wRh ¼ 0.2 wt%, amount of SILP-catalyst mSILP ¼ 0.12 mg, amount of
ert silica 60, 70–230 mesh minert ¼ 0.90 mg, aIL ¼ 10 vol%, IL ¼ [P14666][NTf2], dotted line indicates stable reactor temperature. Fig. 10
Gas-phase 1-butene hydroformylation using different MWCNT-SiO2 supports with varying silica content. Rh-SILP activity (top) is
given in turnover frequency TOF (-). Selectivities Si (bottom) amount of formed n-pentanal (>), 1-butene isomerization (O), aldols (B) and
n/iso selectivity within pentanals (,). Reaction conditions: temperature ¼ 100 C, total molar flow prior to reactor 3.75 mmol min1, partial
pressures pH2 ¼ 4.75 bar, pCO ¼ 4.62 bar, p1-butene ¼ 1.95 bar, amount of active metal mRh ¼ 0.45 mg (0.27 mg for 70% silica), wRh ¼ 0.2 wt%,
amount of SILP-catalyst mSILP ¼ 0.12 mg (0.077 mg for 70% silica), amount of inert silica 60, 70–230 mesh minert ¼ 0.90 mg, aIL ¼ 10 vol%, IL ¼
[P14666][NTf2]. Fig. 10
Gas-phase 1-butene hydroformylation using different MWCNT-SiO2 supports with varying silica content. Rh-SILP activity (top) is
given in turnover frequency TOF (-). Selectivities Si (bottom) amount of formed n-pentanal (>), 1-butene isomerization (O), aldols (B) and
n/iso selectivity within pentanals (,). Continuous hydroformylation Reaction conditions: temperature ¼ 100 C, total molar flow prior to reactor 3.75 mmol min1, partial pressures pH2 ¼ 4.75 bar,
pCO ¼ 4.62 bar, p1-butene ¼ 1.95 bar, amount of active metal mRh ¼ 0.45 mg, wRh ¼ 0.2 wt%, amount of SILP-catalyst mSILP ¼ 0.12 mg, amount of
inert silica 60, 70–230 mesh minert ¼ 0.90 mg, aIL ¼ 10 vol%, IL ¼ [P14666][NTf2], dotted line indicates stable reactor temperature. RSC Advances
Paper
Article. Published on 03 September 2019. Downloaded by evonikindustries on 10/16/2019 5:15:40 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Open Access Article. Published on 03 September 2019. Downloaded by evonikindustries on 10/16/2019 5:15:40 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Fig. 9
Gas-phase 1-butene hydroformylation using MWCNT-SiO2 support (30 wt% SiO2). Rh-SILP activity (top) is given in turnover frequency
TOF (-). Selectivities Si (bottom) indicate amount of formed n-pentanal (>), 1-butene isomerization (O), aldols (B) and n/iso selectivity within
pentanals (,). Reaction conditions: temperature ¼ 100 C, total molar flow prior to reactor 3.75 mmol min1, partial pressures pH2 ¼ 4.75 bar,
pCO ¼ 4.62 bar, p1-butene ¼ 1.95 bar, amount of active metal mRh ¼ 0.45 mg, wRh ¼ 0.2 wt%, amount of SILP-catalyst mSILP ¼ 0.12 mg, amount of
inert silica 60, 70–230 mesh minert ¼ 0.90 mg, aIL ¼ 10 vol%, IL ¼ [P14666][NTf2], dotted line indicates stable reactor temperature. Fig. 10
Gas-phase 1-butene hydroformylation using different MWCNT-SiO2 supports with varying silica content. Rh-SILP activity (top) is
given in turnover frequency TOF (-). Selectivities Si (bottom) amount of formed n-pentanal (>), 1-butene isomerization (O), aldols (B) and
n/iso selectivity within pentanals (,). Reaction conditions: temperature ¼ 100 C, total molar flow prior to reactor 3.75 mmol min1, partial
pressures pH2 ¼ 4.75 bar, pCO ¼ 4.62 bar, p1-butene ¼ 1.95 bar, amount of active metal mRh ¼ 0.45 mg (0.27 mg for 70% silica), wRh ¼ 0.2 wt%,
amount of SILP-catalyst mSILP ¼ 0.12 mg (0.077 mg for 70% silica), amount of inert silica 60, 70–230 mesh minert ¼ 0.90 mg, aIL ¼ 10 vol%, IL ¼
[P14666][NTf2]. g. 9
Gas-phase 1-butene hydroformylation using MWCNT-SiO2 support (30 wt% SiO2). Rh-SILP activity (top) is given in turnover frequency
OF (-). Selectivities Si (bottom) indicate amount of formed n-pentanal (>), 1-butene isomerization (O), aldols (B) and n/iso selectivity within
entanals (,). Open Access Article. Published on 03 September 2019. Downloaded by evonikindustries on 10/16/2019 5:15:40 AM.
This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. When comparing the MWCNT-SiO2 composite materials
with pure MWCNT and SiO2, it becomes obvious that the SiO2
lowers the overall activity compared to pure MWCNT. The pure
silica-based SILP catalyst had a TOF of 625 h1, while the pure
MWCNT based SILP was 400% more active around 2440 h1. The activity of composite materials having different ratios
between MWCNT and SiO2 gradually declined with increasing
SiO2 content. The selectivity pattern did not reveal a clear trend,
with all MWCNT-SiO2 based catalysts yielding n/iso-selectivity
(96 4%), moderate toward n-pentanal (55 7%), and isom-
erization (19 2%). The aldol content varied signicantly
between the different catalysts, being 22 9% on average. Especially higher amounts of silica content show an increased
aldol and therefore decreased n-pentanal formation. We explain
this trend with the higher amount of silanol groups on the
surface, which enhance the acid/base-catalyzed aldol formation. Calcining the silica at an elevated temperature of 600 C for
several hours reduces the number of surface silanol groups
signicantly.5a Due to a combustion temperature in the range of
400–500 C of the MWCNT microtubes, calcining at 600 C was
not possible. The combustion temperature of MWCNT micro-
tubes in air was analyzed with TGA measurements (see Fig. S3†). Acknowledgements The authors gratefully acknowledge nancial support from the
European Commission within the Horizon2020-SPIRE project
ROMEO (grant agreement number 680395). Furthermore, the
authors want to thank Heike Zander, for her support in the
MWCNT-microtube production. References 1 Applied
Homogeneous
Catalysis
with
Organometallic
Compounds, ed. B. Cornils, W. A. Herrmann, M. Beller and
R. Pacielo, Wiley-VCH, Weinheim, 2017. This journal is © The Royal Society of Chemistry 2019 Continuous hydroformylation Reaction conditions: temperature ¼ 100 C, total molar flow prior to reactor 3.75 mmol min1, partial
pressures pH2 ¼ 4.75 bar, pCO ¼ 4.62 bar, p1-butene ¼ 1.95 bar, amount of active metal mRh ¼ 0.45 mg (0.27 mg for 70% silica), wRh ¼ 0.2 wt%,
amount of SILP-catalyst mSILP ¼ 0.12 mg (0.077 mg for 70% silica), amount of inert silica 60, 70–230 mesh minert ¼ 0.90 mg, aIL ¼ 10 vol%, IL ¼
[P14666][NTf2]. This journal is © The Royal Society of Chemistry 2019 27740 | RSC Adv., 2019, 9, 27732–27742 RSC Advances
View Article Online RSC Advances
View Article Online View Article Online RSC Ad
View Article Online Paper process of restructuring of the ionic liquid lm due to changes
in the wettability when substrates and products dissolve.16
Especially the change in aldol content in the effluent stream of
the reactor is a strong indication for the restructuring process. To compare different MWCNT-SiO2 compositions, we calcu-
lated the average values of the steady state for all selectivities, as
well as the turnover frequency (see Fig. 10). Since the sample
consisting of pure MWCNT material showed a steady deactiva-
tion throughout the observed period (see Fig. S8†), the initial
TOF values were considered. stability is low if the materials are not adequately calcined,
which is difficult, because of thermal instability of MWCNTs
above 600 C. WGS and hydroformylation of 1-butene are two promising
case studies, which are part of the ROMEO project, targeting the
development of SILP membrane reactor systems.27 The tech-
nology to combine SILP and tunable MWCNT based microtubes
could be expanded and used for other chemical reactions. Hydroformylation of smaller olens (e.g. propene) is feasible,
while for higher olens (e.g. hexenes or octenes) the pore
condensation inside the monolithic structure can hamper the
overall efficiency. Next, we aim to apply molecular selective
membranes on the microtube surface: a self-supporting reac-
tion system combined with a membrane to separate products in
situ would be a signicant step towards process intensication. Conclusion 2 12 Design Principles of Green Chemistry, ACS Green Chemistry
Institute, http://www.acs.org/greenchemistry, accessed May
2019. Improving large-scale chemical reactions to reduce emissions
and costs is a signicant challenge of today's society. On the one
hand, catalyst immobilization via SILP technology offers an
opportunity to combine the advantages of homogeneous and
heterogeneous catalytic reactions. On the other hand, tunable
MWCNT based microtubes can serve as freestanding support
materials for catalytic reactions and structure the inside of
a reactor. This can lead to avoiding high pressure drops or
packing problems, as they occur, e.g., in packed bed reactors as
well as to better control of mass and heat transport inside the
reactor. In this work, the SILP platform was integrated into
a new tubular geometry based on MWCNT. The ability to tune
microtubes with porous particles, which are known for their
good SILP applicability, is advantageous for incorporating the
SILP system into structured supports and reactors. 3 Multiphase Homogeneous Catalysis, ed. B. Cornils, W. A. Herrmann, I. T. Horv´ath, W. Leitner, S. Mecking, H. Olivier-Bourbigou and D. Vogt, Wiley-VCH, Weinheim, 2005. 4 Supported Ionic Liquids – Fundamentals and Applications, ed. R. Fehrmann, A. Riisager and M. Haumann, Wiley-VCH,
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the WGS reaction is lower for Al2O3 particle integrated micro-
tubes compared to pure Al2O3 powder. Nonetheless, the
combined support shows stable and selective behavior. The
catalytic
activity
of
MWCNT
microtubes
for
the
hydro-
formylation reaction is very high for pure MWCNT microtubes
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The COVID-19 Pandemic and Responses in Nursing Homes: A Cross-Sectional Study in Four European Countries
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To cite this version: Fabrice Mbalayen, Valentine Dutheillet-De-Lamothe, Aude Letty, Solenn Le Bruchec, Manon Pond-
jikli, et al.. The COVID-19 Pandemic and Responses in Nursing Homes: A Cross-Sectional Study in
Four European Countries. International Journal of Environmental Research and Public Health, 2022,
19 (22), pp.15290. 10.3390/ijerph192215290. hal-03888942 Distributed under a Creative Commons Attribution 4.0 International License HAL Id: hal-03888942
https://hal.science/hal-03888942v1
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abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License International Journal of
Environmental Research
and Public Health International Journal of
Environmental Research
and Public Health *
Correspondence: sylvain.gautier@aphp.fr Citation: Mbalayen, F.;
Dutheillet-de-Lamothe, V.; Letty, A.;
Le Bruchec, S.; Pondjikli, M.; Berrut,
G.; Benatia, L.; Ndiongue, B.M.;
Fourrier, M.-A.; Armaingaud, D.;
et al. The COVID-19 Pandemic and
Responses in Nursing Homes: A
Cross-Sectional Study in Four
European Countries. Int. J. Environ. Res. Public Health 2022, 19, 15290. https://doi.org/10.3390/
ijerph192215290
Academic Editors: Paul B. Tchounwou and Florian Fischer
Received: 4 October 2022
Accepted: 14 November 2022
Published: 19 November 2022 Citation: Mbalayen, F.;
Dutheillet-de-Lamothe, V.; Letty, A.;
Le Bruchec, S.; Pondjikli, M.; Berrut,
G.; Benatia, L.; Ndiongue, B.M.;
Fourrier, M.-A.; Armaingaud, D.;
et al. The COVID-19 Pandemic and
Responses in Nursing Homes: A
Cross-Sectional Study in Four
European Countries. Int. J. Environ. Res. Public Health 2022, 19, 15290. https://doi.org/10.3390/
ijerph192215290 Abstract: Background: Studies comparing how the European nursing homes (NHs) handled the first
wave of the COVID-19 pandemic remain scarce. Methods: A cross-sectional study was conducted
during the first wave in a private NHs network in Belgium, France, Germany and Italy. Mortality
rates were estimated, and prevention and control measures were described by country. Data from the
Oxford governmental response tracker project were used to elaborate a “modified stringency index”
measuring the magnitude of the COVID-19 global response. Results: Of the 580 NHs surveyed,
383 responded to the online questionnaire. The COVID-19 mortality rate was similar in France
(3.9 deaths per 100 residents) and Belgium (4.5). It was almost four times higher in Italy (11.9) and
particularly low in Germany (0.3). Prevention and control measures were diversely implemented:
residents’ sectorization was mainly carried out in France and Italy (~90% versus ~30% in Germany
and Belgium). The “modified stringency index” followed roughly the same pattern in each country. Conclusion: This study, conducted in a European network of NHs, showed differences in mortality
rate which could be explained by the characteristics of the residents, the magnitude of the first wave
and the prevention and control measures implemented. These results may inform future European
preparedness plans. Keywords: COVID-19; mortality; nursing home; elderly; first wave Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations. Article
The COVID-19 Pandemic and Responses in Nursing Homes: A
Cross-Sectional Study in Four European Countries Fabrice Mbalayen 1,2, Valentine Dutheillet-de-Lamothe 3
, Aude Letty 4, Solenn Le Bruchec 3, Manon Pondjikli 3,
Gilles Berrut 3,4,5, Lamia Benatia 4, Biné Mariam Ndiongue 4, Marie-Anne Fourrier 4, Didier Armaingaud 4,6,
Loic Josseran 1,2,7, Elisabeth Delarocque-Astagneau 1,2,7 and Sylvain Gautier 1,2,7,* 1
UFR Simone Veil–Santé, Université Versailles Saint-Quentin-en-Yvelines, 1
UFR Simone Veil–Santé, Université Versailles Saint-Quentin-en-Yvelines,
78180 Montigny-le-Bretonneux, France 1
UFR Simone Veil–Santé, Université Versailles Saint-Quentin-en-Yvelines,
78180 Montigny-le-Bretonneux, France 78180 Montigny-le-Bretonneux, France 2
Département Hospitalier d’Épidémiologie et de Santé Publique, Hôpital Raymond-Poincaré, Groupe
Hospitalier Universitaire Université Paris-Saclay, Assistance Publique-Hôpitaux de Paris,
92380 Garches, France 3
Gérontopôle Autonomie et Longévité des Pays de la Loire, 44000 Nantes, France 4
Fondation Korian pour le Bien-Vieillir, 75000 Paris, France
5 4
Fondation Korian pour le Bien-Vieillir, 75000 Paris, France
5 5
Pôle Hospitalo-Universitaire de Gérontologie Clinique, Centre Hospitalier Universitaire de Nantes,
44000 Nantes, France 6
Direction Médical Éthique et Qualité, Groupe Korian, 75000 Paris, France édical Éthique et Qualité, Groupe Korian, 75000 Paris, France 7
Centre de Recherche en Épidémiologie et Santé des Populations, UMR 1018, Université de Versailles-Saint-Quentin-en-Yvelines, Université Paris-Saclay, 75180 Montigny-Le-Bretonneux, France
*
Correspondence: sylvain.gautier@aphp.fr Versailles-Saint-Quentin-en-Yvelines, Université Paris-Saclay, 75180 Montigny-Le-Bretonneux, France
*
Correspondence: sylvain gautier@aphp fr Int. J. Environ. Res. Public Health 2022, 19, 15290. https://doi.org/10.3390/ijerph192215290 Keywords: COVID-19; mortality; nursing home; elderly; first wave 1. Introduction The first wave of the COVID-19 epidemic reached western European countries at the
beginning of the year 2020. Italy, France and Belgium were quickly and strongly hit, while
other countries such as Germany were less affected. However, their overall first wave
epidemic profiles are comparable [1], due to a similar demography of the aging population. This is all the more important since elderly people represented the main at-risk population
for developing severe COVID-19. Transmission risk was also deemed higher among elderly
people living in nursing homes [2]. Estimates suggest that nursing home (NH) residents
may account for 50% or more of COVID-19 deaths in Europe. However, there were some
disparities between western European countries: from 38% in Germany to 66% in Spain
and 50% in Belgium and in France [3]. 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/). https://www.mdpi.com/journal/ijerph Int. J. Environ. Res. Public Health 2022, 19, 15290. https://doi.org/10.3390/ijerph192215290 2 of 8 Int. J. Environ. Res. Public Health 2022, 19, 15290 These disparities may reflect differences in NH characteristics and residents’ profiles,
as some studies provided evidence on their respective role in the burden of COVID-19
death in NH [4,5]. In addition, general prevention and control measures were the only way
to mitigate the COVID-19 impact during the first wave, in the absence of a cure or a vaccine. These measures were issued both at a national level through the different policies decided
by the public health authorities and implemented at the NH level [6]. In addition, previous
viral pandemics (e.g., H1N1) led to national preparedness plans upon which the NHs also
built their own response to the epidemic. They also benefited from their experience gained
during previous seasonal viral epidemics. Thus, measures taken in NHs may have slightly
differed between countries. Studies comparing how the NHs handled all the dimensions of COVID-19 epidemic
in their premises, between European countries, remain scarce. In this context, the European
Korian network of private NHs represented an opportunity to investigate the burden of
the pandemic and the measures implemented, in a comparative way. 2. Methods This observational cross-sectional study was conducted during the first wave of the
epidemic in a European private NHs network in four countries: Belgium, France, Germany
and Italy, composed of 73 NHs, 290 NHs, 177 NHs and 40 NHs, respectively, corresponding
to a total of 51,356 residents. 1. Introduction Thus, the aim of this
study was to describe, on the one hand, the impact of the COVID-19 pandemic in NHs in
terms of mortality and hospitalization and, on the other hand, the responses in a network
of NHs of four European countries. 2.1. Data Collected Data were collected between 14 September 2020 and 27 October 2020 from an online
questionnaire covering the period from 1 February 2020 to 31 July 2020. It was sent to
each NH of the network in the above-mentioned countries and translated from French to
the corresponding language. Information collected focused on the main organizational
and structural characteristics of each NH (number of beds, surface, presence of specific
units such as “Alzheimer unit” . . . ), on the characteristics of the residents (aggregated
data on mean age, dependency level . . . ) and details on the management of the COVID-19
epidemic (hospitalizations requests, prevention and control measures taken . . . ) as well
as COVID-19 death numbers. The dependency level of the residents was assessed using a
different scale in each country: the Katz scale in Belgium [7], the GIR scale in France [8], the
“Begutachtungs-instrument” in Germany [9] and the Barthel index in Italy [10]. COVID-19
deaths were defined as deaths occurring during the first epidemic wave, confirmed by
a PCR-test or suspected by a physician. Among the prevention and control measures
taken by the NHs, information on visitor bans, residents’ containment, sectorization,
implementation of dedicated COVID-19 units, staff and residents’ screening, training in
hygiene practices were collected. The different definitions of the prevention and control
measures implemented are detailed in Supplementary Table S1. 2.3. Statistical Analysis The statistical unit of the study was the NH. Descriptive statistics were performed
to quantify the implementation of the different prevention and control measures for the
epidemic, as well as mortality and hospitalization related to COVID-19 within the “NHs”
for each country of the study. Numbers and proportions were used to describe qualitative
variables and sum or mean (with standard deviation) for quantitative variables. Categorical
and quantitative variables were compared using chi squared tests and one-way ANOVA,
respectively. A p value of less than 0.05 was regarded as statistically significant. The
COVID-19 mortality rate and 95% confidence interval were estimated for each country as
the number of reported COVID-19 deaths over the number of residents. All statistics were
conducted using the free software R, version 3.6.2. 2.2. Other Sources of Data In order to contextualize potential differences between the countries studied, we used
data from the Oxford governmental response tracker project [11] lead by Thomas Hale
et al. In this project, the researchers computed 23 indicators reflecting the daily level of the
different policy responses during the COVID-19 pandemic in 180 countries. From these
indicators, they developed several indices, such as the “stringency index”, which measures
the level of the response at a given time in a given country. This original “stringency index”
is calculated from the nine following response indicators: (1) school closures; (2) workplace
closures; (3) cancellation of public events; (4) restrictions on public gatherings; (5) closures
of public transport; (6) stay-at-home requirements; (7) public information campaigns and
(8) restrictions on internal movements and (9) international travel controls. The “stringency Int. J. Environ. Res. Public Health 2022, 19, 15290 3 of 8 index”, on any given day, is calculated as the mean score of these nine indicators, each
taking a value between 0 and 100, a higher score indicating a stricter response. g
g
g
p
Here, we computed a “modified stringency index”. In order to consider an index more
suited to the context of our study, we added the specific “protection of elderly people”
indicator (H8) and the “testing policy” indicator (H2). Moreover, we did not include
the “stay at home requirements” indicator (C6) and the “public information campaign”
indicator (H1) as we considered them not sufficiently discriminant between the countries
studied. The calculation of this “modified stringency index” was based on the same
calculation process developed by the Oxford team [11]. Finally, we obtained a daily
quantitative bounded variable between 0 to 100 for Belgium, France, Germany and Italy. This index recorded the strictness of government policies over the period from 1 February
to 31 July 2020. The time period considered corresponded to the period in which we
collected the mortality data in the participating NHs. We presented weekly data using the
last available value of a given week. 1 The dependency level of the residents was assessed using the Katz scale in Belgium, the GIR scale in France, the
“Begutachtungs-instrument” in Germany and the Barthel index in Italy. 2 Reporting a COVID-19 cluster close to
the nursing home: 5, missing data for Germany. 3 N.A.: not available. 4 (95% CI): 95% confidence interval. 3. Results Of the 580 NHs of the network surveyed, 383 (66.0%) responded to the online ques-
tionnaire. Participation was 100%, 61.7%, 66.2% and 48.0% in Italy, Belgium, France and
Germany, respectively. Resident mean age was higher in France (88 ± 1.8), lower in Ger-
many (80 ± 6.1) and quite similar in Belgium and Italy (85 ± 3.9 and 84 ± 4.0, respectively)
(Table 1). Resident mean age was significantly different between countries (p < 0.01). The
proportion of dependent residents was higher in Germany (80.0%) and Italy (70.1%) and
lower in Belgium (49.4%) and France (53.7%) (p < 0.01). Table 1. Main characteristics of nursing homes (NHs) surveyed, mortality rates and hospitalization
for COVID-19. CluDe Study, 2020. Table 1. Main characteristics of nursing homes (NHs) surveyed, mortality rates and hospitalization
for COVID-19. CluDe Study, 2020. Belgium
N = 66
France
N = 192
Germany
N = 85
Italy
N = 40
Total number of residents
6422
15,307
8399
4412
Dependent residents 1, n (%)
3173 (49.4)
8227 (53.7)
6723 (80.0)
3091 (70.1)
Partially dependent residents, n (%)
2559 (39.8)
5679 (37.1)
1658 (19.7)
841 (19.1)
Autonomous residents, n (%)
690 (10.7)
841 (5.5)
50 (0.6)
480 (10.9)
Residents’ mean age, mean ± SD
85 ± 3.9
88 ± 1.8
80 ± 6.1
84 ± 4.0
Reporting a COVID-19 cluster close to the NH, n (%)
16 (24.2)
75 (39.0)
27 (33.8) 2
26 (65.0)
Number of NHs with at least one COVID-19 case
44 (66.7)
125 (65.1)
15 (17.6)
22 (55.0)
Number of hospitalization requests for COVID-19 cases
Accepted hospitalization requests, n (%)
224
189 (84.4)
603
416 (68.5)
30
30 (100.0)
N.A. 3
N.A. COVID-19 mortality rate: number of deaths for 100
residents, mean [95% CI] 4
4.5 (2.9–6.2)
3.9 (2.9–4.9)
0.3 (0.05–0.6)
11.9 (8.1–15.9)
1 The dependency level of the residents was assessed using the Katz scale in Belgium, the GIR scale in France, the
“Begutachtungs-instrument” in Germany and the Barthel index in Italy. 2 Reporting a COVID-19 cluster close to
the nursing home: 5, missing data for Germany. 3 N.A.: not available. 4 (95% CI): 95% confidence interval. 1 The dependency level of the residents was assessed using the Katz scale in Belgium, the GIR scale in France, the
“Begutachtungs-instrument” in Germany and the Barthel index in Italy. 2 Reporting a COVID-19 cluster close to
the nursing home: 5, missing data for Germany. 3 N.A.: not available. 3. Results n (%)
Belgium
N = 66
France
N = 192
Germany
N = 85
Italy
N = 40
Visitor ban
66 (100.0)
192 (100.0)
81 (95.3)
40 (100.0)
Residents’ containment 1
66 (100.0)
189 (99.4) 2
15 (18.1) 2
23 (57.5)
Residents’ sectorization 1
25 (37.9)
177 (92.1)
29 (34.5) 3
38 (95.0)
Dedicated COVID-19 unit 1
29 (70.7)
104 (92.0)
9 (60.0)
19 (90.5)
Day staff dedicated to this unit
25
98
8
17
Night staff dedicated to this unit
13
80
8
10
Support by an external hygiene team
62 (93.9)
123 (64.0)
43 (50.6)
19 (47.5)
Standard precautions’ training in the previous 2 years
62 (93.9)
187 (97.3)
80 (94.1)
39 (97.5)
Audit of practices
62 (93.9)
141 (73.4)
79 (94.0) 4
40 (100.0)
Residents’ screening 5
62 (93.9)
179 (93.2)
18 (22.5) 6
34 (85.0)
Staff’ screening
63 (95.5)
184 (95.8)
24 (29.3) 7
33 (82.5)
1 Residents’ containment was defined as confining the residents to their room. Residents’ sectorization was
defined as partitioning areas/spaces of the nursing home premises with clear restrictions on who can access it. A
dedicated COVID-19 unit is an area where COVID-19 positive tested patients are taken care of. COVID-19 unit
was implemented when there was at least one confirmed case of COVID-19 in the nursing home. The number of
COVID-19 units was only reported on NHs where at least 1 case of COVID-19 occurred: 41, 113, 15 and 21 nursing
homes reported at least 1 case of COVID-19 in Belgium, France, Germany and Italy, respectively. 2 Residents’
containment variable: 2 missing data points for France and Germany, respectively. 3 Residents’ sectorization
variable: 1 missing data point for Germany. 4 Audit of practices: 1 missing data point for Germany. 5 In Germany,
a screening was only carried out by the NH among staff members and residents if there were suspicious cases. It
concerned 125 NHs. 6 Residents’ screening: 5 missing data points for Germany. 7 Staff screening: 3 missing data
points for Germany. Containment and Closures Policies Indicators
Overall, the start of implementation of these policies varied between countries, with The proportion of NHs reporting a COVID-19 cluster close to their premises var-
ied from 65.0% in Italy to 24.2% in Belgium. 3. Results The proportion of accepted hospitalization
requests among NHs who reported at least one COVID-19 case in France was lower
(68.5%) than in Germany (100.0%) and Belgium (84.4%) (p < 0.01). The COVID-19 mor-
tality rate as number of COVID-19 deaths per 100 residents was similar in France (3.9,
95% CI = (2.9–4.9)) and Belgium (4.5, 95% CI = (2.9–6.2)), whereas it was almost four times
higher in Italy (11.9, 95% CI = (8.1–15.9)). In Germany, the mortality rate was particularly
low (0.3, 95% CI = (0.05–0.6)). In almost all the NHs of the four countries, visitors were banned from accessing the
nursing home premises (Table 2). Residents were confined in their room in 100%, 99.4%,
18.1% and 57.5% of the nursing homes in Belgium, France, Germany and Italy, respectively. Residents’ sectorization was implemented in circa one-third of nursing homes in Belgium
and Germany and reached 90% or more in France and Italy. In addition, implementation of
COVID-19 units in the nursing homes was heterogeneous between the four countries and
did not always include a dedicated day/night staff. In each country, more than 90% of the
facilities had conducted standard precautions’ training in the previous 2 years. An external
hygiene team was in support in at least half of the NHs. In the same way, more than 90%
(except France at 73.4%) of the NHs reported that an audit of practices had been carried out. Systematic screenings were performed among residents and staff in circa 90% in Belgium,
France and Italy. In Germany, it was carried out in less than one-third of the NHs. Table 2. Characteristics of prevention and control measures implemented in the nursing homes
surveyed. CluDe Study, 2020. Table 2. Characteristics of prevention and control measures implemented in the nursing homes
surveyed. CluDe Study, 2020. 3. Results In the same way, Italy started with
a high level of “protection of elderly people” (“extensive restrictions for isolation and
hygiene in long term care facilities, all non-essential external visitors prohibited”) followed
by Germany 1 week after with an increase in two phases, by France 2 weeks after, and then,
by Belgium 3 weeks after. In addition, the level of the measures varied: regarding “school 3. Results n (%)
Belgium
N = 66
France
N = 192
Germany
N = 85
Italy
N = 40
Visitor ban
66 (100.0)
192 (100.0)
81 (95.3)
40 (100.0)
Residents’ containment 1
66 (100.0)
189 (99.4) 2
15 (18.1) 2
23 (57.5)
Residents’ sectorization 1
25 (37.9)
177 (92.1)
29 (34.5) 3
38 (95.0)
Dedicated COVID-19 unit 1
29 (70.7)
104 (92.0)
9 (60.0)
19 (90.5)
Day staff dedicated to this unit
25
98
8
17
Night staff dedicated to this unit
13
80
8
10
Support by an external hygiene team
62 (93.9)
123 (64.0)
43 (50.6)
19 (47.5)
Standard precautions’ training in the previous 2 years
62 (93.9)
187 (97.3)
80 (94.1)
39 (97.5)
Audit of practices
62 (93.9)
141 (73.4)
79 (94.0) 4
40 (100.0)
Residents’ screening 5
62 (93.9)
179 (93.2)
18 (22.5) 6
34 (85.0)
Staff’ screening
63 (95.5)
184 (95.8)
24 (29.3) 7
33 (82.5)
1 Residents’ containment was defined as confining the residents to their room. Residents’ sectorization was
defined as partitioning areas/spaces of the nursing home premises with clear restrictions on who can access it. A
dedicated COVID-19 unit is an area where COVID-19 positive tested patients are taken care of. COVID-19 unit
was implemented when there was at least one confirmed case of COVID-19 in the nursing home. The number of
COVID-19 units was only reported on NHs where at least 1 case of COVID-19 occurred: 41, 113, 15 and 21 nursing
homes reported at least 1 case of COVID-19 in Belgium, France, Germany and Italy, respectively. 2 Residents’
containment variable: 2 missing data points for France and Germany, respectively. 3 Residents’ sectorization
variable: 1 missing data point for Germany. 4 Audit of practices: 1 missing data point for Germany. 5 In Germany,
a screening was only carried out by the NH among staff members and residents if there were suspicious cases. It
concerned 125 NHs. 6 Residents’ screening: 5 missing data points for Germany. 7 Staff screening: 3 missing data
points for Germany. Table 2. Characteristics of prevention and control measures implemented in the nursing homes
surveyed. CluDe Study, 2020. 3. Results n (%)
Belgium
N = 66
France
N = 192
Germany
N = 85
Italy
N = 40
Visitor ban
66 (100.0)
192 (100.0)
81 (95.3)
40 (100.0)
Residents’ containment 1
66 (100.0)
189 (99.4) 2
15 (18.1) 2
23 (57.5)
Residents’ sectorization 1
25 (37.9)
177 (92.1)
29 (34.5) 3
38 (95.0)
Dedicated COVID-19 unit 1
29 (70.7)
104 (92.0)
9 (60.0)
19 (90.5)
Day staff dedicated to this unit
25
98
8
17
Night staff dedicated to this unit
13
80
8
10
Support by an external hygiene team
62 (93.9)
123 (64.0)
43 (50.6)
19 (47.5)
Standard precautions’ training in the previous 2 years
62 (93.9)
187 (97.3)
80 (94.1)
39 (97.5)
Audit of practices
62 (93.9)
141 (73.4)
79 (94.0) 4
40 (100.0)
Residents’ screening 5
62 (93.9)
179 (93.2)
18 (22.5) 6
34 (85.0)
Staff’ screening
63 (95.5)
184 (95.8)
24 (29.3) 7
33 (82.5)
1 Residents’ containment was defined as confining the residents to their room. Residents’ sectorization was
defined as partitioning areas/spaces of the nursing home premises with clear restrictions on who can access it. A
dedicated COVID-19 unit is an area where COVID-19 positive tested patients are taken care of. COVID-19 unit
was implemented when there was at least one confirmed case of COVID-19 in the nursing home. The number of
COVID-19 units was only reported on NHs where at least 1 case of COVID-19 occurred: 41, 113, 15 and 21 nursing
homes reported at least 1 case of COVID-19 in Belgium, France, Germany and Italy, respectively. 2 Residents’
containment variable: 2 missing data points for France and Germany, respectively. 3 Residents’ sectorization
variable: 1 missing data point for Germany. 4 Audit of practices: 1 missing data point for Germany. 5 In Germany,
a screening was only carried out by the NH among staff members and residents if there were suspicious cases. It
concerned 125 NHs. 6 Residents’ screening: 5 missing data points for Germany. 7 Staff screening: 3 missing data
points for Germany. Containment and Closures Policies Indicators
Overall, the start of implementation of these policies varied between countries, with
Italy always starting sooner (Figure 1). For instance, “workplace closing” took place at
week 7 of the year 2020 in Italy whereas it took place 3 weeks later for Belgium (week 10)
and 4 weeks later for France and Germany (week 11). 3. Results 4 (95% CI): 95% confidence interval. 4 of 8 Int. J. Environ. Res. Public Health 2022, 19, 15290 The proportion of NHs reporting a COVID-19 cluster close to their premises var-
ied from 65.0% in Italy to 24.2% in Belgium. The proportion of accepted hospitalization
requests among NHs who reported at least one COVID-19 case in France was lower
(68.5%) than in Germany (100.0%) and Belgium (84.4%) (p < 0.01). The COVID-19 mor-
tality rate as number of COVID-19 deaths per 100 residents was similar in France (3.9,
95% CI = (2.9–4.9)) and Belgium (4.5, 95% CI = (2.9–6.2)), whereas it was almost four times
higher in Italy (11.9, 95% CI = (8.1–15.9)). In Germany, the mortality rate was particularly
low (0.3, 95% CI = (0.05–0.6)). In almost all the NHs of the four countries, visitors were banned from accessing the
nursing home premises (Table 2). Residents were confined in their room in 100%, 99.4%,
18.1% and 57.5% of the nursing homes in Belgium, France, Germany and Italy, respectively. Residents’ sectorization was implemented in circa one-third of nursing homes in Belgium
and Germany and reached 90% or more in France and Italy. In addition, implementation of
COVID-19 units in the nursing homes was heterogeneous between the four countries and
did not always include a dedicated day/night staff. In each country, more than 90% of the
facilities had conducted standard precautions’ training in the previous 2 years. An external
hygiene team was in support in at least half of the NHs. In the same way, more than 90%
(except France at 73.4%) of the NHs reported that an audit of practices had been carried out. Systematic screenings were performed among residents and staff in circa 90% in Belgium,
France and Italy. In Germany, it was carried out in less than one-third of the NHs. Table 2. Characteristics of prevention and control measures implemented in the nursing homes
surveyed. CluDe Study, 2020. Containment and Closures Policies Indicators Overall, the start of implementation of these policies varied between countries, with
Italy always starting sooner (Figure 1). For instance, “workplace closing” took place at
week 7 of the year 2020 in Italy whereas it took place 3 weeks later for Belgium (week 10)
and 4 weeks later for France and Germany (week 11). In the same way, Italy started with
a high level of “protection of elderly people” (“extensive restrictions for isolation and
hygiene in long term care facilities, all non-essential external visitors prohibited”) followed
by Germany 1 week after with an increase in two phases, by France 2 weeks after, and then,
by Belgium 3 weeks after. In addition, the level of the measures varied: regarding “school Int. J. Environ. Res. Public Health 2022, 19, 15290 5 of 8 closure” indicator, France and Italy implemented the measure at the highest level (“require
closing all school degrees”) whereas Germany and Belgium started at the medium level
(“only some school degrees”). Regarding the “protection of elderly people”, the duration
differed between countries: the measure remained in place at a medium level (“narrow
restrictions for isolation, hygiene in long term care facilities”) during more than 10 weeks
with variations between countries. Resulting from the combination of these indicators, the
“modified stringency index” followed roughly a same pattern, with a prompt increase to
the highest index value (between 70 and 80%) at different weeks and then a stabilization
around an index value estimated at 60% for the four countries until 31 July 2020. W Figure 1. Containment and closure policies indicators and “Modified stringency index” in Belgium
France, Germany, Italy: trends between 1 February and 31 July 2020. * School closure: (0) none
(1) recommend closing; (2) require closing only some levels or categories; (3) require closing all
levels. Workplace closing: (0) none; (1) recommend closing; (2) require closing only some sectors
or categories of workers; (3) require closing for all but essential. Testing policy: (0) none; (1) only
those who both have symptoms meet specific criteria; (2) testing of anyone showing COVID-19
symptoms; (3) open public testing. Cancel public events: (0) none; (1) recommend cancelling; (2
require cancelling. Close public transport: (0) none; (1) recommend closing; (2) require closing
Restrictions on internal movement: (0) none; (1) recommend not to travel between regions or cities
(2) internal movement restrictions in place. 4. Discussion This study, conducted in a large private European network of NHs, showed differ-
ences in mortality rate across the participating countries, with a higher mortality in Italy,
comparable figures in France and Belgium and a much lower mortality in Germany. Resi-
dents’ characteristics also differed: in French NHs, residents were older and less dependent,
when in German NHs, they were younger and more dependent. In addition, prevention
and control measures were largely reported, with disparities between countries on their
completeness of coverage. p
g
The COVID-19 mortality in NHs is expected to be in relation with the magnitude
of the epidemic, the timeliness and stringency of the prevention and control measures
issued at the national level and, at a local level, the measures implemented in the NH
and the characteristics of the premises and the residents. In particular, there are several
hypotheses to explain the higher COVID-19 mortality rate observed in the French NHs
surveyed compared to German ones. First, strategies in detecting COVID-19 cases differed,
with a more systematic approach in France than in Germany. Indeed, in the latter, it was
recommended to test residents and staff only in the occurrence of a suspected case. Second,
the definition of a COVID-19 death varied between the two countries, with a more sensitive
definition used in French NHs [12]. Third, the magnitude of the epidemic was higher in
France, thus increasing the risk of introduction of the virus in NH [13,14]. In the NHs, face
masks were available later in France. A contrario, Germany had built up a stockpile of
protective masks and was implementing a policy of banning the export of its protective
equipment [15]. Finally, mean age of residents in the German NHs was lower, with a
difference of about 8 years, thus decreasing the risk of death. In addition, the proportion
of accepted hospitalization requests may also have affected the mortality in NHs: this
proportion was lower in France than in Germany. However, the number of requests in
France largely exceeded the number of requests in Germany. This raises the question of
the capacity of the NHs to take care of COVID-19 patients, which could differ between
the countries. Indeed, in Germany, it has been shown that the majority of NH managers
reported no deficits in general practitioners’ [16]. In this study, details on the measures put in place in the NHs were collected. Containment and Closures Policies Indicators International travel controls: (0) none; (1) screening
arrivals; (2) quarantine arrivals from some oral regions; (3) ban arrivals from some regions; (4) ban
on all regions or total border closure. Restrictions on gatherings: (0) none; (1) restrictions on very
large gatherings (the limit is above 1000 people); (2) restrictions on gatherings between 101 and
1000 people; (3) restrictions on gatherings between 11 and 100 people; (4) restrictions on gatherings Figure 1. Containment and closure policies indicators and “Modified stringency index” in Belgium,
France, Germany, Italy: trends between 1 February and 31 July 2020. * School closure: (0) none;
(1) recommend closing; (2) require closing only some levels or categories; (3) require closing all
levels. Workplace closing: (0) none; (1) recommend closing; (2) require closing only some sectors
or categories of workers; (3) require closing for all but essential. Testing policy: (0) none; (1) only
those who both have symptoms meet specific criteria; (2) testing of anyone showing COVID-19
symptoms; (3) open public testing. Cancel public events: (0) none; (1) recommend cancelling; (2)
require cancelling. Close public transport: (0) none; (1) recommend closing; (2) require closing. Restrictions on internal movement: (0) none; (1) recommend not to travel between regions or cities;
(2) internal movement restrictions in place. International travel controls: (0) none; (1) screening
arrivals; (2) quarantine arrivals from some oral regions; (3) ban arrivals from some regions; (4) ban
on all regions or total border closure. Restrictions on gatherings: (0) none; (1) restrictions on very
large gatherings (the limit is above 1000 people); (2) restrictions on gatherings between 101 and
1000 people; (3) restrictions on gatherings between 11 and 100 people; (4) restrictions on gatherings Int. J. Environ. Res. Public Health 2022, 19, 15290 6 of 8 of 10 people or less. Protection of elderly: (0) none; (1) recommended isolation, hygiene and visitor
restriction measures in long term care facilities and/or elderly people to stay at home; (2) narrow
restrictions for isolation, hygiene in long term care facilities; (3) extensive restrictions for isolation
and hygiene in long term care facilities, all non-essential external visitors prohibited. Modified
stringency index: composite measure based on nine response indicators rescaled to a value from 0 to
100 (100 = strictest). This index records the strictness of government policies over the period from
1 February to 31 July 2020. 4. Discussion First of
all, in order to limit the risk of introduction of the virus in the premises, visits were banned
in almost all the surveyed NHs from the four countries. To decrease the risk of transmission
inside the NH, residents’ containment measure was widely implemented, whether or not
there was already a case of COVID-19 detected in the premises. However, in Germany and
Italy, this measure was less reported. In Italy, where the majority of residents were hosted
in double rooms, the containment measure could not be possible. Thus, sectorization was
implemented. In Germany, this measure was poorly reported, partly because of a lower
incidence of COVID-19 in this country [14]. In the NHs where at least one COVID-19 case
occurred, NHs set up a COVID-19 unit with dedicated staff. Regarding hygiene practices,
standard precautions’ training and audit of practices were widely carried out. Nevertheless,
the support by an external hygiene team was less reported, while it allows the advice issued
to be updated in a rapidly changing context. All these measures of prevention and control of the epidemic must be put in parallel
with the temporality of the series of measures issued at the national level of each coun-
try, in relation with the specific epidemic context. All the data collected by the Oxford
governmental response tracker project make it possible to appreciate the timing and the Int. J. Environ. Res. Public Health 2022, 19, 15290 7 of 8 magnitude of the policy responses (such as “school closure”, “restriction on gatherings”,
“cancel public events”, “restriction on movement”, “protection of elderly people” . . . ) and
to compare them between the countries considered. The “protection of elderly people”
indicator completes the information on the types of specific measures implemented in the
NHs. Indeed, it shows that the timing was slightly different between the countries as well
as the duration of this measure. In addition, the “modified stringency index”, calculated as
a combination of these indicators, provides an overview of the measures. Despite an earlier
and a more stringent response observed in Italy, the mortality in the surveyed NHs was the
highest. This likely reflects the fact that Italy was the first European country affected by
the epidemic. p
This study has several limits. 4. Discussion Due to its cross-sectional design, we only provided
cumulative mortality estimates and the timing of the prevention and control measures was
not available, which makes it not possible to establish a relation between the mortality
and the measures. Information bias is possible due to the retrospective nature of the data
collection. In addition, different dependency scales were used. The study only concerned
a network of private NHs which are not representative of all the NHs in the countries
considered. In addition, although all the NHs belonging to the network in Italy participated
in the study, the number is limited. However, the COVID-19 mortality estimates and the
magnitude of the difference between France and Germany are comparable to the figures
published by the ECDC Public Health Emergency Team [3]. References 1. COVID-19 Excess Mortality Collaborators. Estimating excess mortality due to the COVID-19 pandemic: A systematic analysis of
COVID-19-related mortality, 2020-21. Lancet 2022, 399, 1513–1536. [CrossRef] y
2. Smith, D.R.M.; Duval, A.; Pouwels, K.; Guillemot, D.; Fernandes, J.; Huynh, B.-T.; Temime, L. COVID-19 surveillance in long-term care facilities: A modelling study. BMC Med. 2020, 18, 386. [C 2. Smith, D.R.M.; Duval, A.; Pouwels, K.; Guillemot, D.; Fernandes, J.; Huynh, B.-T.; Temime, L.; Opatowski, L. Optimizing
COVID-19 surveillance in long-term care facilities: A modelling study. BMC Med. 2020, 18, 386. [CrossRef] [PubMed]
3
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kli
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L 3. ECDC Public Health Emergency Team; Danis, K.; Fonteneau, L.; Georges, S.; Daniau, C.; Bernard-Stoecklin, S.; Domegan, L.;
O’Donnell, J.; Hauge, S.H.; Dequeker, S.; et al. High impact of COVID-19 in long-term care facilities, suggestion for monitoring in
the EU/EEA, May 2020. Eurosurveillance 2020, 25, 2000956. [CrossRef] 4. Cazzoletti, L.; Zanolin, M.E.; Tussardi, I.T.; Alemayohu, M.A.; Zanetel, E.; Visentin, D.; Fabbri, L.; Giordani, M.; Ruscitti, G.;
Benetollo, P.P.; et al. Risk Factors Associated with Nursing Home COVID-19 Outbreaks: A Retrospective Cohort Study. Int. J. Environ. Res. Public Health 2021, 18, 8434. [CrossRef] [PubMed] 5. Brown, K.A.; Jones, A.; Daneman, N.; Chan, A.K.; Schwartz, K.L.; Garber, G.E.; Costa, A.P.; Stall, N.M. Association Between
Nursing Home Crowding and COVID-19 Infection and Mortality in Ontario, Canada. JAMA Intern. Med. 2021, 181, 229–236. [CrossRef] [PubMed] 6. Giri, S.; Chenn, L.M.; Romero-Ortuno, R. Nursing homes during the COVID-19 pandemic: A scoping review of challenges and
responses. Eur. Geriatr. Med. 2021, 12, 1127–1136. [CrossRef] [PubMed] 7. Katz, S.; Downs, T.D.; Cash, H.R.; Grotz, R.C. Progress in Development of the Index of ADL. Gerontologist 1970, 10, 20–30. [CrossRef] [PubMed] 8. Vernerey, D.; Anota, A.; Vandel, P.; Paget-Bailly, S.; Dion, M.; Bailly, V.; Bonin, M.; Pozet, A.; Foubert, A.; Benetkiewicz, M.; et al. Development and validation of the FRAGIRE tool for assessment an older person’s risk for frailty. BMC Geriatr. 2016, 16, 187,
Erratum in: BMC Geriatr. 2016, 16, 212. [CrossRef] [PubMed] 9. Neuer Pflegebedürftigkeits-begriff der Beitrag der Medizinischen Dienste Neuer Pflegebedürftigkeitsbegriff Pflegebedürftigkeit
und Pflegebegutachtung Medizinischer Dienst Bund. Available online: https://md-bund.de/themen/pflegebeduerftigkeit-und-
pflegebegutachtung/neuer-pflegebeduerftigkeitsbegriff-historie.html (accessed on 3 October 2022). 10. Mahoney, F.I.; Barthel, D.W. Functional evaluation: The Barthel Index. Md. State Med. J. 1965, 14, 61–65. y
11. 5. Conclusions Despite these limitations, this study offers a descriptive approach of the impact and
management of the COVID-19 epidemic in NHs in four European countries, mobilizing a
“modified stringency index” measuring the magnitude COVID-19 global response at the
country level. It enriches the lessons-learned process at the European scale. More evidence
is, however, needed to improve the next preparedness plans on emerging diseases dedicated
to the NHs, in a European perspective. Some differences between the NHs surveyed, such
as the characteristics of the residents (age, dependency . . . ) and the measures taken,
underline the necessity to adapt the plans at the local level. In addition, as a perspective,
the stringency of the measures implemented in the NHs should take into account the
magnitude of the local epidemic. Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/ijerph192215290/s1, Supplementary Table S1: Definitions of
preventive and control measures taken in the NHs. Author Contributions: F.M.: methodology, software, formal analysis, writing—original draft, writing—
review and editing, visualization, validation. V.D.-d.-L.: data management, writing—review and
editing, visualization, validation. A.L.: project administration, writing—review and editing, valida-
tion. S.L.B.: data management, writing—review and editing, visualization, validation. M.P.: data
management, writing—review and editing, visualization, validation. G.B.: writing—review and
editing, visualization, validation. L.B.: writing—review, visualization, validation. B.M.N.: writing—
review and editing, visualization, validation. M.-A.F.: project administration, writing—review,
visualization, validation. D.A.: conceptualization, project administration, supervision, writing—
review and editing, visualization, validation. L.J.: project administration, writing—review and
editing, visualization, validation. E.D.-A.: conceptualization, methodology, project administration,
supervision, writing—original draft, writing—review and editing, validation. S.G.: conceptualization,
methodology, project administration, supervision, writing—original draft, writing—review and
editing, validation. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by Korian Foundation for Ageing Well, as part of a collaboration
contract between the company and the affiliated University of the researchers, the University of
Versailles-Saint-Quentin-en-Yvelines (78180, Montigny-le-Bretonneux). No funding number available. 8 of 8 Int. J. Environ. Res. Public Health 2022, 19, 15290 Data Availability Statement: Data were provided by the Korian Foundation for Ageing Well. Conflicts of Interest: The company had no role in the analyses and interpretation of data. None of
the authors received grants directly from the company. References Hale, T.; Angrist, N.; Goldszmidt, R.; Kira, B.; Petherick, A.; Phillips, T.; Webster, S.; Cameron-Blake, E.; Hallas, L.; Majumdar, S.;
et al. A global panel database of pandemic policies (Oxford COVID-19 Government Response Tracker). Nat. Hum. Behav. 2021, 5,
529–538. [CrossRef] [PubMed] 12. Santé Publique France. Signalement de cas de COVID-19 Dans les Etablissements Sociaux et Médico-Sociaux—Guide Pour les
Établissements. Available online: https://www.santepubliquefrance.fr/media/files/01-maladies-et-traumatismes/maladies-
et infections respiratoires/infection a coronavirus/guide signalement de cas de covid 19 dans les etablissements sociaux 12. Santé Publique France. Signalement de cas de COVID-19 Dans les Etablissements Sociaux et Médico-Sociaux—Guide Pour les
Établissements. Available online: https://www.santepubliquefrance.fr/media/files/01-maladies-et-traumatismes/maladies-
et-infections-respiratoires/infection-a-coronavirus/guide-signalement-de-cas-de-covid-19-dans-les-etablissements-sociaux-
et-medico-sociaux_120122 (accessed on 3 October 2022). É et-infections-respiratoires/infection-a-coronavirus/guide-signalement-de-cas-de-covid-19-dans-les-etablissements-sociaux-
et-medico-sociaux_120122 (accessed on 3 October 2022). É 13. Santé Publique France. COVID-19: Point Épidémiologique du 29 mai 2020. Available online: https://www.santepubliquefrance. fr/maladies-et-traumatismes/maladies-et-infections-respiratoires/infection-a-coronavirus/documents/bulletin-national/
covid-19-point-epidemiologique-du-29-mai-2020#:~{}:text=Points%20cl%C3%A9s,nombres%20d\T1\textquoterightactes%20
SOS%20m%C3%A9decins (accessed on 3 October 2022). /
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SOS%20m%C3%A9decins (accessed on 3 October 2022). 14. Robert Koch Institut (RKI). Täglicher Lagebericht des RKI zur Coronavirus-Krankheit-2019 (COVID-19). Available online:
https://www.rki.de/DE/Content/InfAZ/N/Neuartiges_Coronavirus/Situationsberichte/Maerz_2022/2022-03-31-de.pdf?__
blob=publicationFile (accessed on 3 October 2022). p
15. Mitze, T.; Kosfeld, R.; Rode, J.; Wälde, K. Face masks considerably reduce COVID-19 cases in Germany. Proc. Natl. Acad. Sci. USA
2020, 117, 32293–32301. [CrossRef] [PubMed] 16. Kühl, A.; Hering, C.; Herrmann, W.; Gangnus, A.; Kohl, R.; Steinhagen-Thiessen, E.; Kuhlmey, A.; Gellert, P. General practitioner
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Prevención de caídas y lesiones derivadas en adultos mayores hospitalizados: Experiencia de cuidado de enfermería de la aplicación de una guía de buenas prácticas en la Fundación Oftalmológica de Santander (FOSCAL)
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Artículo de investigación científica y tecnológica / Original
ISSN 0123-7047 Impreso
ISSN 2382-4603 Electrónico
Vol. 20(2):174-181, agosto - noviembre 2017
https://doi.org/10.29375/01237047.3246
Prevención de caídas y lesiones derivadas en adultos
mayores hospitalizados: Experiencia de cuidado de
enfermería de la aplicación de una guía de buenas prácticas
en la Fundación Oftalmológica de Santander (FOSCAL)
Prevention of Falls and Injuries Derived in Hospitalized Older
Adults: Nursing Care Experience of the Application of a Guide of
Good Practices in the Ophthalmological Foundation of Santander
(FOSCAL)
Prevenção de quedas e de lesões em idosos, derivadas das
hospitalizações: Experiência dos cuidados na enfermagem a
partir da aplicação de uma guia de boas práticas na Fundação
Oftalmológica de Santander (FOSCAL)
Maribel Esparza-Bohórquez, Enf., MSc.*
Lina María Granados-Oliveros, Enf., MSc. **
Emerson Serrano-Sanmiguel, Enf., Esp. **
Senieth Peñaloza-Jaimes, Enf., MSc. **
Disminuir las caídas en personas mayores en los servicios de
hospitalización, a través de la implementación de la guía de
buenas prácticas en enfermería de la asociación de
enfermeras registradas de Ontario. Metodología: Estudio
descriptivo, prospectivo de implementación de la guía de
Prevención de Caídas y Lesiones Derivadas de las Caídas en
Personas Mayores, entre el II semestre de 2012 y II semestre
de 2014, en 2 servicios de medicina interna de hospitalización
en los cuales se implementó la guía asociación de
enfermeras registradas de Ontario: Prevención de Caídas y
Lesiones derivadas de las caídas en personas mayores.
Resumen
Introducción: Las caídas representan un indicador de la
calidad de cuidado de las instituciones hospitalarias a nivel
mundial y generan un problema de salud pública debido al
gran impacto que ocasionan en el individuo, familia e
institución. Para enfermería es imprescindible contar con
estrategias basadas en la mejor evidencia disponible que
orienten el cuidado y ayuden a la enfermera en la toma de
decisiones que favorezcan la salud de los pacientes más
susceptibles de sufrir este tipo de eventos. Objetivo:
* Enfermera, jefe división de Enfermería, Fundación Oftalmológica de Santander (FOSCAL), Floridablanca, Santander, Colombia.
** Coordinador de hospitalización, Fundación Oftalmológica de Santander (FOSCAL), Floridablanca, Santander, Colombia.
Correspondencia: Emerson Serrano Sanmiguel. Clínica Foscal Internacional, 5 piso, servicio hospitalización, Calle 158 No. 20-95, Floridablanca,
Santander, Colombia. E-mail: emerson.serrano@foscal.com.co
Artículo recibido: 28 de enero de 2014
Aceptado: 07 de abril de 2017
174
Prevención de caídas y lesiones derivadas en adultos mayores hospitalizados: Experiencia de cuidado de enfermería
de la aplicación de una guía de buenas prácticas en la Fundación Oftalmológica de Santander (FOSCAL)
ISSN 0123-7047 Impreso
ISSN 2382-4603 Electrónico
Fase I: Capacitación-definición de población objeto. Fase II:
Implementación-auditoria y seguimiento. Resultados: Se
valoró un total de 2,387 (88.3%) pacientes hospitalizados, se
identificó el riesgo de caída en 1,960 pacientes (82.1%) y se
implementó un plan multifactorial de prevención de caídas en
1,831 (93.4%). 19 usuarios con riesgo presentaron caída con
grado de lesión leve y se disminuyó en un 20% la incidencia
total de caídas con relación al año 2012. Conclusiones: La
implementación de guías de enfermería basadas en la
evidencia genera un efecto positivo en la calidad de los
cuidados, logrando resultados que impactan en la salud y
bienestar de los pacientes. Prevención de caídas y lesiones
derivadas en adultos mayores hospitalizados. [EsparzaBohórquez M, Granado-Oliveros LM, Serrano-Sanmiguel E,
Peñaloza-Jaimes S. Prevención de caídas y lesiones
derivadas en adultos mayores hospitalizados: Experiencia de
cuidado de enfermería de la aplicación de una guía de
buenas prácticas en la Fundación Oftalmológica de
Santander (FOSCAL). MedUNAB 2017; 20(2): 174-181].
Palabras clave: Enfermería Basada en la Evidencia;
Accidentes por Caídas; Prevención de Accidentes; Seguridad
del Paciente; Anciano.
Abstract
Introduction: Falls represent a quality indicator of care in
hospital institutions worldwide and generate a public health
problem due to the great impact they have on the individual,
family and institution. For nursing, it is essential to have
strategies based on the best available evidence to guide care
and help the nurse in making decisions that favor the health of
the most susceptible patients to suffer this type of event.
Objective: To decrease falls in elderly people in inpatient
services, through the implementation of the nursing best
practice guidelines of the registered nurses association of
Ontario. Methodology: This is a descriptive and prospective
study related to the implementation of the prevention of falls
and injuries derived from falls in the older adults guidelines,
between the 2nd semester of 2012 and the 2nd semester of
2014 in 2 inpatient internal medicine services in which the
registered nurses association of Ontario guideline (prevention
of falls and injuries derived from falls in the older adult) was
implemented. Phase I: Training-definition of the target
population. Phase II: Implementation-audit and follow-up.
Results: A total of 2,387 (88.3%) hospitalized patients were
assessed. The risk of falls was identified in 1,960 patients
(82.1%) and a multifactorial fall prevention plan was
implemented in 1,831 (93.4%) of them. 19 users at risk
presented a fall with a slight degree of injury and the total
incidence of falls was reduced by 20% in relation with the year
2012. Conclusions: The implementation of evidence-based
nursing guidelines generates a positive effect on the quality of
care, achieving results that impact the health and well-being
of patients as well as the prevention of falls and injuries
Introducción
La Organización Mundial de la salud (OMS) define el
término caídas como “acontecimientos involuntarios que
hacen perder el equilibrio y dar con el cuerpo en tierra u otra
superficie que lo detenga” (1). Las caídas prevenibles y no
prevenibles son la principal causa de muerte por accidente
derived from them in inpatient older adults. [EsparzaBohórquez M, Granado-Oliveros LM, Serrano-Sanmiguel E,
Peñaloza-Jaimes S Prevention of Falls and Injuries Derived in
Hospitalized Older Adults: Nursing Care Experience of the
Application of a Guide of Good Practices in the
Ophthalmological Foundation of Santander (FOSCAL).
MedUNAB 2017; 20(2): 174-181].
Keywords: Evidence-Based Nursing; Accidental Falls;
Accident Prevention; Patient Safety; Aged.
Resumo
Introdução: As quedas representam um indicador da
qualidade do atendimento das instituições hospitalares em
todo o mundo e geram um problema de saúde pública devido
ao grande impacto que têm sobre o indivíduo, a família e a
instituição. Para a enfermagem, é essencial ter estratégias
baseadas na melhor evidência disponível para orientar os
cuidados e ajudar a enfermeira a tomar decisões que
favoreçam a saúde dos pacientes mais susceptíveis de sofrer
esse tipo de quedas e das suas conseqüências. Objetivo:
Reduzir as quedas nas pessoas idosas nos serviços de
hospitalização, através da implementação das guias de boas
práticas da enfermagem da associação de enfermeiros
registrados de Ontário. Metodologia: Estudo descritivo e
prospectivo da implementação da guia para a Prevenção de
quedas e lesões como consequencia das quedas em idosos,
entre o segundo semestre de 2012 a II semestre de 2014, nos
dois serviços de medicina interna e da hospitalização nas
quais foi implementada a guia associação de enfermeiros
registrados de Ontário: prevenção de quedas e lesões
decorrentes das quedas em pessoas idosas. Fase I:
Formação-definição da população-alvo. Fase I I :
Implementação-auditoria e acompanhamento. Resultados:
Foram avaliados 2.387 (88,3%) pacientes hospitalizados, o
risco de queda foi identificado em 1.960 pacientes (82,1%) e
um plano multi-fatorial para prevenir quedas foi implementado
em 1.831 (93,4%). Dez e nove (19) usuários em risco
apresentaram uma queda com um ligeiro grau de lesão e a
incidência total de quedas foi reduzida em 20% em relação ao
ano de 2012. Conclusões: A implementação das guias de
enfermagem baseadas nas evidências gera um efeito positivo
na qualidade dos cuidados, obtendo resultados que afetam a
saúde e o bem-estar dos pacientes. A prevenção de quedas e
das lesões decorrentes nos idosos hospitalizados. [EsparzaBohórquez M, Granado-Oliveros LM, Serrano-Sanmiguel E,
Peñaloza-Jaimes S. Prevenção de quedas e de lesões em
idosos, derivadas das hospitalizações: Experiência dos
cuidados na enfermagem a partir da aplicação de uma guia de
boas práticas na Fundação Oftalmológica de Santander
(FOSCAL). MedUNAB 2017; 20(2): 174-181].
Palavras-chave: Enfermagem Baseada em Evidências;
Acidentes por Quedas; Prevenção de Acidentes; Segurança do
Paciente; Idoso.
intrahospitalario, especialmente en adultos mayores y para
el año 2015 fue el tercer evento adverso más notificado en la
base de datos de sucesos centinela de la Joint Commission
(2).
Uno de los indicadores que revela la calidad del cuidado de
enfermería en las instituciones de salud a nivel mundial es el
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de caídas, donde la mayoría de los reportes de estos eventos
adversos en instituciones de salud ocasionan algún tipo de
lesión secundaria, generando entre el 4-6% lesiones graves
como: fracturas, hematomas intracraneales, hemorragias y
muerte (3). En áreas de hospitalización, la tasa de caídas
reportada oscila entre el 2.3 y 7 cada 1,000 días paciente(4),
evidenciando que las caídas son un problema de salud
pública que además, genera un gran impacto a nivel del
individuo, la familia y la institución de salud.
A nivel del individuo, las caídas generan en los pacientes,
sentimientos de minusvalía, con el consecuente temor a
caerse de nuevo, llevándolos a una disminución de la
movilidad y de su capacidad funcional. De otra parte, la
estancia hospitalaria aumenta (cercana a los 10-12 días) (5),
como consecuencia del incremento de sus comorbilidades
asociado a una disminución de sus capacidades funcionales
en un entorno desconocido y, en este sentido, se crea la
necesidad de un mayor número de personal entrenado para
su cuidado.
A nivel económico las caídas producen un sobrecosto en la
atención de los pacientes, según la OMS el costo medio para
el sistema sanitario en Finlandia es de US $ 3,611 y en
Australia de US $1,049 (5). Por otro lado, los datos de
Canadá indican que la aplicación de estrategias preventivas
y eficaces con la consecuente reducción de caídas supondría
un ahorro cercano a US $ 120 millones al año(6).
La Fundación Oftalmológica de Santander (FOSCAL) es
una institución de cuarto nivel de atención en salud ubicada
en la ciudad de Bucaramanga (Colombia), con una
capacidad instalada de 293 camas, con un porcentaje
ocupacional del 95% y un promedio días-estancia de 7 días
(7), para el año 2012, se presentaron un total de 82 caídas las
cuales representaban una tasa de 0.82 por mil días estancia y
el 17.22% de los eventos adversos reportados, consideradas
en un 93% de carácter leve, es decir, no presentaron ningún
tipo de lesión secundaria en el paciente y un 6% consideradas
moderadas con lesiones de tipo leve a moderado, el rango de
edad que sufrió el mayor número de caídas fue el de adultos
entre 70 y 79 años, el lugar donde más se cayeron fue de la
cama (40.7%) seguido del baño (25%), el horario de mayor
ocurrencia de caídas es el comprendido entre la medianoche
y las 10 de la mañana, y el servicio de hospitalización que
más casos presentó fue el de medicina interna(8).
El cuidado de enfermería es el eje central en las instituciones
de salud y para proporcionar un cuidado altamente
calificado debe estar basado en el conocimiento científico y
la experiencia que lo apoya, con resultados medibles que
permitan la participación y colaboración del usuario en pro
de la mejoría de su condición de salud. Para enfermería
como profesión es imprescindible contar con estrategias
basadas en la mejor evidencia disponible orientada al
cuidado y ayuda de la enfermera en la toma de decisiones
que favorezcan la salud de los pacientes más susceptibles de
sufrir este tipo de eventos.
Hallazgos previos en la literaturademuestran que aproximadamente entre el 2 y el 10% de los pacientes hospitalizados
presentarán una caída durante su estancia hospitalaria
ocasionada por diversas causas como: edad, morbilidades
propias del paciente, tratamiento médico y el ambiente
hospitalario desconocido para el individuo (9). Estos
eventos tienen un gran impacto en la salud de los pacientes y
las instituciones, haciendo necesaria la implementación de
estrategias que intervengan cada una de las causas y
minimicen la incidencia de caídas y lesiones derivadas de
las caídas en la población más vulnerable a sufrirlas.
Frente a esta problemática, la Asociación de Enfermeras
Registradas de Ontario (RNAO), ha trabajado desde hace
más de 12 años, y, financiado por el gobierno de Ontario
desarrolló el Programa de Directrices de las Mejores
Prácticas de Enfermería (BPG), el cual involucra una
metodología rigurosa que incluye: la identificación del tema
específico de la BPG, una revisión sistemática de la mejor y
actualizada evidencia disponible, la selección de un panel de
expertos (enfermeros, otras profesiones, pacientes y
comunidad) que desarrollen las recomendaciones clínicas,
organizacionales, educativas y a nivel de sistema, derivadas
de la evidencia y la publicación final de la directriz o guía
(10). Actualmente las guías de enfermería basadas en
evidencia son ampliamente utilizadas por más de 60
instituciones a nivel mundial (BPSO). En Suramérica se
están implementado en Chile en instituciones a nivel
asistencial como la clínica los CONDES y académico,
como la universidad de Chile y en Colombia, en dos
instituciones de salud ampliamente reconocidas: La
Fundación Cardioinfantil y FOSCAL.
La guía de “Prevención de Caídas y Lesiones derivadas de las
caídas en personas mayores” de la RNAO fue adoptada por
la FOSCAL con el objetivo de identificar los factores de
riesgo, disminuir la incidencia de caídas y disminuir la
incidencia de caídas con lesión (11) en los adultos mayores
que se hospitalizan en la institución. A partir de la iniciativa
institucional de aplicación de esta guía, la división de
enfermería tuvo como objetivo demostrar la reducción en la
incidencia de caídas y los eventos adversos relacionados con
las mismas en dos servicios hospitalarios de medicina interna
de la Fundación Oftalmológica Carlos Ardila LulleFOSCAL.
Metodología
Tipo de estudio y población
El presente estudio es de tipo descriptivo, prospectivo de
implementación de intervenciones de enfermería basadas en
evidencia, para la prevención de caídas y lesiones derivadas
de las caídas; se llevó a cabo en una institución de cuarto
nivel de atención, en dos servicios de medicina interna, con
una población de usuarios mayores de 65 años, entre el
segundo semestre de 2012 y de 2014. El muestreo se realizó
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a conveniencia y se consideraron dentro del estudio todos
los usuarios hospitalizados mayores de 65 años. Se definió
para el reporte de caídas que se tomarían todas las caídas de
los usuarios de estos dos servicios, independiente si ocurren
o no en el servicio (durante un traslado para apoyos
diagnósticos, cirugía, etc.). Estos datos son consolidados
por el sistema de información electrónica de la institución
en el módulo de seguridad del paciente de la FOSCAL.
Fase I: Capacitación – Definición de Población objeto Determinación de Intervenciones
En el segundo semestre de 2012 se inició el proceso de
implementación de la guía, con la capacitación del personal
así:
Dos enfermeras de la institución se entrenaron como líderes
en implementación de guías RNAO, quienes a su vez
formaron un grupo de líderes integrado por: 12
coordinadores de enfermería y una enfermera de atención
segura. El grupo de líderes analizó la guía y definió como
población sujeto para una fase piloto, a los usuarios de
medicina interna mayores de 65 años de dos servicios de
hospitalización, población identificada a nivel institucional
como la de mayor riesgo de caerse, de igual manera, escogió
intervenciones aplicables en la institución y con la mayor
evidencia científica. Las intervenciones de la guía RNAO
seleccionadas fueron: 1). Valoración del riesgo de presentar
una caída en el momento de ingreso; 2). Valorar el riesgo de
caída tras sufrir una caída, y, por último; 3). Implementación
de intervenciones multifactoriales para la prevención de
caídas.
Posteriormente, se entrenó el personal de enfermería (18
profesionales y 36 auxiliares) de los dos servicios de
medicina interna, específicamente en la guía de prevención
de caídas y lesiones derivadas de las mismas respecto a la
valoración del riesgo de caerse y en las intervenciones a
aplicar según el riesgo en el que se encuentre el usuario. Las
capacitaciones se realizaron de forma mensual por la
rotación de personal con retroalimentación diaria al equipo
de trabajo durante las rondas de enfermería realizadas por el
coordinador del servicio.
Se definieron los grupos de interés, los cuales fueron
conformados por las personas que participan directa e
indirectamente en el proceso de atención y que influyen en el
proceso de implementación, siendo primordiales: la familia
o el cuidador familiar, porque participan activamente
durante todo el proceso educativo y son colaboradores para
el cumplimiento de la indicaciones dadas por enfermería
para minimizar el riesgo; el personal de atención al usuario,
quienes fortalecen el proceso de educación de enfermería
durante la ronda hospitalaria; la oficina de atención segura
participa en la verificación de las estrategias y en el análisis
y valoración posterior a una caída. El personal médico y
paramédico, quienes fortalecen la educación y la
implementación de las medidas de prevención de caídas.
Fase II: Implementación - Auditoria – Seguimiento
Implementación de la guía
Para la clasificación del riesgo se decidió utilizar la escala de
MORSE recomendada en la guía (11). Este instrumento ha
sido validado previamente en población adulta
hospitalizada, que ofrece de una manera rápida una
valoración del riesgo del usuario de presentar una caída (12).
De igual manera, se establecieron las intervenciones
multifactoriales propuestas por la guía para la prevención de
caídas, dentro de las cuales se consideraron: capacitación al
personal, modificación del entorno (cama baja, orden y aseo,
pisos secos, barandas de la cama abajo), acompañamiento
permanente por familiar o cuidador, acompañamiento
permanente por enfermería para actividades de
desplazamiento o durante el baño, información del riesgo
durante cada entrega de turno o traslado del usuario,
colocación de la manilla y adhesivo color fucsia en el
usuario con alto riesgo y diligenciamiento del formato de
indicación a familia y usuario con riesgo de caídas.
En el estudio se diseñaron formatos para la evaluación del
riesgo y la implementación de intervenciones multifactoriales, así como para la valoración del riesgo tras sufrir una
caída. Se realizó prueba piloto de los mismos durante 1 mes
en ambos servicios, con el fin de que todo el personal
pudiera usarlos y se hicieron las adecuaciones necesarias
para su utilización durante el proceso de implementación.
Para la evaluación del riesgo se acordó que los puntajes en la
escala de MORSE, iguales o inferiores a 44 puntos, se
tomarían como Sin Riesgo de sufrir una caída, se les
aplicaría las medidas generales de seguridad y se les
revaloraría el riesgo cada 72 horas. Los puntajes iguales o
superiores a 45 se tomarían como Riesgo Alto de sufrir una
caída y se implementaron medidas especiales para la
prevención, además de informar y educar al familiar sobre el
riesgo y firmar el documento de indicaciones especiales
para los familiares con cada una de la recomendaciones a
implementar, las cuales fueron: acompañamiento
permanente por familiar o cuidador, acompañamiento
permanente por enfermería para actividades de
desplazamiento o durante el baño (hora identificada en la
institución como de mayor incidencia de caídas), cama en
posición baja, uso de brazalete de identificación de riesgo de
caídas, informar en cada turno o traslado del usuario el
riesgo de caídas, modificación del entorno: cama baja, pisos
secos, orden y aseo de habitaciones, dispositivos de ayuda
en buen estado y la solicitud de ayuda permanente al
personal de enfermería en caso de desplazamiento o ingreso
al baño, siendo la educación al personal y familiares, así
como, la implementación de un plan multifactorial, las
intervenciones identificadas como protectoras según
estudios (13,14). Todas estas recomendaciones estaban
descritas en un folleto informativo el cual se le entrega al
familiar para reforzar la educación brindada.
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A nivel institucional se actualizó la guía de prevención de
caídas y lesiones derivadas de las caídas bajo los parámetros
de la RNAO y se diseñó el plegable institucional de
prevención de caídas en el cual se dan recomendaciones a
familiares y pacientes con riesgo de caídas.
Desde octubre de 2012, teniendo en cuenta la rotación de
personal de los servicios, el uso de la escala de valoración
MORSE y la inclusión de las estrategias de intervención, se
capacitó mensualmente al personal de enfermería de los
servicios seleccionados en cuanto al uso de la guía y las
herramientas a utilizar para su implementación, además fue
necesaria capacitación y retroalimentación permanente e
individualizada al personal profesional y auxiliar de
enfermería durante todo el proceso.
Aspectos Éticos
El presente estudio fue clasificado como una investigación
con mínimo riesgo de acuerdo con la Resolución 8430 de
1993, artículo 11 del Ministerio de Salud de Colombia y
como se trata de datos desagregados de los pacientes no se
solicitó consentimiento informado.
Se inició la recolección de los datos en marzo de 2013 y se
fue evaluando mensualmente el proceso de implementación
de la guía en los servicios seleccionados. Se establecieron
responsabilidades directas al personal profesional y auxiliar
de enfermería que sirve de apoyo en los servicios, para la
verificación de la valoración del riesgo al ingreso y para la
verificación de las intervenciones. Se realizó el acompañamiento al personal para la valoración y verificación de las
intervenciones multifactoriales.
Resultados
Se estableció una ronda de prevención de caídas en la cual se
verificaba diariamente a través de un formato: la educación
recibida, la presencia de brazalete de identificación del
riesgo y la modificación del entorno. Se retroalimentó al
personal de forma permanente en temas como: valoración
del riesgo, intervenciones a realizar y verificación del
cumplimiento de las mismas.
A continuación, se presentan los resultados de la fase de
capacitación (Tabla 1). A partir de la implementación de la
guía se encontró que del total de usuarios con edades iguales
o superiores a 65 años admitidos en el servicio de medicina
interna, se estableció el riesgo de presentar una caída al
83.6% de los hospitalizados en 2013 y al 92.3% de usuarios
hospitalizados en 2014 (Tabla 2).
Se analizó la pertinencia de los indicadores descritos por la
RNAO para evaluar la implementación de la guía y la forma
de recolección de los datos para su presentación y
evaluación, definiendo el uso de los siguientes indicadores
propuestos: evaluación del riesgo de caídas en usuarios que
ingresan al servicio, implementación de un plan
multifactorial de prevención de caídas, tasa global de caídas
por 1,000 días estancia.
A medida que avanzó el proceso de implementación, la
valoración del riesgo de caídas en los usuarios hospitalizados en los dos servicios aumentó progresivamente,
estabilizándose en 2014, manteniéndose superior al 90% de
los pacientes hospitalizados (Figura 1). Estos indicadores
son consecuencia de la capacitación, supervisión y
retroalimentación permanente al personal de enfermería en
la realización de la valoración a los usuarios mayores que
Fase III de Evaluación
Tabla 1. Fase de capacitación
Año
Personal de inducción capacitado (enfermeras y auxiliares de enfermería)
2013
204
2014
551
Fuente: Elaboración de los autores
Tabla 2. Evaluación del riesgo de caídas en los adultos mayores que ingresan al servicio
de medicina interna por año.
Año
Total de admitidos
Usuarios con riesgo
Frecuencia
%
2013
1,239
1036
83.6
2014
1,463
1351
92.3
Fuente: Elaboración de los autores
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ingresan a los servicios, actividades que llevan a la
estandarización del procedimiento en sí.
La implementación del plan multifactorial en los usuarios
con riesgo de caerse se aplicó en el 95% de los usuarios
hospitalizados, clasificados con riesgo alto para el año
2,013; se presentó disminución de la adherencia en el
primer trimestre, el cual se fortaleció mediante listas de
verifica-ción, auditoría diaria en los servicios y
retroalimentación permanente de las fallas encontradas
durante las rondas de enfermería, para así lograr una
mejoría en la adherencia del 100% de los usuarios en riesgo
(Figura 2).
La verificación diaria del plan multifactorial consideraba los
ítem (cama en posición baja, orden y aseo, pisos secos,
acompañamiento permanente por familiar o cuidador,
acompañamiento permanente por enfermería para
actividades de desplazamiento o durante el baño,
información del riesgo durante cada entrega de turno o
traslado del usuario, mantener buena iluminación).
Posteriormente, eran incluidos en la medición sólo los
usuarios que cumplían diariamente con el total de criterios
de verificación. Esta estrategia permitió evaluar la
implementación de las medidas seleccionadas, la adherencia
del personal y la importancia de la educación de la familia
Percentage of Patients For Whom a Falls Risk
Assessment Was Completed on Admission
RNAO BPSO® BPG Indicator Report
Clinica FOSCAL
Prevention of Falls and Fall Injuries in the Older Adult
Falls_Pro01: Fall Risk Assessment, New Admissions
100
50
0
Jul '13
Jan '14
Jul '14
Jan '15
Reporting period
Hospitalization-9
Internal Medicine-2
BPSO Median (December 2015)
Fuente: NQuiRE, Registered Nurses’ Association of Ontario, May 23, 2018.
Figura 1. Evaluación semestral del riesgo de caídas en usuarios que ingresan al
servicio de medicina interna 2013-2015.
RNAO BPSO® BPG Indicator Report
Clinica FOSCAL
Percentage of Patients Identified As “At
Risk for Fall” With a Prevention or Injury
Reduction Plan
Prevention of Falls and Fall Injuries in the Older Adult
Falls_Pro03: Fall Prevention
100
50
0
Jul '13
Jan '14
Jul '14
Jan '15
Reporting period
Hospitalization-9
Internal Medicine-2
BPSO Median (December 2015)
Fuente: NQuiRE, Registered Nurses’ Association of Ontario, May 23, 2018.
Figura 2. Implementación de un plan multifactorial de prevención de caídas
servicio de medicina interna por año.
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y/o cuidador permanente en el cumplimiento de normas de
prevención. De igual manera, se encontró que las estrategias
eran aplicadas exclusivamente por el personal de enfermería,
situación que generó la necesidad de integrar al equipo
interdisciplinario que trabaja a favor de la seguridad del
paciente, a través de la socialización de las intervenciones de
la guía, al equipo interprofesional (fisioterapeutas, atención
al usuario, médicos tratantes, personal de aseo, camilleros,
etc) para su aplicación en cada actividad durante su
intervención asistencial.
revaloración posterior del paciente y la revisión inmediata
por el personal líder de seguridad y el coordinador de
enfermería para identificar los factores causales e
intervenirse inmediatamente y socializar con el personal las
fallas encontradas para evitar que se presentara una nueva
caída. Estos datos coinciden con los encontrado por MiakeLye et al (15),en los cuales los programas para prevención de
caídas con múltiples componentes reducen en un 30% el
riesgo relativo de sufrir una caída.
A partir de la determinación del riesgo de caídas y la
aplicación del plan multifactorial se observó un descenso de
la tasa de caídas en el año 2014 (Figura 3). Se encontró que
se presentaron 12 caídas en el año 2013 y 7 caídas en el año
2014, para un total de 19 caídas en los dos servicios, todas
clasificadas como severidad en el desenlace leve (Tabla 3).
Discusión
De igual manera la tasa de caídas del año 2014 fue menor en
comparación con los datos obtenidos de 2012 (0.82 *1,000
días) y de 2013, observándose una diferencia de casi un 20%
de la tasa global del año 2012 cuando no se había iniciado la
implementación de la guía de la RNAO en los servicios.
Todos los usuarios que presentaron caídas al hacer el análisis
se identificaban con alto riesgo en la escala de MORSE al
ingreso. Al presentarse las caídas una de las estrategias era la
Tabla 3. Total de caídas por año
Año
Caídas
Días de Estancia
2013
12
11,436
2014
7
11,431
Fuente: Elaboración de los autores.
Mediante la implementación de la guía basada en la
evidencia se logró consolidar un grupo de trabajo en los
servicios seleccionados y a su vez protocolizar el
procedimiento para así aplicar las actividades encaminadas
a disminuir las caídas y sus complicaciones en los usuarios
fortaleciendo la política de seguridad de la institución.
El tener un instrumento definido (16) para la evaluación del
riesgo al ingreso de los usuarios a la institución permite a
enfermería lograr mayor empoderamiento y liderazgo en la
prevención de caídas y aumento en la identificación del
riesgo en todos los usuarios hospitalizados.
De acuerdo con los resultados y con otros estudios (17,18),
es de gran importancia la identificación de los factores y la
valoración del riesgo de sufrir una caída mediante un
instrumento e implementar un plan multifactorial que
incluya la educación al personal de enfermería, equipo
multidisciplinario e intervenciones relacionadas con el
manejo del ambiente hospitalario con el fin de disminuir la
incidencia de caídas y lesiones derivadas de las caídas en los
pacientes.
RNAO BPSO® BPG Indicator Report
Clinica FOSCAL
Rate of Falls, With or Without Injury, in
Patients per 1000 Patient Days
Prevention of Falls and Fall Injuries in the Older Adult
Falls_Out01: Falls Rate
4
2
0
May '13
Sep '13
Jan '14
May '14
Sep '14
Jan '15
Reporting period
Hospitalization-9
Internal Medicine-2
BPSO Median (December 2015)
Fuente: NQuiRE, Registered Nurses’ Association of Ontario, May 23, 2018.
Figura 3. Tasa de global de caídas por 1,000 días de estancia en el servicio de
medicina interna por año
180
Prevención de caídas y lesiones derivadas en adultos mayores hospitalizados: Experiencia de cuidado de enfermería
de la aplicación de una guía de buenas prácticas en la Fundación Oftalmológica de Santander (FOSCAL)
ISSN 0123-7047 Impreso
ISSN 2382-4603 Electrónico
Mediante la aplicación de las intervenciones del plan
multifactorial se identificó la importancia del familiar como
cuidador principal en la adecuada adherencia de las
actividades planteadas.
Referencias
Conclusiones
Se estableció como cultura organizacional la valoración del
riesgo de caídas de los usuarios que ingresan a la institución
lográndose una adherencia del 95% en los servicios
seleccionados.
La implementación de las guías basadas en la evidencia
permitió disminuir la tasa de caídas en los servicios
seleccionados mediante la aplicación de los cuidados
estandarizados en el plan multifactorial fortaleciendo la
política de seguridad institucional y, a pesar de que se
presentaron eventos adversos no se encontraron secuelas
dado que la mayoría de los eventos ocurridos fueron leves.
La evaluación del riesgo de sufrir una caída a través de un
instrumento, es uno de los componentes principales en la
prevención, porque permite la identificación de factores de
riesgo que predisponen a los adultos mayores a sufrir una
caída. Al evaluar el riesgo se logra determinar el tipo de
intervenciones que se pueden aplicar para minimizarlo y así
poder generar una disminución en la presentación.
La implementación de la guía de prevención de caídas y
lesiones derivadas de las caídas en adultos mayores de 65
años, requiere de la participación decidida de la enfermera
en la planeación y ejecución del cuidado teniendo en cuenta
factores físicos y ambientales del paciente y en las
herramientas o estrategias a implementar para conseguir los
resultados que impacten en la salud de los pacientes y en los
estándares de calidad de las instituciones.
Responsabilidades éticas
Protección de personas y animales. Los autores declaran que
para esta investigación no se han realizado experimentos en
seres humanos ni en animales.
Confidencialidad de los datos. Los autores declaran que
han seguido los protocolos de su centro de trabajo sobre la
publicación de datos de pacientes.
Derecho a la privacidad y consentimiento informado. Los
autores declaran que en este artículo no aparecen datos de
pacientes.
Conflicto de intereses
Los autores declaran no tener conflictos de interés.
181
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_and_Fall_Injuries_in_the_Older_Adult.pdf
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15. Miake-Lye I, Hempel S, Ganz D, Shekelle P. Inpatient fall
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Audiovisual Integration During Joint Action: No Effects for Motion Discrimination and Temporal Order Judgment Tasks
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Frontiers in psychology
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cc-by
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ORIGINAL RESEARCH
published: 04 February 2020
doi: 10.3389/fpsyg.2020.00079 Edited by:
Andreas K. Engel,
University Medical Center
Hamburg-Eppendorf, Germany
Reviewed by:
Valerio Santangelo,
University of Perugia, Italy
Valter Prpic,
De Montfort University,
United Kingdom
*Correspondence:
Basil Wahn
basilwahn@gmail.com Edited by:
Andreas K. Engel,
University Medical Center
Hamburg-Eppendorf, Germany Reviewed by:
Valerio Santangelo,
University of Perugia, Italy
Valter Prpic,
De Montfort University,
United Kingdom *Correspondence:
Basil Wahn
basilwahn@gmail.com Keywords: multisensory integration, social cognition, joint action, motion, temporal, task co-performance Specialty section:
This article was submitted to
Cognitive Science,
a section of the journal
Frontiers in Psychology Audiovisual Integration During Joint
Action: No Effects for Motion
Discrimination and Temporal Order
Judgment Tasks In daily life, humans constantly process information from multiple sensory modalities (e.g.,
visual and auditory). Information across sensory modalities may (or may not) be combined
to form the perception of a single event via the process of multisensory integration. Recent research has suggested that performing a spatial crossmodal congruency task
jointly with a partner affects multisensory integration. To date, it has not been investigated
whether multisensory integration in other crossmodal tasks is also affected by performing
a task jointly. To address this point, we investigated whether joint task performance also
affects perceptual judgments in a crossmodal motion discrimination task and a temporal
order judgment task. In both tasks, pairs of participants were presented with auditory
and visual stimuli that might or might not be perceived as belonging to a single event. Each participant in a pair was required to respond to stimuli from one sensory modality
only (e.g., visual stimuli only). Participants performed both individual and joint conditions. Replicating earlier multisensory integration effects, we found that participants’ perceptual
judgments were significantly affected by stimuli in the other modality for both tasks. However, we did not find that performing a task jointly modulated these crossmodal
effects. Taking this together with earlier findings, we suggest that joint task performance
affects crossmodal results in a manner dependent on how these effects are quantified
(i.e., via responses time or accuracy) and the specific task demands (i.e., whether tasks
require processing stimuli in terms of location, motion, or timing). Keywords: multisensory integration, social cognition, joint action, motion, temporal, task co-performance 1. INTRODUCTION Humans constantly process information from several sensory modalities (e.g., touch, vision,
audition). This information may (or may not) be combined to form a unitary percept via the process
of multisensory integration. Previous research has investigated several factors that could affect this
integration process ranging from where and when the stimuli occur (Meredith et al., 1987; Meredith
and Stein, 1996; Guski and Troje, 2003; Holmes and Spence, 2005) to the attentional demands
placed on the observer (Bertelson et al., 2000; Vroomen et al., 2001; Alsius et al., 2005; Santangelo
and Spence, 2008, 2009; Santangelo and Macaluso, 2012; Vercillo and Gori, 2015; Wahn and König,
2015a,b, 2016; Wahn et al., 2017b). Received: 19 July 2019
Accepted: 13 January 2020
Published: 04 February 2020 Citation: Importantly, past work has also demonstrated that the effect
of multisensory integration varies with the nature of the task. For
instance, as discussed above, when judging the spatial direction
of two auditory stimuli, irrelevant and incongruent visual stimuli
have a negative effect on performance. Note, when the task is
reversed, and one is required to determine the direction of two
visual stimuli, incongruent auditory motion has no impact on
performance. In general, the explanation for this asymmetry
is that multisensory integration is preferentially biased toward
the modality that provides the most reliable signal for the
task at hand, in this case, spatial direction. In other words,
vision provides a more reliable spatial signal than does audition,
a point we are all too familiar with when we are trying to
determine in a group whose phone is ringing; it is only when
we see a person move that we localize the sound. Critically,
this advantage of a visual signal over an auditory signal reverses
when the task is to judge when, rather than where, two events
have occurred. This was demonstrated by Morein-Zamir et al. (2003) who asked participants to judge which of two visual
stimuli appeared first on a computer screen. They found that
performance improved when an auditory click trailed the second
visual stimulus, as if the second visual event was pulled toward
the trailing auditory click. As the paradigm used by Soto-Faraco
et al. (2002) can be readily adapted to that of Morein-Zamir
et al. (2003), we examined if a joint task manipulation affects
both dynamic spatial judgments and temporal judgments. That
is, adapting the paradigm used by Soto-Faraco et al. (2002)
to that of Morein-Zamir et al. (2003) only involves minimal
changes (i.e., instead of judging the direction of two rapidly
presented stimuli, participants are required to judge the temporal
order of two stimuli), allowing for the specific targeting of the
question of whether temporal judgments are affected by joint
task performance in a within-subject design. In doing so, we can
also assess whether potential effects of joint task performance
are comparable both in situations where vision affects auditory
judgments (i.e., sound direction) and when audition affects visual
judgments (i.e., visual timing). Citation: demonstrated that social factors can have a significant impact
on how individuals perceive an isolated visual event (Sebanz
et al., 2003, 2006; Knoblich and Sebanz, 2006; Böckler et al.,
2012; Karlinsky et al., 2017; Vesper et al., 2017). Also, people
in everyday life routinely perform multisensory tasks with, or
in the presence of, others. For instance, when eating a meal
with a friend, visual, tactile, smell, and taste information are
combined; and when attending a concert both visual (e.g., seeing
the musicians) and auditory information (e.g., hearing the music)
are processed in the presence of others. p
p
To the best of our knowledge, the first study (Heed et al.,
2010) that investigated the relationship between multisensory
integration and joint task performance involved a tactile spatial
localization task. Heed et al. (2010) required participants to
indicate the location of a tactile stimulus while a visual stimulus
was presented simultaneously in either the same (congruent)
or different (incongruent) location. Past work has demonstrated
that when participants perform this type of task alone, responses
to the tactile stimulus are slower and less accurate if the visual
stimulus appears at an incongruent location (Spence et al., 2004). Heed et al. (2010) found that this congruency effect was reduced
when performing the task jointly, as the participant performing
the tactile task “off-loaded” attending to the visual distractor to
their partner. As a potential mechanism to explain this effect,
Heed et al. (2010) suggested that the participant performing the
tactile task co-represented (Sebanz et al., 2003) the partner’s task
and could hence better filter out the visual distractors from their
own task representation. The process of co-representation (i.e.,
that co-actors take into account each other’s tasks) has been
proposed to occur automatically whenever co-actors perform
tasks jointly (Sebanz et al., 2003) and to form the basis for more
complex joint actions (Vesper et al., 2010). In a recent study, we
replicated the finding by Heed et al. (2010) in a joint audiovisual
congruency task (Wahn et al., 2017a). That is, we found that
the negative effect of an incongruent visual stimulus on sound
localization was reduced for participants performing the task
jointly. Relatedly, Sellaro et al. (2018) found that such a division
of labor of tasks also reduced interference in a purely visual
picture–word interference task (for a recent review about the
benefits of labor division in joint tasks, see Wahn et al., 2018). Citation: Wahn B, Dosso JA and Kingstone A
(2020) Audiovisual Integration During
Joint Action: No Effects for Motion
Discrimination and Temporal Order
Judgment Tasks. To date, however, only a handful of studies have investigated whether social factors (e.g.,
performing a task with another person) affect multisensory integration (Heed et al., 2010; Wahn
et al., 2017a). This comes something as a surprise given that past work on joint action has Front. Psychol. 11:79. doi: 10.3389/fpsyg.2020.00079 Front. Psychol. 11:79. doi: 10.3389/fpsyg.2020.00079 February 2020 | Volume 11 | Article 79 Frontiers in Psychology | www.frontiersin.org Audiovisual Integration During Joint Action Wahn et al. Soto-Faraco et al. (2002) introduced an audiovisual motion
congruency task that is conceptually very similar but qualitatively
distinct from the crossmodal congruency task with static
stimuli (Soto-Faraco et al., 2002, 2004), but to date it has
only been tested with individual, isolated participants. In the
typical audiovisual motion task, a participant receives visual
and auditory stimuli that either move together in the same
direction (congruent presentations) or in opposite directions
(incongruent presentations). The critical task is to judge the
movement direction of the auditory stimuli. Results indicated
that participants often failed to correctly identify the direction
of sound motion when the direction of the visual motion was
incongruent (e.g., leftward auditory motion and rightward visual
motion). We viewed this paradigm as a logical next step to test
whether joint task performance affects audiovisual integration
using a task involving more ecologically valid stimuli (i.e., motion
stimuli). That is, the audiovisual motion congruency task used
by Soto-Faraco et al. (2002) represented only a minimal change
(i.e., static stimuli are replaced by moving stimuli) relative to our
earlier study using a spatial audiovisual congruency task (Wahn
et al., 2017a). demonstrated that social factors can have a significant impact
on how individuals perceive an isolated visual event (Sebanz
et al., 2003, 2006; Knoblich and Sebanz, 2006; Böckler et al.,
2012; Karlinsky et al., 2017; Vesper et al., 2017). Also, people
in everyday life routinely perform multisensory tasks with, or
in the presence of, others. For instance, when eating a meal
with a friend, visual, tactile, smell, and taste information are
combined; and when attending a concert both visual (e.g., seeing
the musicians) and auditory information (e.g., hearing the music)
are processed in the presence of others. Frontiers in Psychology | www.frontiersin.org February 2020 | Volume 11 | Article 79 Citation: Finally, it is worth noting that While the above research has demonstrated that performing
a task jointly does affect audiovisual and visuotactile integration
in a spatial congruency task (Heed et al., 2010; Wahn et al.,
2017a), it has not been investigated whether the effect of joint
performance on multisensory integration can be generalized to
other situations, particularly whether the results extend beyond
the presentation of two solitary, static stimulus events. The
stimuli one routinely encounters in everyday life are normally in
motion because we are often in motion (e.g., walking, moving
our head, and shifting our gaze several times a second) and
the world around us is in motion, too (e.g., living animals
move, water flows, and trees sway in the wind). An important
extension of the previous work then is to test if multisensory
integration with moving stimuli is affected by joint performance. That is, investigating this question would be informative as
to whether multisensory integration of moving stimuli is also
affected by joint task performance or whether the effect of joint
task performance is specific to stationary spatial stimuli. February 2020 | Volume 11 | Article 79 Frontiers in Psychology | www.frontiersin.org 2 Wahn et al. Audiovisual Integration During Joint Action there is currently uncertainty in the literature as to whether
joint task performance on multisensory integration affects how
quickly people respond, their accuracy of response, or both. For instance, Heed et al. (2010) used inverse efficiency scores
(i.e., a combined measure of response times and accuracy)
to analyze their data, rendering the speed/accuracy question
equivocal. Wahn et al. (2017a) analyzed response times and
accuracy separately and found that joint task performance only
affected response times. As crossmodal congruency effects on
response times are often vulnerable to alternative explanations
that do not demand an explanation in terms of multisensory
integration (e.g., a race model explains why two congruent
signals may result in faster responses time than either of them
alone, see Miller, 1982; Stevenson et al., 2014) we aimed to test
whether performing a task jointly affects perceptual judgments
(i.e., perceptual accuracy) rather than response times. Crucially,
in both tasks mentioned above (i.e., the motion discrimination
task and temporal order judgment task), crossmodal effects were
quantified via response accuracies. FIGURE 1 | Experimental setup. Response keys were marked by green
stickers on the keyboards. 2.1. Participants Nineteen pairs of students (32 female and 6 male, M =
19.58 years, SD = 1.44 years) of the University of British
Columbia participated in the present study. The participants
provided their written informed consent to participate in
this study and received course credits as compensation for
their participation. Citation: To summarize, the current work aims to extend previous
research on multisensory integration and joint task performance
(Heed et al., 2010; Wahn et al., 2017a) in three ways: (1)
through the use of moving rather than static stimuli in a
crossmodal congruency task, (2) by investigating temporal
crossmodal effects, and (3) by using tasks that quantify
crossmodal effects with regard to response accuracy (rather than
response time). FIGURE 1 | Experimental setup. Response keys were marked by green
stickers on the keyboards. was counterbalanced across pairs so that half of the pairs started
with the motion discrimination task and the remaining half
began with the temporal order judgment task. In the following
section we describe the procedure for each task separately. As a
point of note, the participant performing the auditory motion
discrimination task also performed the visual temporal order
judgment task. The reasoning for this design choice was that, in
both tasks, the crossmodal effects were expected to occur, and we
planned to correlate the size of the crossmodal effects and the size
of social effects across tasks. 2.3.1. Motion Discrimination Task Pairs of participants were seated next to each other, 60 cm from a
computer screen (resolution: 1920 x 1000 pixels, 64.13 x 33.40
visual degrees, 60 Hz refresh rate, model: ACER V243H, 24
inches) so that when they looked straight ahead they could see
the left or right edge of the computer screen, respectively. The
auditory stimuli were received via speakers (model: Dell A215)
placed next to the computer screen. The speakers were positioned
at a height so that the middle of the speakers would align with
the middle of the screen and were about 80 cm apart from each
other. In front of each participant, a QWERTY keyboard was
positioned for making responses (see Figure 1 for an overview
of the experimental setup). The experiment was run on an Apple
Mac Mini (2012 model), and we used its internal sound card to
play the auditory stimuli. In the motion discrimination task, two beeps (duration: 50 ms)
were presented, one from each speaker, one after the other in
a rapid sequence (interstimulus interval: 100 ms) to create the
apparent perception that stimuli were moving either in the left
or right direction. The frequency of the two beeps was randomly
selected out of a set of three frequencies (450, 500, and 550
Hz). Simultaneously with the presentation of the beeps, two
flashes (duration: 50 ms) were presented that moved either in the
same direction (congruent presentation) or opposite directions
(incongruent presentation) of the auditory stream. The flashes
(radius: 1.34 visual degrees) were presented at a distance of 15
visual degrees from a fixation dot (radius: 0.53 visual degrees) that
was positioned in the center of the computer screen. In control
trials, the flashes and beeps were presented asynchronously (for
an overview of stimuli combinations see Figure 2). That is, in
the asynchronous trials, the presentation of the flashes began
300 ms after the second beep was presented. For each trial, we 2.3. Experimental Procedure FIGURE 2 | Stimuli combinations for the motion discrimination task: Auditory
and visual stimuli could either synchronously (upper left) or asynchronously
(upper right) move in the same direction or opposite directions (lower left and
lower right). If stimuli were presented asynchronously, the first flash was
presented 300 ms after the second auditory stimulus. randomly selected whether stimuli were presented synchronously
or asynchronously. y
y
One of the participants in the pair was required to perform
the same auditory motion discrimination task as in the original
study by Soto-Faraco et al. (2002). That is, they were required to
indicate the movement direction of the beeps. For participants
that sat on the left, they indicated the motion direction using the
“A” key (for leftward motions) and the adjacent “S” (for rightward
motions). For participants that sat on the right, they used the
“K” (for leftward motions) and the adjacent “L” (for rightward
motions). Responses were performed on the two keyboards
placed in front of the participants (i.e., participants sitting on
the left used the left keyboard, whereas participants sitting on
the right used the right keyboard). While participants performed
this task, they were also required to fixate the central dot on the
computer screen. We did not explicitly instruct participants to
turn their heads to the computer screen but often observed this
to be the case as it is a more natural head position to fixate the
central dot. To directly align their heads with the center of the
screen, participants likely turned their heads by about 25 degrees. To ensure that participants maintained fixation, similar to Heed
et al. (2010), there were a small number of catch trials (11 %),
in which the central fixation dot would briefly flash (50 ms)
and no other stimuli were presented. When this happened, the
participant was required to press the “space” key if they sat on
the left and the “enter” key if she/he sat on the right (“fixation
control task”). Participants were instructed to prioritize accuracy
over speed for their responses. As a point of note, any of the keys
would end a trial regardless of the required task. At the beginning of each block, the block type was announced
on the screen (“Joint Block,” “Individual Auditory Block,” or
“Individual Visual Block”), and participants were asked to contact
the experimenter. 2.3. Experimental Procedure Each pair of participants performed the motion discrimination
task and the temporal order judgment task. The order of tasks February 2020 | Volume 11 | Article 79 Frontiers in Psychology | www.frontiersin.org 3 Audiovisual Integration During Joint Action Wahn et al. FIGURE 2 | Stimuli combinations for the motion discrimination task: Auditory
and visual stimuli could either synchronously (upper left) or asynchronously
(upper right) move in the same direction or opposite directions (lower left and
lower right). If stimuli were presented asynchronously, the first flash was
presented 300 ms after the second auditory stimulus. on the seating position, either the “enter” or “space” key would
be required for the fixation control task and either the “A” and
“S” or the “K” and “L” keys would be required for indicating the
moving direction of the visual stimuli. Participants performed their assigned tasks either alone or
jointly (see Figure 3). When they were alone in the room, they
sat in the same seat that they occupied when performing the
task jointly. The seating positions of participants performing the
different tasks were counterbalanced across pairs. As a point of note, as it has been done in earlier studies (Heed
et al., 2010; Wahn et al., 2017a), in the data results section we only
considered the response data of the participant performing the
auditory motion discrimination task, as the crossmodal effects
were expected to occur in the auditory motion discrimination
task (i.e., the visual stimuli were expected to influence the
auditory motion discrimination but not vice versa). Testing
involved
two
sets
of
three
blocks:
visual
discrimination performed alone, auditory motion discrimination
performed alone (by the other participant), and visual and
auditory discrimination tasks performed simultaneously by
the two participants together. The order of the conditions in a
set was randomly selected and then repeated. Each block had
56 trials, composed of 8 fixation control trials and 48 motion
discrimination task trials. Each block was composed of an
equal number of trials for each combination of the factor levels
of Synchrony (synchronous, asynchronous) and Congruency
(congruent, incongruent) trials (e.g., 12 synchronous congruent
trials and 12 synchronous incongruent trials). After the last
required response on a trial, the program automatically
continued to the next trial following a 1,000 ms break. Frontiers in Psychology | www.frontiersin.org 2.3. Experimental Procedure The experimenter would then make the
necessary setup adjustment (e.g., ask one of the participants to
wait outside of the experimental room). The experimenter waited
outside of the experimental room throughout testing. The experiment was programmed using Python 2.7.3. It took
about 20 min to complete. 2.3.2. Temporal Order Judgment Task A trial is completed after
both participants pressed a key. FIGURE 4 | Stimuli combinations for the temporal order judgment task: two
auditory and two visual stimuli were presented. The first visual and auditory
stimulus was presented at the same time. The second auditory stimulus was
either presented together with the second flash (baseline trial) or 100 ms after
the second flash (trailing trial). in the motion discrimination task above, the flashes remained on
the screen after stimulus onset and only disappeared after the
participants’ responses. Also note that we deviated with regard
to a few design choices from the original study by Morein-Zamir
et al. (2003). The original study selected SOAs from a larger set
with a wider range (12, 24, 36, 48, 72, 96, and 144 ms). We
selected SOAs from a smaller range and set (25, 50, 75, and 100
ms) to reduce the overall number of trials. Moreover, the original
study included several types of trailing trials (ranging from 75 to
600 ms). We only used one type of trailing trials, for which the
crossmodal effect was the strongest in the original study (100 ms). As for the motion discrimination task above, each participant
in a pair was assigned to perform a task in one of the sensory
modalities. In particular, one participant in the pair was required
to indicate whether the upper or lower flash occurred first (visual
temporal order judgment task), which was the same task as
performed by participants in the original study (Morein-Zamir
et al., 2003). If the participant was sitting on the left, she/he was
required to use the “A” key to indicate that the top flash came first
and the “Z” key to indicate that the bottom flash occurred first. If the participant was sitting on the right, she/he was required
to use the “K” key to indicate that the top flash occurred first
and the “M” key to indicate that the bottom flash occurred first. While performing the visual temporal order judgment task, the
participant was also required to maintain fixation on the central
fixation dot. As for the motion discrimination task above, during
a small number of trials (3%), the participant was also required to
perform the fixation control task. That is, the central fixation dot
would briefly flash (50 ms) and no other stimuli were presented
during such a trial. 2.3.2. Temporal Order Judgment Task 2.3.2. Temporal Order Judgment Task
In the temporal order judgment task, two flashes (radius: 1.34
visual degrees, 5 ms) were presented in a rapid temporal
sequence. The time between the flash presentations was
randomly selected for each trial out of a set of four stimulus
onset asynchronies (SOAs): 25, 50, 75, and 100 ms. One flash
was presented below and one above the fixation dot (radius:
0.53 visual degrees) at a distance of 15 visual degrees. Whether
the top or the bottom flash was presented first varied randomly
between trials. Simultaneously with the first flash, a click sound
(impulse tone, 5 ms) was presented as well. Depending on the
type of trial, a second click was presented simultaneously with
the second flash (baseline trial) or the second click trailed behind
the second flash by 100 ms (trailing trial) (for an overview of
all stimuli combinations, see Figure 4). One of the clicks was
presented from the left loudspeaker and the other from the right
loudspeaker. Baseline and trailing trials were selected randomly, The other participant in the pair was required to perform the
fixation control task and indicate the movement direction of the
flashes (“visual motion discrimination task”). Again, depending February 2020 | Volume 11 | Article 79 4 Wahn et al. Audiovisual Integration During Joint Action FIGURE 3 | Example of a synchronous congruent trial for the joint condition: Participants receive a flash and beep presented on the left side followed by a flash and
beep presented on the right side. In this example pair, the left participant is required to indicate the flash direction (left or right) while the participant sitting on the right
is required to indicate the beep direction (left or right). Arrows indicate that participants were also required to fixate the center of the screen. A trial is completed after
both participants pressed a key. FIGURE 3 | Example of a synchronous congruent trial for the joint condition: Participants receive a flash and beep presented on the left side followed by a flash and
beep presented on the right side. In this example pair, the left participant is required to indicate the flash direction (left or right) while the participant sitting on the right
is required to indicate the beep direction (left or right). Arrows indicate that participants were also required to fixate the center of the screen. Frontiers in Psychology | www.frontiersin.org 2.3.2. Temporal Order Judgment Task Also, for this task, we did not explicitly
instruct participants to turn their heads toward the computer
screen but often observed this to be the case as it is a more natural
head position to fixate the central dot. For these fixation control
task trials, depending on the seating position, the participant was
required to press “space” (sitting of the left) or “enter” (sitting on
the right). FIGURE 4 | Stimuli combinations for the temporal order judgment task: two
auditory and two visual stimuli were presented. The first visual and auditory
stimulus was presented at the same time. The second auditory stimulus was
either presented together with the second flash (baseline trial) or 100 ms after
the second flash (trailing trial). as were which speaker delivered which sound. Given earlier
findings by Morein-Zamir et al. (2003), we expected crossmodal
effects to occur in the trailing trials as the trailing click should
affect the temporal perception of the second flash, “pulling” the
perception of the two flashes apart. As a point of note, the stimuli presentations of the clicks (left
and right) and flashes (top and bottom) were deliberately chosen
to be orthogonal to avoid any spatial crossmodal influences
between stimuli. Moreover, contrary to the stimuli presentations February 2020 | Volume 11 | Article 79 Frontiers in Psychology | www.frontiersin.org 5 Audiovisual Integration During Joint Action Wahn et al. auditory motion discrimination task and visual temporal order
judgment task), we used baseline data from the conditions where
no crossmodal effects were expected to occur. For the auditory
motion discrimination task in particular, we used the data from
the incongruent asynchronous presentations when a participant
performed their task alone in the room. For the visual temporal
order judgment task, we used the baseline trials with the longest
SOA (100 ms) when a participant was alone in the room. For
both these situations, and for the fixation control tasks, we set
the inclusion criteria to a performance above 70%. The other participant in the pair was required to indicate
which of the clicks occurred first (auditory temporal order
judgment task). If the participant was sitting on the left, pressing
the “A” key would be required to indicate that the click played on
the left speaker occurred first and the adjacent “S” key when the
click played on the right speaker occurred first. 3.1. Auditory Motion Discrimination Task 3.1. Auditory Motion Discrimination Task
Based on Soto-Faraco et al. (2002), we expected the factors
Congruency and Synchrony to interact. The rationale is that
the performance difference between incongruent and congruent
presentations should be larger for the synchronous than the
asynchronous presentations because it is during the synchronous
presentations that the incongruent visual signals should distract
participants from accurately judging the sound motion. For
social factors to influence this crossmodal effect, we would
expect a significant interaction between the factors Congruency,
Synchrony, and Social Condition. The experiment took about 40 min to complete. It was
programmed in Python 2.7.3. Frontiers in Psychology | www.frontiersin.org 2.3.2. Temporal Order Judgment Task Four of these were trials for the fixation control task. Half of the remaining trials were trailing trials, and the other
half were baseline trials. After responses were made, the program
automatically continued to the next trial after a 1,000 ms break. Again, the experimenter waited outside of the experimental
room throughout testing, making the necessary setup adjustment
between blocks. 2.3.2. Temporal Order Judgment Task If the participant
was sitting on the right, the “K” (for left) and “L” (for right)
were required. While performing the auditory temporal order
judgment task, the participant was also required to maintain
fixation at the central dot and to also perform the fixation
control task. As for the motion discrimination task above, all
participants were instructed to prioritize accuracy over speed for
their responses for all tasks. We aimed to match the sample size of our current study to the
sample size of earlier studies investigating social manipulations
in crossmodal tasks, which was 11 in the case of Heed et al. (2010) and 12 in the case of Wahn et al. (2017a). Moreover, we
counterbalanced the seating position and task order across pairs
such that we have an equal number of pairs for each combination
of these factors. We also sought to have a sample of participants
that were able to accurately perform the motion discrimination
task, temporal order judgment task, and fixation control task. Our data collection ran until all these criteria were fulfilled for a
sample size of 12 pairs (21 females and 3 males, M = 19.67 years,
SD = 1.68 years). An additional 7 pairs (11 females, 3 males, M =
19.42 years, SD = 1.03 years) did not fulfill our inclusion criteria. The fixation control task was performed at a high accuracy in
the final sample both in the motion discrimination experiment
(M = 97% correct, SD = 4.81%) and temporal order judgment
experiment (M = 96% correct, SD = 3.08%). As for the motion discrimination task above and in line with
earlier studies investigating the effect of joint task performance
on multisensory integration (Heed et al., 2010; Wahn et al.,
2017a), we only considered the response data of participants
performing the visual temporal order judgment task since
crossmodal effects were only expected to occur in this task. Indeed, like Morein-Zamir et al. (2003), we did not collect trailing
visual stimuli to assess the influence of visual signals on auditory
temporal order judgments. As before, the experiment was divided into two sets of three
blocks: one block for each participant to perform the visual or
auditory temporal order judgment task while alone in the room
and one block for the two tasks to be performed simultaneously
while together in the room (see Figure 5). Each block contained
136 trials. 2.4. Data Pre-processing For our data analysis later on, in line with earlier studies and
as noted in the task procedure, we only considered data of the
participants performing the tasks in which crossmodal effects
were expected to occur (i.e., the auditory motion discrimination
task and visual temporal order judgment task). y
y
Figure 6 (upper panels) displays the response accuracy for
all combinations of these factors, including the task order. On a descriptive level, we observed large differences between
congruent and incongruent presentations for the synchronous
trials, suggesting that the crossmodal effects that found in the
original study Soto-Faraco et al. (2002) were replicated in the
present study. With regard to our social manipulation, we
observed that the difference between congruent and incongruent
presentations for synchronous trials was not modulated by
whether a task was performed alone or jointly. Lastly, we did
not observe any order effects (i.e., that the pattern of results for
performing the motion discrimination task first or second did
not differ). To briefly confirm this expectation, at least for the motion task
(as an analysis for the auditory temporal order judgment task is
not feasible as noted above), we assessed the performance in the
visual motion discrimination task for the synchronous individual
condition and found a high accuracy performance regardless of
the type of presentations (congruent: M = 0.92 vs. incongruent:
M = 0.90). We also found that there was no significant difference
between congruent and incongruent presentations [t(11) = –1.16,
p = 0.269], suggesting that there were no crossmodal effects
present in the visual motion discrimination task. In the following,
only data from the auditory motion discrimination and visual
temporal order judgment task were considered. To confirm whether these observations were significant,
we analyzed participants’ performance using a four-factorial
ANOVA with the response accuracy as the dependent variable. To assess participants’ general performance accuracy for the
two tasks we primarily considered for the analysis (i.e., the February 2020 | Volume 11 | Article 79 6 Wahn et al. Audiovisual Integration During Joint Action FIGURE 5 | Example of baseline trial in the joint condition: Participants first receive a flash presented at the bottom and simultaneously a click sound on the left. Then,
a second flash is presented (the first one remains on the computer screen) simultaneously with a second click on the right. 2.4. Data Pre-processing In particular with regard to crossmodal effects, there was no
significant interaction effect between the factors Congruency
and Synchrony [F(1, 10) = 1.18, p = 0.302], suggesting there
were no crossmodal effects present from the perspective of the
response times. In sum, we replicated earlier crossmodal effects by Soto-
Faraco et al. (2002), finding that for synchronous presentations
visual stimuli affected auditory motion judgments more than for
asynchronous presentations. We did not find that performing a
task jointly modulated this effect. In fact, our calculated Bayes
factor suggests that the null hypothesis that there is no effect is
considerably more likely than the alternative hypothesis. As the absence of a significant interaction effect between the
factors Congruency, Synchrony, and Condition [F(1, 10) = 0.11, p
= 0.746] suggested that performing a task jointly did not affect
audiovisual integration, we also computed a Bayes factor using
the R package “BayesFactor” (Morey et al., 2015) for this effect
to assess how much more likely the null hypothesis was relative
to the alternative hypothesis. We found a Bayes factor of 0.30,
meaning that our data was 1/0.30 or 3.33 times more likely under
the null hypothesis than the alternative hypothesis. 3.2. Visual Temporal Order Judgment Task
For analyzing the data of the visual temporal order judgment
task, we followed the same analysis procedure as in the
original study by Morein-Zamir et al. (2003). That is, using a
logistic regression, we fitted psychometric curves to each of the
participants’ responses, separately for each condition. Based on
these fits, we extracted for each participant the just noticeable
difference (JND). These JNDs were used as a dependent variable
for our further analyses. To test for the possibility of whether an auditory stimulus
moving toward where the participant was sitting or away from
the participant may have interacted with our Social Condition
factor, we repeated the above ANOVA with the additional factor
Auditory Moving Direction (Away and Toward). Apart from
the effects already found above (i.e., a significant main effects
for the factors Congruency [F(1, 10) = 65.55, p < 0.001, η2
G =
0.54] and Asynchrony [F(1, 10) = 41.60, p < 0.001, η2
G = 0.34]
and a significant interaction effect between these two effects To replicate the crossmodal effect found by Morein-Zamir
et al. (2003), we expected a main effect for the factor Timing
with the levels baseline and trailing. 2.4. Data Pre-processing In this example seating arrangement, the
left participant is required to indicate which click came first and the right participants is required to indicate which flash came first. Arrows indicate that participants
were also required to fixate the center of the screen. A trial is completed after both participants pressed a key. FIGURE 5 | Example of baseline trial in the joint condition: Participants first receive a flash presented at the bottom and simultaneously a click sound on the left. Then,
a second flash is presented (the first one remains on the computer screen) simultaneously with a second click on the right. In this example seating arrangement, the
left participant is required to indicate which click came first and the right participants is required to indicate which flash came first. Arrows indicate that participants
were also required to fixate the center of the screen. A trial is completed after both participants pressed a key. [F(1, 10) = 184.27, p < 0.001, η2
G = 0.49], no other effects in the
ANOVA were significant (all ps > 0.068). The three within-subject factors were Congruency (Incongruent
and Congruent), Synchrony (Synchrony and Asynchrony), and
Social Condition (Alone and Joint). The between-subject factor
was the Task Order (First and Second). We found main effects
for the factors Synchrony [F(1, 10) = 41.60, p < 0.001, η2
G =
0.43] and Congruency [F(1, 10) = 65.55, p < 0.001, η2
G = 0.63]. Replicating crossmodal effects from the original study (Soto-
Faraco et al., 2002), we found an interaction effect between
these two factors [F(1, 10) = 184.27, p < 0.001, η2
G = 0.58]. Given that our ANOVA only involved factors with two levels,
there were no follow-up tests required since the interaction
effect already tested the pairwise comparison. To describe
this interaction in more detail, the difference in performance
accuracies between congruent and incongruent presentations
was significantly larger for synchronous presentations than for
asynchronous presentations. All other effects in the ANOVA were
not significant (all ps > 0.129). Apart from assessing response accuracy, we also repeated the
same ANOVA above for the response times as the dependent
variable. We found no significant effects for this ANOVA. Frontiers in Psychology | www.frontiersin.org 2.4. Data Pre-processing That is, we expected that
participants were significantly better at judging which of the
two flashes occurred first for trailing trials compared to baseline
trials. For a social effect to occur, we expected an interaction
effect between the factors Social Condition (Alone, Joint) and
Timing (Baseline, Trailing). Plotting the averaged JNDs as a February 2020 | Volume 11 | Article 79 Frontiers in Psychology | www.frontiersin.org 7 Audiovisual Integration During Joint Action Wahn et al. FIGURE 6 | Results overview for the motion discrimination task (upper two panels) and the temporal order judgment task (lower two panels) when the respective task
is performed first (left column) or second (right column). Error bars are standard error of the mean in all panels. FIGURE 6 | Results overview for the motion discrimination task (upper two panels) and the temporal order judgment task (lower two panels) when the respective task
is performed first (left column) or second (right column). Error bars are standard error of the mean in all panels. function of these factors, including Task Order (see Figure 6,
lower panels), we observed large differences between baseline and
trailing trials, suggesting that we replicated the earlier crossmodal
effect. Yet, the size of this crossmodal effect (i.e., the difference
in JND between baseline and trailing trials) did not appear
to be modulated by whether the task was performed jointly
or alone. However, there was the suggestion that the effect of
the Social Condition was modulated by Task Order, with the
JNDs somewhat raised for the joint condition compared to the
individual condition when the temporal order judgment task was
performed first. were no interaction effects, including, most importantly, no
significant Social Condition x Timing interaction effect, [F(1, 10)
= 1.06, p = 0.327]. For this interaction, we again computed a
Bayes factor to assess how more likely the null hypothesis is
compared to the alternative hypothesis given the present data. We observed a Bayes factor of 0.47, meaning that our data were
1/0.47 or 2.15 times more likely under the null hypothesis than
the alternative hypothesis. In sum, we replicated the crossmodal effect found by Morein-
Zamir et al. (2003). As for the motion discrimination task
above, we found that audiovisual integration was not affected by
performing the task jointly rather than alone. Frontiers in Psychology | www.frontiersin.org 2.4. Data Pre-processing In fact, the null
hypothesis that there is no effect was more than two times more
likely than the alternative hypothesis. To assess whether these observations were statistically
significant, we performed a three factorial ANOVA with the
JNDs as the dependent variable and the within-subject factors
Social Condition (Alone, Joint) and Timing (Baseline, Trailing)
and the between-subject factor Task Order. We found a main
effect of Timing [F(1, 10) = 39.05, p < 0.001, η2
G = 0.24]. All
the other effects were not significant (all ps > 0.124). There Similar to the auditory motion discrimination task, we also
tested whether the click starting position (either starting on the
participant’s side or opposite side) interacted with our social
manipulation. For this purpose, we repeated the ANOVA above February 2020 | Volume 11 | Article 79 Frontiers in Psychology | www.frontiersin.org 8 Audiovisual Integration During Joint Action Wahn et al. with the additional factor Auditory Starting Position (Same and
Opposite). We again found a significant main effect of Timing
[F(1, 10) = 67.02, p < 0.001, η2
G = 0.26]. Moreover, we found
a significant main effect of Auditory Starting Position [F(1, 10)
= 7.70, p = 0.012, η2
G = 0.03] and a significant interaction
effect between Timing and Auditory Starting Position [F(1, 10) =
7.84, p = 0.019, η2
G = 0.03]. Yet, none of the effects involving
the factor Social Condition (Alone and Joint) were significant
(all ps > 0.155). A major difference between the present task and crossmodal
congruency tasks investigated earlier is that crossmodal effects
in the present study were quantified in both tasks via perceptual
judgments (i.e., response accuracies), while in the earlier studies
effects were quantified (at least in part) with response times. Moreover, in the audiovisual crossmodal congruency task
investigated earlier (Wahn et al., 2017a), the effect of task
co-performance was also only present for the response times
while response accuracies were unaffected. Given that response
accuracies were also not affected by joint task performance
in the present study for both tasks, one could also suggest
that performing a task jointly specifically affected crossmodal
effects quantified with response times while performance
accuracies remain unaffected. An outstanding question for future
investigation is whether those past response time crossmodal
effects reflect multisensory integration at all or merely the speed
at which one of the signals reaches the response threshold. 2.4. Data Pre-processing The fact that no social effect has been observed in response
accuracy suggests that there may not be any social effect on
multisensory integration. with the additional factor Auditory Starting Position (Same and
Opposite). We again found a significant main effect of Timing
[F(1, 10) = 67.02, p < 0.001, η2
G = 0.26]. Moreover, we found
a significant main effect of Auditory Starting Position [F(1, 10)
= 7.70, p = 0.012, η2
G = 0.03] and a significant interaction
effect between Timing and Auditory Starting Position [F(1, 10) =
7.84, p = 0.019, η2
G = 0.03]. Yet, none of the effects involving
the factor Social Condition (Alone and Joint) were significant
(all ps > 0.155). (
p
)
Given that we replicated earlier crossmodal effects in both
tasks, we also correlated the sizes of these effects across tasks. For each task we averaged the data across the levels of all
factors except for Timing in the temporal order judgment
task and Congruency in the motion discrimination task. For the temporal order judgment task, we then computed
the difference between the baseline and trailing condition. For
the
motion
discrimination
task,
we
computed
the
difference between the congruent and incongruent condition. Correlating these differences, we found a moderately sized
correlation, which was not significant [r
= –0.39, t(10)
= –1.35, p = 0.204]. For this correlation, we found a
Bayes factor of 0.80, meaning that our data were 1/0.80 or
1.15 times more likely under the null hypothesis than the
alternative hypothesis. An alternative explanation for the divergent findings between
the present data and previous work may rest with the difference
in task demands between the present study and earlier studies. In particular, in earlier studies participants were required to
localize static stimuli whereas in the present study participants
were required to judge the movement direction of stimuli and
their temporal order. Given this difference between tasks, one
possibility could be that participants are only able to “off-load”
stimuli to a co-actor if the task primarily involves static spatial
stimuli (as in the earlier studies Heed et al., 2010; Wahn et al.,
2017a) while this is not possible for moving stimuli or temporal
stimuli. In other words, stimuli may be required to be spatial and
static for co-actors to be able to “off-load” these stimuli to other
co-actors. 2.4. Data Pre-processing Possibly, the mechanism of task co-representation,
which was suggested to have allowed participants to filter out
distracting stimuli in earlier studies; (Heed et al., 2010; Wahn
et al., 2017a) could be specific to static spatial stimuli. Future
studies could test this proposal by investigating whether joint
task performance also does not affect multisensory integration in
other tasks requiring spatial processing of moving stimuli (e.g., in
an audiovisual bounce-inducing effect; Sekuler, 1997; Grassi and
Casco, 2009) or temporal processing (e.g., in the sound-induced
flash illusion; Shams et al., 2002). February 2020 | Volume 11 | Article 79 Frontiers in Psychology | www.frontiersin.org REFERENCES Meredith, M. A., Nemitz, J. W., and Stein, B. E. (1987). Determinants of
multisensory integration in superior colliculus neurons. I. Temporal factors. J. Neurosci. 7, 3215–3229. doi: 10.1523/JNEUROSCI.07-10-03215.1987 Alsius, A., Navarra, J., Campbell, R., and Soto-Faraco, S. (2005). Audiovisual
integration of speech falters under high attention demands. Curr. Biol. 15,
839–843. doi: 10.1016/j.cub.2005.03.046 Meredith, M. A. and Stein, B. E. (1996). Spatial determinants of multisensory
integration in cat superior colliculus neurons. J. Neurophysiol. 75, 1843–1857. doi: 10.1152/jn.1996.75.5.1843 Bertelson, P., Vroomen, J., De Gelder, B., and Driver, J. (2000). The ventriloquist
effect does not depend on the direction of deliberate visual attention. Percept. Psychophys. 62, 321–332. doi: 10.3758/BF03205552 Miller, J. (1982). Divided attention: evidence for coactivation with redundant
signals. Cogn. Psychol. 14, 247–279. doi: 10.1016/0010-0285(82)90010-X Böckler, A., Knoblich, G., and Sebanz, N. (2012). Effects of a coactor’s focus of
attention on task performance. J. Exp. Psychol. Hum. Percept. Perform. 38:1404. doi: 10.1037/a0027523 Morein-Zamir, S., Soto-Faraco, S., and Kingstone, A. (2003). Auditory capture
of vision: examining temporal ventriloquism. Cogn. Brain Res. 17, 154–163. doi: 10.1016/S0926-6410(03)00089-2 Morey, R. D., Rouder, J. N., and Jamil, T. (2015). Bayesfactor: Computation of Bayes
Factors for Common Designs. R package version 0.9. 12-2 [Computer software]. Available online at: https://CRAN.R-project.org/package=BayesFactor Grassi, M., and Casco, C. (2009). Audiovisual bounce-inducing effect: Attention
alone does not explain why the discs are bouncing. J. Exp. Psychol. Hum. Percept. Perform. 35:235. doi: 10.1037/a0013031 Guski, R., and Troje, N. F. (2003). Audiovisual phenomenal causality. Percept. Psychophys. 65, 789–800. doi: 10.3758/BF03194815 Santangelo, V., and Macaluso, E. (2012). “spatial attention and audiovisual
processing,” in The New Handbook of Multisensory Processes, ed B. E. Stein
(Cambridge, MA: The MIT Press), 359–370. Heed, T., Habets, B., Sebanz, N., and Knoblich, G. (2010). Others’ actions
reduce crossmodal integration in peripersonal space. Curr. Biol. 20, 1345–1349. doi: 10.1016/j.cub.2010.05.068 Santangelo, V., and Spence, C. (2008). Crossmodal attentional capture in
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155–165. doi: 10.1080/13506280701453540 Holmes, N. P., and Spence, C. (2005). Multisensory integration: space, time and
superadditivity. Curr. Biol. 15, R762–R764. doi: 10.1016/j.cub.2005.08.058 Santangelo, V., and Spence, C. (2009). Crossmodal exogenous orienting improves
the accuracy of temporal order judgments. Exp. Brain Res. 194, 577–586. doi: 10.1007/s00221-009-1734-4 Karlinsky, A., Lam, M. Y., Chua, R., and Hodges, N. J. (2017). Whose turn is
it anyway? the moderating role of response-execution certainty on the joint
simon effect. Psychol. Res. 83, 833–841. 4. DISCUSSION February 2020 | Volume 11 | Article 79 Frontiers in Psychology | www.frontiersin.org 9 Wahn et al. Audiovisual Integration During Joint Action integration in other tasks that involve more richer audiovisual
stimuli (than static spatial stimuli). Another difference to
consider, which is specific to the temporal order judgment task
in relation to the spatial congruency tasks in earlier studies
(Heed et al., 2010; Wahn et al., 2017a), concerns the direction
of the crossmodal effects (i.e., whether stimuli in the visual
sensory modality influence processing stimuli in a different
sensory modality or vice versa). That is, in the temporal order
judgment task crossmodal effects were present for stimuli in the
visual sensory modality (i.e., auditory information influenced
visual processing) whereas in the earlier studies crossmodal
effects were either present in the auditory or tactile sensory
modalities due to an influence of visual stimuli. Hence, one
could raise the possibility that joint task performance only
affects multisensory integration in tasks, where the visual sensory
modality is affecting processing in other sensory modalities
but not vice versa. Yet, given that we did not find an
effect of joint task performance for the audiovisual motion
discrimination task—a task in which visual stimuli affect auditory
processing—this proposal may only apply to static stimuli in
crossmodal tasks. demands (i.e., whether tasks require processing stimuli in terms
of location, motion, or timing). ACKNOWLEDGMENTS We want to thank Michelle Tomaszewski, Jane Kim, and
Katherine Chow with their help with the data collection. 4. DISCUSSION In the present study, we replicated earlier crossmodal effects
in a motion discrimination task (Soto-Faraco et al., 2002) and
temporal order judgment task (Morein-Zamir et al., 2003);
auditory judgments of motion were affected by visual input,
and visual judgments of timing were affected by auditory
input. In the case of the motion discrimination task, we
found that participants’ performances were significantly worse
when incongruent rather than congruent visual information was
presented. For the temporal order judgment task, participants
were better at judging the order of the flashes if a click trailed
the second flash. These findings demonstrate that these known
crossmodal effects were robust, persisting despite the many
design changes we made, the most profound ones being that we
introduced social situations where two participants performed
their respective tasks together. Related to the difference in task demands between the present
study and those earlier, whether joint task performance affects
multisensory integration or not may also depend on the strength
of the integration of the investigated multisensory effect. In
particular, for the motion discrimination task, the integrated
moving stimuli may more strongly be integrated (and hence less
susceptible to effects of joint task performance) as the received
stimuli provide more cues (i.e., spatial and motion information)
to be integrated. Similarly, for the temporal order judgment
task, the mere fact of presenting two audiovisual stimuli may
have resulted in a stronger integration of stimuli that is less
susceptible to the effects of joint task performance. Future
studies could test this proposal by investigating whether joint
performance task performance also does not affect multisensory On this last score, despite the introduction of joint task
situations, and contrary to previous findings that a joint
performance modulated crossmodal spatial congruency effects
(Heed et al., 2010; Wahn et al., 2017a), we found that our robust
crossmodal motion and timing effects were not modulated by
joint task performance. These findings highlight the importance
of investigating the effects of social factors across a range of
crossmodal paradigms. In other words, as we had noted in
the introduction, one cannot assume that because social factors
impact crossmodal performance in one particular paradigm,
social factors will affect crossmodal performance in all situations. Below, we speculate why the present tasks are resistant to
social manipulations and how one might test our proposals in
the future. FUNDING In summary, we successfully replicated earlier crossmodal
temporal and motion effects, in which participants were required
to perform perceptual judgments. Yet, the present work fails
to find evidence that joint task performance modulates these
replicated crossmodal effects. Given that earlier studies found
an effect of joint task performance for crossmodal spatial
congruency tasks (Heed et al., 2010; Wahn et al., 2017a), we
suggest that the effect of joint task performance on crossmodal
tasks could potentially depend on how crossmodal effects are
quantified (i.e., via responses times or accuracies) and task We acknowledge the support of a DFG research fellowship (WA
4153/2-1) awarded to BW, and a NSERC Discovery grant and
SSHRC Insight grant awarded to AK. We acknowledge the support of a DFG research fellowship (WA
4153/2-1) awarded to BW, and a NSERC Discovery grant and
SSHRC Insight grant awarded to AK. DATA AVAILABILITY STATEMENT The datasets collected for this study are available on request to
the corresponding author. ETHICS STATEMENT The studies involving human participants were reviewed and
approved by University of British Columbia’s ethics committee. The participants provided their written informed consent to
participate in this study. AUTHOR CONTRIBUTIONS BW, JD, and AK: Study design, wrote the manuscript and revised
the manuscript. BW: Programmed the experiments and analyzed
the data. REFERENCES doi: 10.1007/s00426-017-0901-7 Sebanz, N., Bekkering, H., and Knoblich, G. (2006). Joint action: bodies and
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general mechanisms for coordinating with others. Front. Psychol. 7:2039. doi: 10.3389/fpsyg.2016.02039 Copyright © 2020 Wahn, Dosso and Kingstone. 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
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Tear fluid and complement activation products in tears after ocular surgery
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BMC ophthalmology
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Abstract Purpose Due to technological advancements, surgical invasiveness has been reduced. However, cataract surgery
has been implicated in causing postoperative inflammation, including dry eye syndrome. The innate immune system
may be involved in postoperative inflammation, and complement activation could potentially play a crucial role
in defense against pathogens, homeostasis, and wound healing. To investigate changes in the tear film complement
activation products (CAPs) and ocular surface after vitrectomy combined with cataract surgery. Methods Forty-three patients (23 women; median age, 69 years) were enrolled in this prospective study and under-
went phacoemulsification and vitrectomy. We measured Schirmer’s test (ST) and CAPs in the tears at baseline (the day
before surgery), 4 days and 1 month after the surgery. Tears were collected in microtubes. The CAPs in the tear fluid
were analyzed by cytometric bead array. Results The median ST (8.5 mm) at baseline increased to 16 mm at 4 days ( P < 0.001) and 10 mm at 1 month
(P = 0.44). The C3a levels (1202 pg/ml) at baseline increased to 2753 pg/ml at 4 days (P < 0.001), and 1763 pg/ml
at 1 month (P = 0.049). The C4a levels (476 pg/ml) at baseline increased to 880 pg/ml at 4 days (P < 0.001), and 657 pg/
ml at 1 month (P = 0.013). The C5a levels (22.6 pg/ml) at baseline increased to 470.9 pg/ml at 4 days (P < 0.001),
and 38.3 pg/ml at 1 month (P = 0.0048). The surgical eyes were divided into the short ST group (≦ 10 mm, n = 22)
and long ST group (> 10 mm, n = 21) based on the preoperative ST values. At 1 month postoperatively, the C3a levels
were 2194 pg/ml in the preoperative short ST group and 1391 pg/ml in the long ST group, with significantly higher
C3a concentrations in the short ST group (P < 0.001). Conclusions The CAPs levels in tears increased after vitrectomy combined with cataract surgery. A preoperative defi-
cit in tear secretion might induce prolonged complement activation and delayed recovery of ocular surface param-
eters postoperatively. Keywords Tears, Schirmer’s test, Complement activation products, Ocular surface, Tear, Inflammation BMC Ophthalmology BMC Ophthalmology Maehara et al. BMC Ophthalmology (2023) 23:329
https://doi.org/10.1186/s12886-023-03037-6 Open Access © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the
original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or
other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this
licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco
mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Introduction In developed countries, the number of cataract patients is
increasing due to the growing elderly population. There is
also a rise in the prevalence of dry eye among older indi-
viduals[1–3]. Ophthalmologists often perform cataract
surgery on patients with dry eye. While advancements
in technology have made surgery less invasive, certain
factors such as light exposure during surgery, disinfect-
ant toxicity, and evaporation from the ocular surface can *Correspondence:
Tetsuju Sekiryu
sekiryu@fmu.ac.jp
1 Department of Ophthalmology, Fukushima Medical University School
of Medicine, Hikarigaoka 1, Fukushima 960‑1247, Japan
2 Department of Immunology, Fukushima Medical University School
of Medicine, Fukushima, Japan *Correspondence:
Tetsuju Sekiryu
sekiryu@fmu.ac.jp
1 Department of Ophthalmology, Fukushima Medical University School
of Medicine, Hikarigaoka 1, Fukushima 960‑1247, Japan
2 Department of Immunology, Fukushima Medical University School
of Medicine, Fukushima, Japan © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the
original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or
other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this
licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco
mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Tear fluid and complement activation
products in tears after ocular surgery
Hiroki Maehara1, Koki Norikawa1, Keiichiro Tanaka1, Yutaka Kato1, Akihito Kasai1, Tomoko Omori2
Takeshi Machida2, Hideharu Sekine2 and Tetsuju Sekiryu1* Hiroki Maehara1, Koki Norikawa1, Keiichiro Tanaka1, Yutaka Kato1, Akihito Kasai1, Tomoko Omori2,
Takeshi Machida2, Hideharu Sekine2 and Tetsuju Sekiryu1* Open Access T Maehara et al. BMC Ophthalmology (2023) 23:329 Page 2 of 6 contribute to postoperative dry eye [4–8]. This can be
problematic for both surgeons and patients, but there is
no established treatment for it.h underwent combined vitrectomy and cataract surgery. Therefore, in this study, we examined tear secretion and
analyzed the changes in levels of C3a, C4a, and C5a in
tears of these patients following the surgical procedures. The innate immune system may play a role in post-
operative inflammation, and complement, an impor-
tant component of this system, is involved in protecting
against foreign pathogens, maintaining homeostasis, and
promoting wound healing [9–11]. Complement activa-
tion occurs through three pathways: the classical, lec-
tin, and alternative pathways. Complement is associated
with various acute inflammatory processes and helps in
defending the host against infections [9–11]. The classi-
cal pathway of complement activation is initiated by IgM
and IgG immunoglobulins. This leads to the cleavage
of C4 and C2, resulting in the activation of C3 and C5. Additionally, the classical pathway can also be activated
through the Lectin pathway without the involvement of
immunoglobulins. The alternative pathway, on the other
hand, operates independently of antibodies and can rap-
idly activate complement even on the ocular surface. When tissue is damaged, it contains proteases that pro-
mote the activation of C3 and C5 [10]. C3 is then cleaved
to generate C3a and C3b, while C5 is activated to form
C5a and C5b. Ultimately, the complement pathway forms
a membrane attack complex (MAC), which leads to cell
lysis and death [11]. C3a is known to act as a mediator
in inflammatory reactions, affecting polymorphonuclear
leukocytes, vasoconstriction, and causing leakage. It is
responsible for increased vascular permeability [12]. C5a has been found to mobilize neutrophils and acti-
vate basophils, monocytes, and neutrophils, resulting in
increased vascular permeability [12, 13]. In ophthalmol-
ogy, the complement system is involved in age-related
macular degeneration [14–18], bacterial keratitis [19],
and allergic conjunctivitis [20]. In Sjögren’s syndrome
and dry eye, tear fluid secretion is decreased, caus-
ing inflammation of the ocular surface, which has been
reported to be associated with complement activation
products (CAPs) [21, 22]. Patients and methodsh The study protocol (ID: 30,046) was approved by the
Institutional Review Board of Fukushima Medical Uni-
versity, Fukushima, Japan. The research was conducted
in accordance with the principles of the Declaration of
Helsinki. Written informed consent was obtained from
all patients after providing them with a detailed explana-
tion of the study procedures and potential consequences
of participation. The study included consecutive patients
who underwent combined vitrectomy and cataract sur-
gery for epiretinal membranes and macular holes at
Fukushima Medical University Hospital. No patients had
received any treatments for dry eye prior to the study. The surgeries were performed by 2 experienced surgeons
(YK and AK). Preoperative use of dry eye treatments was
not reported among the patients.h The exclusion criteria consisted of a history of previ-
ous eye surgeries, the presence of severe conjunctival
chalasis, superior limbic keratoconjunctivitis, lid-wiper
epitheliopathy, or pterygium. Other exclusion criteria
involved systemic diseases that could potentially impact
the condition of the ocular surface, such as type 2 diabe-
tes, rheumatoid arthritis, and Sjögren’s syndrome. Vitrectomy combined with cataract surgeryli Levofloxacin 1.5% eye drops (Pfizer, New York, NY, USA)
were applied 4 times daily for 3 days preoperatively in all
cases. Immediately before surgery, 0.25% polyvinyl alco-
hol iodine (Rohto Nitten, Nagoya, Japan) was applied,
and the surgical field was disinfected according to the
package insert. Vitrectomy combined with cataract surgery was per-
formed using the Constellation Vision System (Alcon
Japan Pharma, Tokyo, Japan). Cataract surgery was
performed through a 2.4-mm corneal incision, and an
intraocular lens was inserted. A 25-gauge pars plana vit-
rectomy was performed under a non-contact wide-view-
ing system (Resight Fundus Imaging System; Carl Zeiss
Meditec Japan Co., Ltd, Tokyo, Japan). During the sur-
gery, saline was applied to the surgical field to keep the
ocular surface hydrated. Postoperatively, the puls plan-
ner wound was sutured with 8–0 Vicryl (Mani, Tochigi,
Japan) in all cases.l In wound healing, hemostasis, debris clearance, and
tissue reconstruction are necessary. At the site of wound
healing, platelet aggregate at the wound to establish
hemostasis. Degranulation of platelets then induces C3
[12], which generates C3a and C5a that cause migra-
tion, activation, and aggregation of mast cells, polymor-
phonuclear cells, and macrophages [23, 24], all involved
in tissue clearance. This cellular response after comple-
ment activation may be associated with the wound-
healing process in intraocular surgery. Investigating the
relationship between the complement activated proteins
and ocular surface parameters postoperatively may pro-
vide clues to inflammation. We hypothesized that post-
operative inflammation would increase in patients who Postoperative eye drops were instilled, i.e., levofloxacin
1.5% eye drop for 2 weeks, 0.1% fluorometholone (Rohto
Nitten) for 4 weeks, and bromfenac ophthalmic solution
0.1% (Nisshin, Yamagata, Japan) for 12 weeks postopera-
tively in all cases. Page 3 of 6 Maehara et al. Vitrectomy combined with cataract surgeryli BMC Ophthalmology (2023) 23:329 Table 1 Changes of clinical dates during follow-up period
IQR interquartile range, ST schirmer’s test
P value vs
baseline
P value vs 4 days
base line [Median (IQR)]
ST (mm)
8.5 (4–15)
-
-
C3a (pg/ml) 1202 (860–2304)
-
-
C4a (pg/ml) 476 (196–742)
-
-
C5a (pg/ml) 22.6 (15.6–36.0)
-
-
4 days [Median (IQR)]
ST (mm)
16 (7.5–20)
< 0.001
-
C3a (pg/ml) 2753 (1710–
4453)
< 0.001
-
C4a (pg/ml) 880 (479–1464)
< 0.001
-
C5a (pg/ml) 470.9 (98.4–
1061.4)
< 0.001
-
1 month [Median (IQR)]
ST (mm)
10 (5–15)
0.44
< 0.001
C3a (pg/ml) 1763 (1234–
2464)
0.049
< 0.001
C4a (pg/ml) 657 (295–974)
0.013
0.0011
C5a (pg/ml) 38.3 (18.0–66.8)
0.0048
< 0.001 Table 1 Changes of clinical dates during follow-up period The Schirmer’s test and tear fluid collection
and measurements The Schirmer’s test (ST) on the day before surgery (base-
line) and 4 days and 1 month postoperatively to investi-
gate lacrimal fluid secretion. Tear fluid collection was
carried out using a non-invasive method during the
evaluation of the ocular surface, following established
procedures mentioned in previous reports [25]. The same
investigator (HM) collected the tear fluid in all cases. To
avoid the effect of eye drops on the ocular surface meas-
urements, tear collection was performed at least 1 h after
the eye drops were instilled. The patient reclined on a
bed in a supine position, while the clinician utilized a
micropipette (Drummond Scientific, Broomall, PA, USA)
to collect 2 μl of tear fluid from the outer edges of the
eyelids. The collected tear fluid was immediately stored
at -80° C until analysis. The collected tears were meas-
ured using the BD CBA Human Anaphylatoxin Kit (BD
Biosciences, San Jose, CA, USA) for CAPs (C3a, C4a,
and C5a), according to the manufacturer’s instructions
[17–19]. IQR interquartile range, ST schirmer’s test Statistical analysish The Steel–Dwass tests were used to compare preopera-
tive measurements to those during the follow-up period. Correlation coefficients were performed to identify the
association of changes in ST. P < 0.05 was considered sta-
tistically significant. Statistical analyses were performed
using JMP16 software (SAS Institute, Cary, NC, USA). 4 days postoperatively and decreased at 1 month. The dif-
ferences in the C3a levels between baseline and 4 days
and between 4 days and 1 month postoperatively was sig-
nificant (P < 0.001, P = 0.049, respectively) (Table 1). i
The C4a levels at baseline, 4 days, and 1 month post-
operatively were, respectively, 476 pg/ml (IQR, 196–742),
880 pg/ml (IQR, 479–1,464), and 657 pg/ml (IQR, 295–
974) in the surgical eyes. The C4a levels increased signifi-
cantly at 4 days (P < 0.001) and 1 month postoperatively
(P = 0.013) compared to baseline (Table 1). Results A total of 43 patients (23 women, 20 men) with a median
age of 69 years (interquartile range [IQR]: 64–73 years)
underwent combined vitrectomy and cataract surgery in
43 eyes. The surgical procedure was performed on 1 eye
per patient. Among the eyes, 30 had an epiretinal mem-
brane, while the remaining 13 had a macular hole. The
median duration of the surgical procedure was 60.0 min
(IQR: 49.8–79 min). Table 1 summarizes the clinical data
during the follow-up period. All parameters were meas-
ured at baseline, 4 days, and 1 month postoperatively, in
the surgical eyes. The C5a level at baseline, 4 days, and 1 month postop-
eratively were, respectively, 22.6 pg/ml (IQR, 15.6–36.0),
470.9 pg/ml (IQR, 98.4–1,061.4), and 38.3 pg/ml (IQR,
18.0–66.8) in the surgical eyes. The C5a levels increased
at 4 days (P < 0.001) and 1 month (P = 0.0048) postopera-
tively (Table 1). The correlation between ST and CAPs in tear fluid was
examined (Fig. 1). This figure demonstrated a negative
correlation between C3a levels and baseline ST values
(P = 0.015). The ST (mm) values at baseline, 4 days, and 1 month
postoperatively were 8.5 (IQR, 4–15), 16 (IQR, 7.5–20),
and 10 (IQR, 5–15), respectively, in the surgical eyes. The
ST values increased 4 days after the surgery compared to
baseline (P < 0.001) and decreased 1 month postopera-
tively compared to baseline (P = 0.44). To further investigate the relationship between C3a
and ST, we conducted an additional analysis. The sur-
gical eyes were divided into the short ST group (≦
10 mm, n = 22) and long ST group (> 10 mm, n = 21)
based on the preoperative ST values. A significant dif-
ference was seen in the C3a level between the short and
long ST groups (short ST, long ST, 1602 pg/ml [IQR,
960–3193], 882 pg/ml [IQR, 624–1677]) (P = 0.041). In The C3a levels at baseline, 4 days, and 1 month post-
operatively were 1202 pg/ml (IQR, 860–2304), 2753 pg/
ml (IQR, 1710–4453), 1763 pg/ml (IQR, 1234–2464),
respectively, in the surgical eyes. The C3a levels increased Maehara et al. BMC Ophthalmology (2023) 23:329 Page 4 of 6 Fig. 1 The correartion schirmer’s test and complement activation products. The correlation between Schirmer’s test (ST) and complement
activation products (CAPs). There is statistically different between C3a and Schilmer’s test at base line (a). ST schirmer’s test, IQR interquartile range Funding We have no conflicts of interest in this study and have received no financial
support. Discussion The sample size used in the study was too small to
establish a clear relationship between dry eye and
the surgical impact after the operation. Additionally,
we employed absorbable sutures to close the vitrec-
tomy wound as a preventive measure against postop-
erative infection. However, it is important to note that
the biodegradable products (specifically glycol ether)
from these sutures can potentially be toxic to the cor-
nea and conjunctiva, leading to inflammation. It is
worth mentioning that the sutures were fully absorbed
within 2 weeks in all cases, indicating that the impact
of the biodegradable materials might be minimal after
1 month. An increase in tear volume after surgery may
lead to dilution of proteins in the tear fluid, poten-
tially impacting individuals with pre-existing dry eye
conditions. Finally, we did not investigate the relation
between the complement system and inflammatory
cytokines and cellular immunity. Further studies are
needed to determine the role of innate immunity in dry
eye. In our study, we measured the levels of CAPs in tears
and the ST after performing vitrectomy with cata-
ract surgery. Four days after the surgery, we observed
a significant increase in ST compared to the baseline
measurement. Additionally, the levels of C3a, C4a, and
C5a also showed an increase at the 4-day postoperative
mark compared to the baseline levels. These elevated
levels of CAPs remained high even at the 1-month
follow-up.The development of inflammation after cata-
ract surgery is a significant concern for surgeons in this
field. Experts have discussed this issue extensively, but
there is currently no established protocol for effectively
managing postoperative inflammation in patients. Additionally, the innate immune system plays a cru-
cial role in the process of wound healing [26]. However,
excessive activation of the complement system can lead
to undesirable reactions [27, 28]. Previous experimental
studies have indicated a potential association between
complement activity and unfavorable postoperative
responses [28, 29]. Based on this, we hypothesized that
ocular surface parameters might be linked to comple-
ment activity during the postoperative period.h Availability of data and materials Availability of data and materials
Date are available from Hiroki Maehara (hmaehara@fmu.ac.jp) for researchers
who meet the criteria for accessto confidential data. Authors’ contributions H,M and T.S wrote the main manuscript text. All authors reviewed the
manuscript. Results BMC Ophthalmology (2023) 23:329 Page 5 of 6 Page 5 of 6 Page 5 of 6 Acknowledgements
l
bl Acknowledgements
Not applicable. This could be associated with reduced CAPs and tear
production. In the short ST group examined in this
study, the levels of C3a in tear fluid had not returned to
their preoperative values even after 1 month of surgery. This indicates that a short ST may contribute to post-
operative inflammation. Applying artificial tears to eyes
with a short ST could potentially decrease persistent
complement activation and alleviate ocular symptoms
associated with tear film instability. Measuring ST can
help predict postoperative inflammation, and if a short
ST is detected, appropriate anti-inflammatory meas-
ures and tear fluid replacement may be beneficial. This
study is also the first to measure complement activa-
tion products in tear fluid. We believe that this research
will serve as a foundation for future investigations into
inflammation in tear fluid. Results The others had no difference between ST
and CAPs (b—i) Fig. 1 The correartion schirmer’s test and complement activation products. The correlation between Schirmer’s test (ST) and complement
activation products (CAPs). There is statistically different between C3a and Schilmer’s test at base line (a). The others had no difference between ST
and CAPs (b
i) Fig. 1 The correartion schirmer’s test and complement activation products. The correlation between Schirmer’s test (ST) and complement
activation products (CAPs). There is statistically different between C3a and Schilmer’s test at base line (a). The others had no difference between ST
and CAPs (b—i) [IQR, 881–1872] at 1 month (P = 0.032) (Table 2). The
levels of C4a and C5a in both the short and long ST
groups showed an increase at 4 days after surgery and
then decreased to the preoperative levels after 1 month
(Table 2). the short ST group, the C3a level increased (2210 pg/
ml; [IQR, 1506–4328]) at 4 days and remained high
(2194 pg/ml; [IQR, 1348–3192]) at 1 month, while in
the long ST group, the elevated C3a level 2063 pg/ml;
[IQR, 1986–5775] at 4 days decreased to 1391 pg/ml; Table 2 Relationship between complement activation products and ST during follow-up period
ST
hi
’
IQR i
il
Median(IQR)
Short ST group
P value vs
baseline
P value vs
4 days
Long ST group
P value vs
baseline
P value vs
4 days
P value of
short ST vs
Long ST
C3a (pg/ml)
base line
1602 (960–3193)
-
-
887 (624–1677)
-
-
< 0.001
4 days
2210 (1506–
4328)
0.012
-
3063 (1986–
5775)
< 0.001
-
0.29
1 month
2194 (1348–
3192)
0.059
0.076
1391 (881–1872) 0.11
< 0.001
< 0.001
C4a (pg/ml)
base line
386 (171–831)
-
-
519 (241–723)
-
-
0.97
4 days
762 (451–2061)
< 0.001
-
911 (534–1360)
< 0.001
-
0.95
1 month
453 (275–876)
0.46
0.0025
620 (295–1030)
0.12
< 0.001
0.60
C5a (pg/ml)
base line
23.4 (16.4–40.4)
-
-
21.2 (11.7–27.6)
-
-
0.42
4 days
470.9 (93.8–
1153.3)
< 0.001
-
565.7 (155.3–
670.9)
< 0.001
-
0.70
1 month
37.0 (16.8–55.3)
0.043
< 0.001
45.6 (22.2–
128.1)
0.056
< 0.001
0.53 Table 2 Relationship between complement activation products and ST during follow-up period Maehara et al. BMC Ophthalmology (2023) 23:329 Maehara et al. Abbreviation Abbreviation
CAPs
complement activation products
ST
Schirmer’s test Abbreviation
CAPs
complement activation products
ST
Schirmer’s test Conclusioni The increase in C3a, C4a, and C5a levels 4 days after
surgery were considered a common tissue response to
surgical injury [30]. The preoperative C3a level in tear
fluid showed a negative correlation with the ST value. The levels of C3a and C5a remained elevated imme-
diately after surgery and persisted for 1 month in the
short ST group. This suggests that postoperative com-
plement activation through the alternative pathway
may be prolonged in eyes with low basal tear fluid
secretion. Recent proteome analyses have also indi-
cated the involvement of the complement system in dry
eye [31, 32].h Our findings indicate that there was an increase in
CAPs after surgery. A reduction in tear secretion before
the surgery can lead to prolonged complement activa-
tion and a slower recovery of ocular surface parameters
following combined vitrectomy and cataract surgery. Publisher’s Note
S
N Springer Nature remains neutral with regard to jurisdictional claims in pub-
lished maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in pub-
lished maps and institutional affiliations. y
12. Bjork J, Hugli TE, Smedegard G. Microvascular effects of anaphylatoxins
C3a and C5a. J Immunol. 1985;134:1115–9. 13. Foreman KE, Vaporciyan AA, Bonish BK, et al. C5a induced expression of
P-selectin in endothelial cells. J Clin Investig. 1994;94(3):1147–55. 14. Klein RJ, Zeiss C, Chew EY, et al. Complement factor H polymorphism in
age-related macular degeneration. Science. 2005;15:385–9. 15. Hageman GS, Anderson DH, Johnson LV, et al. A common haplotype
in the complement regulatory gene factor H(HF1/CFH) predisposes
individuals to age-related macular degeneration. Proc Natl Acad Sci USA. 2005;102:7227–32. 16. Kato Y, Oguchi Y, Omori T, et al. Complement activation products and
cytokines in pachychoroid neovasculopathy and neovascular age-related
macular degeneration. Invest Ophthalmol Vis Sci. 2020;61:39. 17. Omori T, Oguchi Y, Machida T, et al. Evidence for activation of lection
and classical pathway complement components in aqueous humor
of neovascular age-related macular degeneration. Ophthalmic Res. 2020;63:252–8. •
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? Choose BMC and benefit from: 18. Tanaka K, Oguchi Y, Omori T, et al. Changes in complement activation
products after anti-VEGF injection for choroidal neovascularization in
age-related macular degeneration and pachychoroid disease. Sci Rep. 2021;11:8464. 19. Zaidi TS, Zaidi T, Pier GB. References Starr CE, Gupta PK, Farid M, Beckman KA, Chan CC, Yeu E, et al. An
algorithm for the preoperative diagnosis and treatment of ocular surface
disorders. J Cataract Refract Surg. 2019;45:669–84. g
j
30. Harada T, Sakagami M, Sano M, Matsunaga T. Measurement of anaphyla-
toxin activity during surgery. Acta Otolaryngol. 1993;501(suppl):88–91. 31. Chen X, Rao J, Zheng Z, Yu Y, Lou S, Liu L, et al. Integrated tear proteome
and metabolome reveal panels of inflammatory-relates molecules
via key regulatory pathways in dry eye syndrome. J Proteome Res. 2019;5:2321–30. 7. Shimabukuro M, Maeda N, Koh S, Abe K, Kobayashi R, Nishida K. Effects
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y y
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8. Gomes JAP, Azar DT, Baudouin C, et al. TFOS DEWSII iatrogenic report. Ocul Surf. 2017;15:511–38. 32. Boehm N, Funke S, Wiegand M, Wehrwein N, Pfeiffer N, Grus FH. Altera-
tions in the tear proteome of dry eye patients–a matter of the clinical
phenotype. Invest Ophthalmol Vis Sci. 2013;54:2385–92. https://doi.org/
10.1167/iovs.11-8751. 9. Fujita T, Matsushita M, Endo Y. The lectin-complement pathway – its role
in innate immunity and evolution. Immunol Rev. 2004;198:185–202. 10. Gotze O. The alternate pathway of activation. In: Rother KTG, editor. The
complement system. Berlin: Springer; 1985. p. 154–68. 11. Imagawa DK, Osifchin NE, Paznekas WA. Consequences of cell membrane
attack by complement: release of arachidonate and formation of inflam-
matory derivatives. Proc Natl Acad Sci USA. 1983;80:6647–51. Received: 23 January 2023 Accepted: 11 June 2023 Received: 23 January 2023 Accepted: 11 June 2023 23. Leibovich SJ, Ross R. The role of the macrophage in wound repair. A
study with hydrocortisone and antimacrophage serum. Am J Pathol. 1975;78:71–100. 24. Brown EJ. Phagocytosis. Bio Essays. 1995;17:109–17.f 25. Zakaria N, Grasdoff SV, Wouters K, Rozema J, Koppen C, Lion E, et al. Human tears reveal insights into corneal neovascularization. PLOS One
2012;5(7):e36451. Competing interests 22. Yazdani M, Benedikte K, Rootwelt H, Shahdadfar A, Utheim P, Utheim
TP. Tear metabolomics in dry eye disease: a review. Int J Mol Sci. 2019;20:3755. The authors declare no competing interests. Consent for publication
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Sjogren syndrome patients with dry eye syndrome by two-dimen-
sional-nano-liquid chromatography coupled with tandem mass spec-
trometry. Sci Rep. 2014;4:5772. References 1. Arita R, Mizoguchi T, Kawashima M, Fukuoka S, Koh S, Shirakawa R, et al. Am J Ophthalmol. 2019;207:410–8. 1. Arita R, Mizoguchi T, Kawashima M, Fukuoka S, Koh S, Shirakawa R, et al. Am J Ophthalmol. 2019;207:410–8. 26. Schäffer M, Barbul A. Lymphocyte function in wound healing and follow-
ing injury. Br J Surg. 1998;85:444–60. https://doi.org/10.1046/j.1365-2168.
1998.00734.x. 2. Dana R, Bradley JL, Guerin A, Pivneva I, Stillman I, Evans AM, Schaumberg
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DA. Estimated prevalence and incidence of dry eye disease based on
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disease prevalence by age, sex and geographic characteristics in China: a
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4. Hardten DR. Dry eye disease in patients after cataract surgery. Cornea. 2008;27:855. 29. Anitua E, Fuente M, Muruzabal F, Merayo-Lloves J. Development and
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of the pathogenic factors in patients after cataract surgery. Cornea. 2007;26(9 Suppl 1):S16–20. 6. Starr CE, Gupta PK, Farid M, Beckman KA, Chan CC, Yeu E, et al. An
algorithm for the preoperative diagnosis and treatment of ocular surface
disorders. J Cataract Refract Surg. 2019;45:669–84. 6. Ethics approval and consent to participate The Institutional Review Board of Fukushima Medical University, Fukushima,
Japan, approved the study protocol (ID: 30046). The research was conducted
in accordance with the tenets of the Declaration of Helsinki, and all patients
provided written informed consent after they received a detailed explana-
tion of the study protocols and possible consequences associated with
participation. Maehara et al. BMC Ophthalmology (2023) 23:329 Page 6 of 6 Maehara et al. BMC Ophthalmology (2023) 23:329 Publisher’s Note
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N Role of neutrophils, MyD88-mediated neu-
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Immunol. 1985;76:473–6.
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https://www.researchsquare.com/article/rs-3420261/latest.pdf
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English
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The up-regulation of PAK2 indicates unfavorable prognosis in patients with serous epithelial ovarian cancer and contributes to paclitaxel resistance in ovarian cancer cells.
|
Research Square (Research Square)
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cc-by
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Ting Shuang
(
shuangting87@163.com
) Ting Shuang
(
shuangting87@163.com
) g S ua g (
s ua gt g8 @ 63.co
)
The First Affiliated Hospital of Xi'an Jiaotong University
https://orcid.org/0000-0002-6119-6989
Shiyun Wu
The First Affiliated Hospital of Xi'an Jiaotong University
Yifei Zhao
The First Affiliated Hospital of Xi'an Jiaotong University
Yanqi Yang
Xi'an Jiaotong University
Meili Pei
The First Affiliated Hospital of Xi'an Jiaotong University
https://orcid.org/0009-0003-9170-5320 Research Article Keywords: Serous epithelial ovarian cancer, chemo-resistance, PAK2, prognosis, ceRNA
Posted Date: October 16th, 2023
DOI: https://doi.org/10.21203/rs.3.rs-3420261/v1
License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Read Full License Page 1/24 Page 1/24 Page 1/24 Abstract The main challenge in the treatment of ovarian cancer has been the development of resistance to
chemotherapy. Previous studies have reported over-expression of PAK2 in various cancers through
different mechanisms. The objective of this study was to investigate whether up-regulation of PAK2
contributes to chemo-resistance and poor prognosis in ovarian cancer. Bioinformatics analysis was
initially employed and revealed a significant up-regulation of PAK2 in ovarian malignant tumors
compared to adjacent tissues, particularly in patients with stage III-IV disease compared to those with
stage I-II disease (P = 0.0056). High expression of PAK2 was associated with decreased OS, but not DFS,
in ovarian cancer patients. Immunohistochemistry demonstrated positive expression of PAK2 in chemo-
resistant serous EOC tissues, predominantly localized in the cytoplasm, which correlated with poor OS
and DFS. In vitro studies indicated that inhibition of PAK2 expression in A2780/Taxol cells resulted in
reduced colony formation, increased apoptosis, and impaired cell migration. Finally, RNA Binding Protein
Immunoprecipitation (RIP) Assay and luciferase reporter assays were employed which confirmed that lnc-
SNHG1 acts as a competing endogenous RNA (ceRNA) by binding to miR-216b-5p and subsequently
modulating the expression of PAK2. In conclusion, our study confirmed that PAK2 may serve as a
predictive marker for chemo-resistance and poor prognosis in ovarian cancer and could potentially be
targeted therapeutically to overcome chemo-resistance. Introduction Ovarian cancer ranks as the seventh most prevalent form of cancer and is the leading cause of death
among gynecological cancers in women worldwide[1]. Projections based on cancer statistics indicate
that there will be an estimated 19,710 new cases of ovarian cancer and 13,270 deaths related to ovarian
cancer in the United States by the year 2023 [2].Approximately 70% of ovarian cancer patients are
diagnosed at an advanced stage, highlighting the importance of chemotherapy with platinum and
paclitaxel following cytoreductive surgery as the primary treatment approach. However, a significant
challenge in improving survival rates for ovarian cancer lies in the fact that at least 70% of patients
eventually develop resistance to chemotherapy. This resistance has been a major hindrance in recent
decades, with even developed countries reporting a 5-year survival rate of only 47%[3]. Consequently,
finding a solution to this urgent problem remains a critical concern in the clinical management of ovarian
cancer patients. P21 activated kinases (PAKs) are a group of serine/threonine kinases that display a high level of
evolutionary conservation. These kinases can be activated by different upstream signals, particularly Rac
and Cdc42, which are part of the Rho family of small G proteins. The activation of PAKs by these signals
regulates numerous crucial signaling pathways and cellular functions. Importantly, abnormal expression
of PAKs has been observed in various tumors, where they contribute to the remodeling of the
cytoskeleton, cell motility, cell proliferation, differentiation, apoptosis, mitosis, and angiogenesis[4, 5]. PAKs play a significant role in the initiation and progression of tumors. Therefore, investigating targeted Page 2/24 Page 2/24 therapies that specifically inhibit PAKs shows promise for the development of innovative approaches in
tumor treatment. There are six isoforms of PAK kinase (PAK1-6) that have been identified in mammals, and they can be
categorized into two groups. Group I consists of PAK1, PAK2, and PAK3, while Group II consists of PAK4,
PAK5, and PAK6 [6]. Among these isoforms, PAK1 has been extensively studied in various cancers,
including breast cancer, colorectal cancer, and ovarian cancer [7–10].However, unlike PAK1, PAK2 has a
dual function and can regulate both cell survival and cell death pathways, depending on the milieu. The
activation of the full-length PAK2 gene promotes cell survival by phosphorylating Bad, which reduces the
interaction between Bad and Bcl-2 or Bcl-x(L), leading to an increased association between Bad and 14-3-
3tau and ultimately promoting cell survival[11]. Introduction However, proteolytic activation of PAK-2p34 leads to
apoptosis[12]. Li X’s study reported that highly expressed PAK2 mediates chemotherapeutic resistance in
human breast invasive ductal carcinoma by negatively regulating caspase-7 activity [13]. Cho HJ et al
conducted a study that revealed the role of RhoGDI2 in promoting tumor growth, malignant progression,
and chemo-resistance in gastric cancer. The study also demonstrated the involvement of PAK2 in
mediating these effects in gastric cancer cells.[14]. The role of PAK2 in ovarian cancer chemo-resistance
is not as well understood. In our previous research, we demonstrated that PAK2 is a direct target of miR-
134, and our findings suggest that the repression of miR-134 and subsequent up-regulation of PAK2 may
contribute to the development of paclitaxel resistance in ovarian cancer cells[15]. This study aims to investigate the expression of PAK2 in chemo-resistant and chemo-sensitive serous
EOC patients using tissue microarray and bioinformatics analysis. Additionally, the study aims to
examine the disparity in PAK2 expression levels among serous EOC patients at different FIGO stages and
to determine if high expression of PAK2 is associated with poor OS or DFS in these patients. In vitro
studies will also be conducted to assess the impact of inhibited expression of PAK2 on paclitaxel
sensitivity in A2780/Taxol cells. We previously reported that lnc-SNHG1 acted as a sponge for miR-216b-
5p in ovarian cancer cells and this interaction played a crucial role in regulating the sensitivity of ovarian
cancer cells to paclitaxel. It has been predicted that PAK2 is a direct target of miR-216b-5p. Therefore, this
study aims to further investigate whether lnc-SNHG1 functions as a ceRNA by sequestering miR-216b-5p
and subsequently modulating the expression of PAK2. Cell proliferation assay Following transfection, the cells were placed in 96 well plates at a density of 5 × 103 cells/well and
incubated at 37℃ with 5% CO2 for different time intervals of 0, 24, 48, and 72 hours. The proliferation of
the cells was assessed using the CCK-8 assay, following the instructions provided by the manufacturer
(Dojindo, Kumamoto, Japan). The absorbance of each well was measured at a wavelength of 450nm
using a spectrophotometer (XFLUOR4 Version: V 4.51). Cell apoptosis A2780/Taxol cells were cultured in a 6-well plate with a density of 2×105 cells/2mL and incubated for 12
hours. After transfection with PAK2-siRNA-3 and siRNA NC for 48 hours, the A2780/Taxol cells were
exposed to a paclitaxel-containing medium with a concentration of 5 µM. Following a 24-hour incubation,
the cells were collected and apoptosis analysis was performed. This involved washing the cells twice
with PBS and staining them with the Annexin V-FITC cell apoptosis detection kit (BD, New Jersey, USA). Flow cytometry was utilized to measure apoptosis levels. Cell culture and transfection In this study, cell culture was employed to investigate the behavior of A2780/Taxol cells, which are
resistant to the paclitaxel, as well as the parental A2780 cells. The cells were cultured in RPMI 1640
medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. The A2780/Taxol
cells were maintained in the same medium but with the addition of 800ng/mL paclitaxel, which was
removed one week prior to the experiments. SiRNAs targeting PAK2 (and lnc-SNHG1) were synthesized by
GeneCreate Biotech (Wuhan, China) and named as PAK2-siRNA-1, PAK2-siRNA-2, and PAK2-siRNA-3 (lnc- Page 3/24 SNHG1-siRNA-1, lnc-SNHG1-siRNA-2, lnc-SNHG1-siRNA-3). These siRNAs were transfected into
A2780/Taxol cells using Lipofectamine 3000 at a concentration of 100 nM (Invitrogen, CA, USA). Also,
miR-216-5p-mimic and miRNA mimic NC synthesized by GeneCreate Biotech (Wuhan, China) were
transfected into A2780/Taxol cells at a concentration of 100 nM using Lipofectamine 3000 (Invitrogen,
CA, USA) according to the manufacturer's instructions. All cells were cultured under standard conditions
of 37°C, 5% CO2, and saturated humidity. The transfected cells were cultured for 48 hours before the
subsequent experiments were conducted. Wounding healing assay After transfection, A2780/Taxol cells in the logarithmic growth phase were harvested and plated onto 6-
well plates at a density of 5×105 cells per well. Once the cells reached full confluence, a uniform scratch
was created using a 200ul sterile pipette tip. The cells were then gently rinsed three times with PBS to
remove any detached cells. To aid visualization, a mark was made along the edge of the scratch on the
bottom of the plate. The medium was replaced and the cells were incubated at 37℃ in a 5% CO2
incubator. Microscopic images were captured at 0h, 24h, and 48h time points using the microscope
(Olympus, Tokyo, Japan). Test for the ability of clone formation The cells were transfected and subsequently distributed into 6-well plates at a density of 1000 cells per
well. These plates were then placed in a 5% CO2 incubator at a temperature of 37°C for one night. The
following day, the cells in each group were exposed to 5 µM paclitaxel for a duration of 24 hours. After
the treatment, the cells were cultured in a fresh and complete medium and incubated for a period of two
weeks. Subsequently, the cells were stained with 0.1% crystal violet for 30 minutes, washed three times Page 4/24 Page 4/24 with PBS, and subjected to statistical analysis to assess colony formation. The observed differences in
colony formation were then compared. Western Blot The cellular protein was collected using RIPA buffer (ThermoFisher Scientific) and its concentration was
determined using a BCA Protein Array kit (Biosharp, Guangzhou, China). Subsequently, samples from
each group containing 150 µg of protein were mixed with 6 × SDS loading buffer and boiled for 10
minutes. The protein samples were then subjected to electrophoresis on 12% sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (SDS-PAGE) gels, and the resolved proteins were transferred onto
polyvinylidene difluoride (PVDF) membranes using electro-transfer. Following blocking with 5% skimmed
milk, the membranes were incubated with anti-PAK2 (Abcam, Cambridge, UK, 1:500) and anti-GAPDH
antibodies (Abcam, Cambridge, UK, 1:1000), and subsequently with HRP-conjugated anti-rabbit
secondary antibodies (Abcam, Cambridge, UK, 1:2000). The protein bands were visualized using an ECL
system and quantified using Chemi-Doc XRS imaging software (Bio-Rad). RIP The Magna RIP™RNA-Binding Protein Immunoprecipitation Kit, manufactured by Millipore in
Massachusetts, USA, was utilized to conduct the RIP assay in A2780/Taxol cells. In this assay, cell
lysates were combined with magnetic beads that were conjugated with either IgG or a human anti-Ago2
antibody in RIP buffer. Subsequently, the precipitated RNA was purified in preparation for quantitative
real-time polymerase chain reaction (qRT-PCR). Gene expression The total RNAs of all cell groups were isolated using the Trizol reagent (Invitrogen, CA, USA) following the
provided protocol. The cDNA was synthesized using the PrimeScriptVR RT reagent Kit (Takara, Dalian,
China). The expression levels of the PAK2, lnc-SNHG1and miR-216b-5p were determined by qRT-PCR
using the Bio-Rad S1000 with Bestar SYBR Green RT-PCR Master Mix (Toyobo). The Actin gene of the
human species was employed as a control. The relative gene expression was quantified using the 2−ΔΔCt
method (Livak and Schmittgen 2001). Each sample was analyzed in triplicate. RIP ssue microarray and immunohistochemistry The ovarian cancer tissue microarray used in this study was obtained from Shanghai Outdo Biotech,
consisting of 2 cases of benign ovarian tumors and 152 cases of ovarian cancer. A follow-up period of
5–9 years was conducted for all cancer patients. Among the ovarian cancer tissues, 92 cases of serous
EOC were selected, with 31 cases classified as chemo-resistant and 61 cases as chemo-sensitive. Ethical
approval for this study was obtained from the ethics committee of the First Affiliated Hospital of Xi'an
Jiao tong University (approval number: 2020-G143) and the ethics committee of Shanghai Outdo
Biotechnology Company (approval number: YB M-05-02). Immunohistochemical staining of PAK 2 was
performed using a rabbit anti-human polyclonal antibody from Abcam. The staining intensity of
cytoplasmic PAK2 expression was categorized into four levels: no staining (scored as 0), light yellow
(scored as 1), brown yellow (scored as 2), and dark brown (scored as 3). Additionally, the percentage of
positive cells was determined: less than 5% (scored as 0), 5–25% (scored as 1), 26–50% (scored as 2),
51–75% (scored as 3), and more than 75% (scored as 4). The final score was calculated by multiplying
the staining intensity score with the positive cell rate score. Scores ranging from 0 to 2 were considered
negative (-), 3 to 4 were classified as weakly positive (+), 5 to 8 were categorized as slightly positive (++),
and 9 to 12 were considered strongly positive (+++). A score of 7 or higher indicated high expression of
PAK2. To minimize reading errors, two independent observers assessed and scored the samples. Luciferase activity assay A2780/Taxol cells were cultured in 96-well culture plates at a density of 2 × 104 cells per well. The cells
were then transfected with luciferase reporter constructs (400 ng), miR-216b-5p mimics (500 nM), and the
internal control vector pRL-TK, pRL-SV40, or pRL-CMV (Promega, WI, USA) at a ratio of 20:1 (reporter
construct:control vector) using Lipofectamine 3000 (Invitrogen) according to the manufacturer's
instructions. After 5 hours, the transfection medium was replaced with a medium containing 6 µM
curcumin (Sigma-Aldrich, MO, USA) dissolved in 100% dimethyl sulfoxide (DMSO) (Sigma-Aldrich, MO,
USA). The luciferase activity was measured 48 hours after transfection using the Dual-Luciferase
Reporter Assay System (Promega, WI, USA). Page 5/24 Bio-informatics analysis The Cancer Genome Atlas (TCGA) is a cancer genomics project that utilizes genome analysis technology
through large-scale sequencing (https://portal.gdc.cancer.gov/). In this study, datasets of RNAseq for
ovarian cancer patients from the TCGA database were specifically chosen and examined. The Gene
Expression Omnibus (GEO) is an international gene expression database that was established in 2000
and is currently maintained by the National Biotechnology Information Center
(https://www.ncbi.nlm.nih.gov/geo/). Both the TCGA and GEO databases were utilized in the analysis to
investigate the correlation between PAK2 gene expression and the clinical significance of ovarian cancer. (https://www.ncbi.nlm.nih.gov/geo/). Both the TCGA and GEO databases were utilized in the analysis to
investigate the correlation between PAK2 gene expression and the clinical significance of ovarian cancer. Tissue microarray and immunohistochemistry Statistical Analysis The statistical analysis was conducted using the SPSS package (version 19.0, IBM SPSS, IL, USA). The
data were presented as mean ± standard deviation. The relationship between PAK2 and
clinicopathological features of EOC patients was analyzed using the chi-square test. Additionally, a
multivariate logistic regression analysis model was employed to predict the chemo-resistance of EOC
based on the aforementioned factors. Furthermore, the multivariate Cox proportional hazard model was
utilized to analyze the prognostic factors of overall survival (OS) and disease-free survival (DFS) in EOC
patients. Finally, Kaplan-Meier analysis was employed to estimate the OS and DFS for the patients. The
cytology experiment was analyzed using GraphPad Prism software. Cell scratching and apoptosis were
analyzed using Student's t-test, while CCK8 and colony formation were analyzed using Two-way ANOVA. The qPCR test was analyzed using One-way ANOVA. Page 6/24 Page 6/24 Results
1. Differential expression of PAK2 in patients with serous
EOC Firstly, the RNASEQ dataset for ovarian cancer was obtained from the TCGA and GTEx databases. The
dataset consists of 376 samples classified as cancer and 88 samples classified as adjacent to cancer. The RPKM data of PAK2 was logged using the base 2 logarithm (log2). The box plot was generated using
the ggplot package in the R programming language, with the samples grouped into cancer and adjacent
categories. The result showed there is a notable up-regulation of PAK2 expression in ovarian cancer
tissues comparison to adjacent tissues (Fig. 1a). The table below uses the GEO database (with 51 data sets in total) and performs regression analysis
through R language, obtaining the following table. The GSE number is GSE165808. The results indicate a
significant correlation between age and prognosis in the single-factor regression analysis, suggesting
that individuals over the age of 60 have a higher mortality rate. Furthermore, both univariable and
multivariable analyses demonstrate a strong correlation between the expression levels of PAK2 and
prognosis. Specifically, higher expression levels of PAK2 are associated with a higher mortality rate
(Table 1). Additionally, we utilized the tissue microarray technique to assess the expression of the PAK2 protein in
ovarian cancer patients. Given that EOC constitutes approximately 90% of all ovarian malignant tumors,
with serous epithelial ovarian cancer accounting for approximately 75% of EOC cases, our study
specifically investigates the chemo-resistance characteristics of this particular subtype of ovarian cancer. To this end, we included a total of 92 serous EOC tissue samples, which were categorized into 31 chemo-
resistant cases and 61 cases of chemo-sensitive tumors. The expression of PAK2 was observed to be
significantly elevated in chemo-resistant serous EOC tissues, predominantly localized in the cytoplasm,
with a substantial expression ratio of 68% (Table 2, Fig. 1b). Conversely, it exhibited low expression in
chemo-sensitive serous EOC tissues, with a comparatively lower expression ratio of 23% (Table 2,
Fig 1c) Further, the study examined the expression levels of PAK2 in ovarian cancer patients using the FIGO
staging system, which classifies patients into four stages: Stage I, Stage II, Stage III, and Stage IV. Violin
plots were generated using the R programming language packages ggplot2 and ggsignif to visualize the
expression patterns in each stage. The data for this analysis was sourced from the GEO database,
specifically the GSE9891 dataset. Results
1. Differential expression of PAK2 in patients with serous
EOC The distinctions between the four groups were determined using the
WILCOX test, and the p-value was calculated directly. The results illustrate there are significant disparities
between stage I and II(P = 0.027), highly significant disparities between stage I and III(P = 0.00021), and
extremely significant disparities between stage I and IV(P = 0.0054). Also, the observed disparity in PAK2
expression levels between stage I-II and stage III-IV ovarian cancer is highly statistically significant (P =
0.0056). The aforementioned findings were presented in Fig. 1d. Page 7/24 Page 7/24 Table 1
The relationship between PAK2 and prognosis of ovarian cancer patients using regression
analysis. Characteristics
Univariable analyses
Multivariable analyses
HR (95% CI)
P-value
HR (95% CI)
P-value
Diagnosed age
(> 60 vs. ≤60)
2.757
(1.19,6.387)
0.018
*
1.766
(0.6542 ,4.767)
0.262
Stage
(I-II vs. III-IV)
0.8512
(0.327,2.215)
0.741
0.603
(0.2215 ,1.642)
0.322
Expression
(low vs. high)
4.105
(1.687,9.99)
0.00186
**
3.413
(1.2135,9.597)
0.02
* Table 1 Page 8/24 Table 2
The expression of PAK2 in serous EOC P
9 24
ab e
The expression of PAK2 in serous EOC
Case
PAK2 cytoplasm
expression ( ≦ 6)
PAK2 cytoplasm
expression ( ≧ 7)
n
n
n%
n
n%
P
Age (years)
0.2875
Postmenopausal(≧50)
51
29
0.57
22
0.43
Premenopausal (< 50)
41
28
0.68
13
0.32
Tumor stage (FIGO)
0.0343
Ⅰ-Ⅱ
20
17
0.85
3
0.15
Ⅲ-IV
72
41
0.57
31
0.43
Histological grade (n =
86)
0.7593
H
13
9
0.69
4
0.31
L
73
45
0.62
28
0.38
Nodal status(n)
0.1892
Negative
58
39
0.67
19
0.33
Positive
34
18
0.53
16
0.47
Chemo-resistance
<
0.0001
Y
31
10
0.32
21
0.68
N
61
47
0.77
14
0.23
Metastasis
0.1892
Y
28
14
0.5
14
0.5
N
64
43
0.67
21
0.33
2. Risk factors related to chemo-resistance of patients with
serous EOC
In this study, a multivariate logistic regression analysis was employed to examine the relationship
between various clinical features and chemo-resistance in patients with serous EOC. The clinical features
considered in the analysis included PAK2 expression, age, nodal status, metastasis, and FIGO stage. The
forward stepwise selection method was utilized to identify significant variables. The findings revealed 2. Risk factors related to chemo-resistance of patients with
serous EOC In this study, a multivariate logistic regression analysis was employed to examine the relationship
between various clinical features and chemo-resistance in patients with serous EOC. Results
1. Differential expression of PAK2 in patients with serous
EOC The clinical features
considered in the analysis included PAK2 expression, age, nodal status, metastasis, and FIGO stage. The
forward stepwise selection method was utilized to identify significant variables. The findings revealed Page 9/24 Page 9/24 that the differential expression of PAK2 (P༜0.0001) and the nodal status (P = 0.011) were identified as
factors associated with chemo-resistance in patients with serous EOC(Table 3). Table 3
Analysis of multiple variables to determine the factors linked to the resistance of
chemotherapy in serous EOC
B
S.E. Sig. Exp(B)
95.0% C.I.for EXP(B)
Lower
Upper
PAK2 expression
2.065
0.576
0.0001
7.888
2.553
24.37
Age
-0.612
0.577
0.278
0.542
0.175
1.679
Nodal status
1.83
0.716
0.011
6.232
1.531
25.373
Metastasis
0.197
0.699
0.797
1.217
0.309
4.789
FIGO stage
0.665
0.921
0.479
1.944
0.32
11.828
3 The outcome of the multivariate Cox regression model analysis for OS and DFS of serous EOC patien 3. The outcome of the multivariate Cox regression model analysis for OS and DFS of serous EOC patients We applied multivariate Cox regression analysis to estimate the prognostic factors for the OS and DFS of
serous EOC patients. The clinical features including chemo-resistance, expression of PAK2, age, nodal
status, metastasis, and FIGO stage. It was indicated that chemo-resistance(P༜0.0001), FIGO stage (P =
0.003) was independent prognostic factors for the DFS (Table 4), while the Chemo-resistance (P༜0.0001),
PAK2 expression (P = 0.019) and FIGO stage (P = 0.006)were related to the OS(Table 5). Table 4
Multivariable Cox regression analyses were conducted to assess the impact of
multiple variables on disease-free survival. B
SE
Sig. Exp(B)
95.0% CI for Exp(B)
Lower
Upper
Chemo-resistance
3.832
0.636
0.0001
46.135
13.271
160.382
PAK2 expression
0.312
0.284
0.273
1.366
0.783
2.383
Age
-0.111
0.252
0.661
0.895
0.546
1.468
Nodal status
0.143
0.343
0.677
1.153
0.589
2.259
Metastasis
0.168
0.33
0.611
1.183
0.62
2.258
FIGO stage
1.073
0.367
0.003
2.925
1.424
6.007 Table 4 Page 10/24 Page 10/24 Table 5
Multivariable Cox regression analyses were conducted to evaluate the influence on
overall survival. B
S.E. Sig. Exp(B)
95.0% C.I.for EXP(B)
Lower
Upper
Chemo-resistance
1.637
0.321
0.0001
5.142
2.743
9.64
PAK2 expression
0.705
0.3
0.019
2.023
1.124
3.642
Age
-0.361
0.279
0.196
0.697
0.403
1.205
Nodal status
0.64
0.367
0.081
1.897
0.925
3.893
Metastasis
0.473
0.35
0.177
1.604
0.808
3.185
FIGO stage
2.042
0.744
0.006
7.709
1.795
33.111
4. Results
1. Differential expression of PAK2 in patients with serous
EOC The influence of high PAK2 expression on the OS and DFS of patients with ovarian cancer. Table 5 Table 5 4. The influence of high PAK2 expression on the OS and DFS of patients with ovarian cancer. 4. The influence of high PAK2 expression on the OS and DFS of patients with ovarian cancer. Data for PAK2 expression was sourced from the GEO database mentioned above and consisted of 51
data points. Based on the PAK2 expression values for each sample, a cutoff value of 52.92 was
determined using X-tile plots. This threshold was then used to categorize patients into two groups: high
expression and low expression. Patients with expression values above the cutoff were classified as the
high expression group, while those with values below were classified as the low expression group. The R
package "survival" was utilized to create survival curves, which estimated the likelihood of survival based
on factors such as survival time, survival status, and various groupings. These curves were employed to
illustrate the survival probabilities of both the low expression and high expression groups. It is clear that the OS probability of patients in the low expression group is significantly higher than that
of patients in the high expression group. The Log Rank (Mantel-Cox) test yielded a p value of 0.0008,
indicating a highly significant difference. Therefore, the prognosis of the PAK2 high expression group is
poor (Fig. 2a). The statistical analysis revealed that patients with low expression of PAK2 have a higher
probability of survival without disease progression compared to patients with high expression. However,
the Log Rank (Mantel-Cox) test showed a p-value of 0.061, suggesting that the observed difference is not
statistically significant (Fig. 2b). The analysis of PAK2 expression in the tissue microarray revealed that patients with high levels of PAK2
expression exhibited significantly lower OS compared to those with low levels of PAK2 expression (P <
0.0001) (Fig. 2c). Additionally, patients with high PAK2 expression demonstrated reduced DFS (P <
0.0001) (Fig. 2d). 5. The sensitivity of A2780/Taxol cells to paclitaxel could be enhanced by the use of siRNA targeting
PAK2 Page 11/24 Page 11/24 Firstly, it was observed that the expression of PAK2 was higher in A2780/Taxol cells compared to the
parental A2780 cells, both at the protein and RNA levels (Fig. 3A-B). To investigate the role of PAK2 in the
sensitivity of ovarian cancer cells to paclitaxel, A2780/Taxol cells were transfected with three PAK2-
siRNAs (PAK2-siRNA-1, PAK2-siRNA-2, PAK2-siRNA-3) along with a negative control siRNA (NC-siRNA). The efficiency of transfection was assessed using qPCR and western blot analysis, revealing that PAK2-
siRNA-3 had the highest efficiency (Fig. 3A-B). 4. The influence of high PAK2 expression on the OS and DFS of patients with ovarian cancer. Subsequently, the A2780/Taxol cells transfected with
PAK2-siRNA-3 exhibited decreased cell proliferation compared to the NC-siRNA group and the un-
transfected A2780/Taxol cells (Fig. 3C). Functional assays were performed after transfection with PAK2-
siRNA-3 (NC-siRNA) and treatment with paclitaxel for 24 hours. The colony formation assay confirmed
that PAK2 deficiency led to a reduction in the number of colonies (Fig. 3D). The cell apoptosis assay
demonstrated that PAK2 silencing accelerated apoptosis (Fig. 3E). The wound healing assay revealed a
decrease in cell migration after PAK2 knockdown (Fig. 3F). Collectively, these findings indicate that
decreased expression of PAK2 can enhance paclitaxel sensitivity in A2780/Taxol cells. Firstly, it was observed that the expression of PAK2 was higher in A2780/Taxol cells compared to the
parental A2780 cells, both at the protein and RNA levels (Fig. 3A-B). To investigate the role of PAK2 in the
sensitivity of ovarian cancer cells to paclitaxel, A2780/Taxol cells were transfected with three PAK2-
siRNAs (PAK2-siRNA-1, PAK2-siRNA-2, PAK2-siRNA-3) along with a negative control siRNA (NC-siRNA). The efficiency of transfection was assessed using qPCR and western blot analysis, revealing that PAK2-
siRNA-3 had the highest efficiency (Fig. 3A-B). Subsequently, the A2780/Taxol cells transfected with
PAK2-siRNA-3 exhibited decreased cell proliferation compared to the NC-siRNA group and the un-
transfected A2780/Taxol cells (Fig. 3C). Functional assays were performed after transfection with PAK2-
siRNA-3 (NC-siRNA) and treatment with paclitaxel for 24 hours. The colony formation assay confirmed
that PAK2 deficiency led to a reduction in the number of colonies (Fig. 3D). The cell apoptosis assay
demonstrated that PAK2 silencing accelerated apoptosis (Fig. 3E). The wound healing assay revealed a
decrease in cell migration after PAK2 knockdown (Fig. 3F). Collectively, these findings indicate that
decreased expression of PAK2 can enhance paclitaxel sensitivity in A2780/Taxol cells. 6. Lnc-SNHG1 functioned as a ceRNA by binding miR-216b-5p, consequently modulating the expression
of PAK2. 6. Lnc-SNHG1 functioned as a ceRNA by binding miR-216b-5p, consequently modulating the expression
of PAK2. Previously we demonstrated negative association between the up-regulation of lnc-SNHG1 and the down-
regulation of miR-216b-5p with chemo-resistance in patients diagnosed with serous EOC. The lnc-SNHG1
was observed to function as a ceRNA with miR-216b-5p, playing a crucial role in modulating the
sensitivity of ovarian cancer cells to paclitaxel[16]. We in this study further confirm that PAK2 was a direct target of miR-216b-5p. Through the utilization of
starBase v2.0, it was observed that miR-216b-5p possesses a binding site within the 3' untranslated
region (3'UTR) of PAK2. Subsequently, luciferase reporter assays were conducted using A2780/Taxol
cells. The findings demonstrated a significant decrease in fluorescence intensity when miR-216b-5p-
mimics and PAK2-WT were co-transfected, compared to the co-transfection of miR-216b-5p-mimics and
PAK2-MUT. Conversely, there was no significant change observed when miR-216b-5p-mimics were co-
transfected with PAK2-MUT, as compared to NC-mimics + PAK2-MUT (Fig. 4A-B). These results suggest
an interaction between miR-216b-5p-mimics and PAK2-WT. To further validate the direct interaction
between miR-216b-5p and PAK2, RIP analysis was performed. The results of RIP-qPCR indicated a
significant enrichment of miR-216b-5p in A2780/Taxol cells when MS2-tagged wild-type PAK2 (PAK2-WT
MS2) was present, compared to the mimic NC (Fig. 4C-D). Collectively, these findings provide evidence
that miR-216b-5p can directly bind to PAK2. Moreover, A2780/Taxol cells were subjected to transfection with lnc-SNHG1-siRNA (NC siRNA), and the
expression of PAK2 was assessed at both the RNA and protein levels. The results revealed that a
decrease in lnc-SNHG1 led to a corresponding down-regulation of PAK2. Conversely, when miR-216b-5p
was over-expressed through transfection with miR-216b-5p mimics, the expression of PAK2 was down-
regulated at both the RNA and protein levels (Fig. 4E, I). Page 12/24 Page 12/24 Page 12/24 In order to investigate the role of lnc-SNHG1 as a ceRNA by binding to miR-216b-5p and subsequently
modulating the expression of PAK2, we conducted a series of experiments using A2780/Taxol cells. We
designed the groups including A2780/Taxol wild-type as control(group1), A2780/Taxol cells transfected
with NC-siRNA + miR-216b-5p inhibitor (group2), NC-siRNA + NC inhibitor (group 3), lnc-SNHG1-siRNA-2 +
miR-216b-5p inhibitor (group 4), lnc-SNHG1-siRNA-2 + NC inhibitor (group 5), PAK2 siRNA-3 + miR-216b-5p
inhibitor (group 6), PAK2 siRNA-3 + NC inhibitor (group 7). The expression levels of lnc-SNHG1 and miR-
216b-5p were validated (Fig. 4F and G). Comparing Group 2 and Group 3, we observed a significant
increase in the expression of the target gene PAK2 after knockdown of miR-216b-5p. 6. Lnc-SNHG1 functioned as a ceRNA by binding miR-216b-5p, consequently modulating the expression
of PAK2. Comparing Group 2
and Group 4, we concluded that simultaneous knockdown of miR-216b-5p and lnc-SNHG1 led to a
significant decrease in the expression of PAK2. Furthermore, comparing Group 2 and Group 6, we found
that simultaneous knockdown of miR-216b-5p and PAK2 resulted in a significant decrease in the
expression of PAK2 at both the RNA and protein levels (Fig. 4H, J) Discussion However, the PAK2 in ovarian cancer chemo-resistance is not well understand. In this present study, the RNASEQ dataset showed there is a notable upregulation of PAK2 expression in
ovarian cancer tissues comparison to adjacent tissues. Also, the observed disparity in PAK2 expression
levels between stage I-II and stage III-IV ovarian cancer is statistically significant. Consistant with our
result, there was a study claimed overexpression of PAK2 was observed in oral squamous cell carcinoma
(OSCC) in comparison to normal mucosa. Furthermore, the expression of PAK2 was found to be elevated
in higher grades of OSCC. Additionally, following TPF (docetaxel, cisplatin, and fluorouracil) therapy, an
increase in PAK2 expression was observed in OSCC [26]. What’s more, according to Liu R's findings, there
is a notable disparity in the expression of PAK2 between breast cancer cells and normal mammary
epithelial cells. Additionally, it was observed that the presence of PAK2 was strongly associated with the
probability of breast cancer metastasis and mortality[27]. Furthermore, our study revealed that PAK2 exhibited high expression levels in chemo-resistant serous
epithelial ovarian cancer (EOC) patients, while demonstrating relatively low expression in chemo-sensitive
individuals. Additionally, the over-expression of PAK2 was significantly associated with chemo-resistance
in serous EOC patients, indicating a poor OS and DFS prognosis. This finding is consistent with a study
published previously, which observed that both PAK2 and pSer20PAK2 immunostainings were
predominantly localized in the cytoplasm of tumor cells in gastric cancer tissues, exhibiting significantly
higher expression levels compared to normal gastric mucosa. Moreover, patients displaying over-
expression of PAK2 and pSer20PAK2 proteins were strongly correlated with unfavorable clinicopathologic
variables[28]. Another report also indicated a significant elevation of PAK2 expression in gastric cancer
tissues compared to adjacent tissues, with an increased level of PAK2 being associated with cell
proliferation and disease progression[29]. Similarly, PAK2 was found to be significantly over-expressed in
head and neck cancer (HNC), which was associated with a poor clinical outcome for patients. Furthermore, elevated PAK2 expression contributed to cell proliferation, aerobic glycolysis, and chemo-
resistance through the PAK2-c-Myc-PKM2 axis[30]. Through in vitro analysis, we observed that the downregulation of PAK2 expression in paclitaxel-resistant
ovarian cancer cells (A2780/Taxol) resulted in a decrease in colony formation, increased apoptosis, and
inhibited cell migration. Discussion Currently, the primary approaches for managing ovarian cancer involve surgical resection and
chemotherapy. Surgical resection is the preferred treatment modality, with the objective of removing
tumor tissue to the greatest extent possible. However, due to the inherent characteristics of ovarian
cancer and its propensity for metastasis, complete eradication of all tumor tissues through surgical
resection is often challenging, resulting in an elevated risk of recurrence and metastasis. While
chemotherapy can effectively control tumor growth, it can also induce drug resistance and diminish
treatment efficacy. Consequently, investigating the mechanisms underlying chemotherapy resistance in
ovarian cancer and identifying novel therapeutic approaches have emerged as prominent areas of
research. The abnormal expression and activity of PAKs in tumors have been extensively investigated and are
closely associated with the initiation, progression, and prognosis of tumors[17–19].Studies have
demonstrated that the upregulation of PAKs in different types of tumors is linked to increased
proliferation and invasion of tumor cells. PAKs can facilitate cell cycle progression and stimulate tumor
cell proliferation by modulating the activities of cyclins and signaling pathways involved in cell
proliferation. Furthermore, PAKs can also regulate cytoskeletal reorganization and cell migration, thereby
promoting the invasion and metastasis of tumor cells[20]. Among the PAK family, PAK1 was the most widely studied in different cancers [21, 22]. PAK1 was also
found to be over-expressed and activitied in ovarian cancer [8, 10, 23] and endometrial cancer[24].The
previous study found a significant association between an increased extent of PAK1 staining and
unfavorable overall survival outcomes in ovarian cancer patients who experienced disease recurrence
following chemotherapy treatment[8].However, PAK2 was less well studied in ovarian cancer. Siu MK et al
reported the migration and invasion of ovarian cancer cells were decreased when PAK1 and PAK2 were
knocked down[10]. There once was a report claimed the PAK2 is involved in the motility of ovarian cancer
cells. Inhibiting PAK2 with a chemical inhibitor or siRNA transfection reduces the migration of ovarian Page 13/24 Page 13/24 cancer cells on collagen. The results suggest that collagen's effect on migration is mediated, at least in
part, by PAK2.They suggested PAK2 should be further explored as a pro-metastatic gene in ovarian
cancer[25]. However, the PAK2 in ovarian cancer chemo-resistance is not well understand. cancer cells on collagen. The results suggest that collagen's effect on migration is mediated, at least in
part, by PAK2.They suggested PAK2 should be further explored as a pro-metastatic gene in ovarian
cancer[25]. Discussion These findings align with a previous study conducted by Eleonora Sementino,
which demonstrated that inhibiting the PAK2 gene led to a significant reduction in the occurrence and
delayed development of pleural and peritoneal malignant mesotheliomas in mice. In vitro experiments
also revealed that the removal of the PAK2 gene decreased the viability, migration, clonogenicity, and
spheroid formation of malignant mesothelioma cells[31]. Additionally, another study discovered that
PAK2 has the ability to interact with caspase-7 and phosphorylate it, thereby inhibiting cellular apoptosis
and contributing to the development of chemotherapeutic resistance in human breast invasive ductal
carcinoma[13]. Furthermore, research findings indicate that breast cancer cells with lower levels of PAK2
exhibit increased sensitivity to apoptosis induced by anticancer drugs, while heightened PAK2 activity Page 14/24 Page 14/24 disrupts the apoptotic response, leading to anchorage-independent survival, growth, and resistance to
apoptosis induced by anticancer drugs[32]. Recent discoveries have shown that exosomes released by
leukemic cells contain PAK2, and the transfer of these exosomes promotes the proliferation of endothelial
cells [33]. Finally, building upon our previous demonstration that lnc-SNHG1 functions as a ceRNA with
miR-216b-5p and plays a crucial role in modulating the sensitivity of ovarian cancer cells to the
chemotherapeutic agent paclitaxel, our current study further demonstrates that lnc-SNHG1 acts as a
ceRNA by binding to miR-216b-5p and subsequently modulating the expression of PAK2. In conclusion, the findings of this study demonstrate that PAK2 mRNA is significantly upregulated in
ovarian cancer tissues compared to adjacent tissues. This upregulation of PAK2 is associated with a
higher mortality rate in patients. Furthermore, the overexpression of PAK2 protein is linked to chemo-
resistance in serous EOC and is indicative of poor OS and DFS outcomes. In vitro experiments also
revealed that downregulating PAK2 expression in paclitaxel-resistant ovarian cancer cells resulted in a
decrease in colony formation, increased apoptosis, and inhibited cell migration. Additionally, it was
discovered that lnc-SNHG1 acts as a ceRNA by binding to miR-216b-5p and subsequently modulating the
expression of PAK2. Funding This study is supported by the National Natural Science Foundation of China (Grant No. 81702555) and
author Ting Shuang has received research support from it. This study is in part by the Key Research and
Development Program of Shannxi (Program No. 2023YBSF-555) and author Mei Li Pei has received
research support from it. ceRNA, competing endogenous RNA
OS, overall survival
DFS, disease-free survival
RIP: RNA Binding Protein Immunoprecipitation ceRNA, competing endogenous RNA
OS, overall survival
DFS, disease-free survival RIP: RNA Binding Protein Immunoprecipitation Competing Interests The authors have no relevant financial or non-financial interests to disclose. Abbreviations EOC, epithelial ovarian cancer Author Contributions Author Contributions Page 15/24 Page 15/24 Page 15/24 Ting Shuang was involved in study design, execution and manuscript drafting. Meili Pei was involved in
the study design, acquisition of data. Shiyun Wu, Yifei Zhao, and Yanqi Yang contributed to the analysis
of data. All authors commented on previous versions of the manuscript. All authors read and approved
the final manuscript. Consent for publication Not applicable. Data Availability The datasets generated during and analysed during the current study are available from the
corresponding author on reasonable request. Competing interests The authors declare no conflicts of interest. The authors declare no conflicts of interest. Ethics approval The study was approved by the ethics committee of the First Affiliated Hospital of the Xi’an Jiao tong
University (approval number: 2020-G143) and the ethics committee of Shanghai Outdo Biotech Company
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insights into tumour biology and immune modulation. World J Gastroenterol, 24(33):3709–23. 23. References Schraml P, Schwerdtfeger G, Burkhalter F, Raggi A, Schmidt D, Ruffalo T, King W, Wilber K, Mihatsch
MJ, Moch H. Combined array comparative genomic hybridization and tissue microarray analysis
suggest PAK1 at 11q13.5-q14 as a critical oncogene target in ovarian carcinoma. Am J Pathol. 2003;163(3):985–92. 23. Schraml P, Schwerdtfeger G, Burkhalter F, Raggi A, Schmidt D, Ruffalo T, King W, Wilber K, Mihatsch
MJ, Moch H. Combined array comparative genomic hybridization and tissue microarray analysis
suggest PAK1 at 11q13.5-q14 as a critical oncogene target in ovarian carcinoma. Am J Pathol. 2003;163(3):985–92. Page 17/24 24. Siu MK, Kong DS, Ngai SY, Chan HY, Jiang L, Wong ES, Liu SS, Chan KK, Ngan HY, Cheung AN. p21-
Activated Kinases 1, 2 and 4 in Endometrial Cancers: Effects on Clinical Outcomes and Cell
Proliferation. PLoS ONE, 10(7):e0133467. 25. Flate E, Stalvey JR. Motility of select ovarian cancer cell lines: effect of extra-cellular matrix proteins
and the involvement of PAK2. Int J Oncol, 45(4):1401–11. 26. Xiao Y, Deng WW, Yang LL, Li H, Yu GT, Zhang WF, Sun ZJ. Overexpression of p21-activated kinase 2
is correlated with high-grade oral squamous cell carcinomas. Future Oncol, 14(11):1091–100. 27. Liu R, Wang X, Chen GY, Dalerba P, Gurney A, Hoey T, Sherlock G, Lewicki J, Shedden K, Clarke MF. The prognostic role of a gene signature from tumorigenic breast-cancer cells. N Engl J Med. 2007;356(3):217–26. 28. Gao C, Ma T, Pang L, Xie R. Activation of P21-activated protein kinase 2 is an independent prognostic
predictor for patients with gastric cancer. Diagn Pathol, 9:55. 28. Gao C, Ma T, Pang L, Xie R. Activation of P21-activated protein kinase 2 is an independent prognostic
predictor for patients with gastric cancer. Diagn Pathol, 9:55. 29. Liu H, Shin SH, Chen H, Liu T, Li Z, Hu Y, Liu F, Zhang C, Kim DJ, Liu K et al. CDK12 and PAK2 as novel
therapeutic targets for human gastric cancer. Theranostics, 10(14):6201–15. 29. Liu H, Shin SH, Chen H, Liu T, Li Z, Hu Y, Liu F, Zhang C, Kim DJ, Liu K et al. CDK12 and PAK2 as novel
therapeutic targets for human gastric cancer. Theranostics, 10(14):6201–15. 30. Gupta A, Ajith A, Singh S, Panday RK, Samaiya A, Shukla S. PAK2-c-Myc-PKM2 axis plays an
essential role in head and neck oncogenesis via regulating Warburg effect. Cell Death Dis, 9(8):825. 30. 33. Edlinger L, Berger-Becvar A, Menzl I, Hoermann G, Greiner G, Grundschober E, Bago-Horvath Z, Al-
Zoughbi W, Hoefler G, Brostjan C et al. Expansion of BCR/ABL1(+) cells requires PAK2 but not PAK1.
Br J Haematol, 179(2):229–41. References Gupta A, Ajith A, Singh S, Panday RK, Samaiya A, Shukla S. PAK2-c-Myc-PKM2 axis plays an
essential role in head and neck oncogenesis via regulating Warburg effect. Cell Death Dis, 9(8):825. 31. Sementino E, Kadariya Y, Cheung M, Menges CW, Tan Y, Kukuyan AM, Shrestha U, Karchugina S, Cai
KQ, Peri S et al. Inactivation of p21-Activated Kinase 2 (Pak2) Inhibits the Development of Nf2-
Deficient Tumors by Restricting Downstream Hedgehog and Wnt Signaling. Mol Cancer Res,
20(5):699–711. 32. Marlin JW, Eaton A, Montano GT, Chang YW, Jakobi R. Elevated p21-activated kinase 2 activity
results in anchorage-independent growth and resistance to anticancer drug-induced cell death. Neoplasia. 2009;11(3):286–97. 33. Edlinger L, Berger-Becvar A, Menzl I, Hoermann G, Greiner G, Grundschober E, Bago-Horvath Z, Al-
Zoughbi W, Hoefler G, Brostjan C et al. Expansion of BCR/ABL1(+) cells requires PAK2 but not PAK1. Br J Haematol, 179(2):229–41. Figures Figures Page 18/24 Page 18/24 Page 18/24 Page 18/24 Figure 1
Differential expression of PAK2 within ovarian cancer patients. a. The RNASEQ dataset comprises 376
samples categorized as ovarian cancer and 88 samples categorized as adjacent to cancer. The analysis
revealed a significant up-regulation of PAK2 expression in ovarian cancer tissues compared to adjacent
tissues. b-c. The expression of PAK2 was predominantly localized in the cytoplasm, with a substantial
expression ratio of 68% in chemo-resistant serous EOC tissues. Conversely, it exhibited low expression in
chemo-sensitive serous EOC tissues, with a comparatively lower expression ratio of 23%. d. The data
utilized in this analysis was obtained from the GEO database, specifically from the GSE9891 dataset. The
differentiation between the four groups was determined through the implementation of the WILCOX test. The findings demonstrate that there are significant differences between stage I and II (P=0.027), highly
significant differences between stage I and III (P=0.00021), and extremely significant differences between
stage I and IV (P=0.0054). Additionally, the observed variation in PAK2 expression levels between stage I-II
and stage III-IV ovarian cancer is highly statistically significant (P=0.0056). Figure 1 Differential expression of PAK2 within ovarian cancer patients. a. The RNASEQ dataset comprises 376
samples categorized as ovarian cancer and 88 samples categorized as adjacent to cancer. The analysis
revealed a significant up-regulation of PAK2 expression in ovarian cancer tissues compared to adjacent
tissues. b-c. The expression of PAK2 was predominantly localized in the cytoplasm, with a substantial
expression ratio of 68% in chemo-resistant serous EOC tissues. Conversely, it exhibited low expression in
chemo-sensitive serous EOC tissues, with a comparatively lower expression ratio of 23%. d. The data
utilized in this analysis was obtained from the GEO database, specifically from the GSE9891 dataset. The
differentiation between the four groups was determined through the implementation of the WILCOX test. The findings demonstrate that there are significant differences between stage I and II (P=0.027), highly
significant differences between stage I and III (P=0.00021), and extremely significant differences between
stage I and IV (P=0.0054). Additionally, the observed variation in PAK2 expression levels between stage I-II
and stage III-IV ovarian cancer is highly statistically significant (P=0.0056). Differential expression of PAK2 within ovarian cancer patients. a. The RNASEQ dataset comprises 376
samples categorized as ovarian cancer and 88 samples categorized as adjacent to cancer. The analysis
revealed a significant up-regulation of PAK2 expression in ovarian cancer tissues compared to adjacent
tissues. b-c. The expression of PAK2 was predominantly localized in the cytoplasm, with a substantial
expression ratio of 68% in chemo-resistant serous EOC tissues. Conversely, it exhibited low expression in
chemo-sensitive serous EOC tissues, with a comparatively lower expression ratio of 23%. d. The data
utilized in this analysis was obtained from the GEO database, specifically from the GSE9891 dataset. The
differentiation between the four groups was determined through the implementation of the WILCOX test. The findings demonstrate that there are significant differences between stage I and II (P=0.027), highly
significant differences between stage I and III (P=0.00021), and extremely significant differences between
stage I and IV (P=0.0054). Additionally, the observed variation in PAK2 expression levels between stage I-II
and stage III-IV ovarian cancer is highly statistically significant (P=0.0056). Page 19/24 Figure 2
Survival curves were analyzed based on the expression patterns of PAK2 in ovarian cancer patients. a-b
The data for PAK2 expression was obtained from the GEO database, which included 51 data points. Figure 1 Patients were categorized into high expression and low expression groups based on whether their
expression values were above or below a predetermined cutoff. The survival probability of patients in th
low expression group was found to be significantly higher than that of patients in the high expression
group (a). Patients with low expression of PAK2 had a higher likelihood of survival without disease
progression compared to those with high expression, although the difference was not statistically
significant (P=0.061) (b). c. In the tissue microarray analysis, it was observed that patients with high
levels of PAK2 expression had significantly lower OS compared to those with low levels of expression
(P<0.0001). d. Furthermore, patients with high PAK2 expression exhibited reduced DFS (P<0.0001). Figure 2 Survival curves were analyzed based on the expression patterns of PAK2 in ovarian cancer patients. a-b. Th d t f
PAK2
i
bt i
d f
th GEO d t b
hi h i
l d d 51 d t
i t Survival curves were analyzed based on the expression patterns of PAK2 in ovarian cancer patients. a-b. The data for PAK2 expression was obtained from the GEO database, which included 51 data points. Patients were categorized into high expression and low expression groups based on whether their
expression values were above or below a predetermined cutoff. The survival probability of patients in the
low expression group was found to be significantly higher than that of patients in the high expression
group (a). Patients with low expression of PAK2 had a higher likelihood of survival without disease
progression compared to those with high expression, although the difference was not statistically
significant (P=0.061) (b). c. In the tissue microarray analysis, it was observed that patients with high
levels of PAK2 expression had significantly lower OS compared to those with low levels of expression
(P<0.0001). d. Furthermore, patients with high PAK2 expression exhibited reduced DFS (P<0.0001). Page 20/24 Figure 3
The sensitivity of A2780/Taxol cells to paclitaxel is enhanced through the inhibition of PAK2. a. In
of mRNA expression, PAK2 was significantly up-regulated in A2780/Taxol cells compared to paren
A2780 cells Among the PAK2 siRNA variants PAK2 siRNA 3 exhibited the highest interference effic Page 21/24
Figure 3
The sensitivity of A2780/Taxol cells to paclitaxel is enhanced through the inhibition of PAK2. a. In terms
of mRNA expression, PAK2 was significantly up-regulated in A2780/Taxol cells compared to parental
A2780 cells. Figure 1 Among the PAK2-siRNA variants, PAK2-siRNA-3 exhibited the highest interference efficiency. b. At the protein level, Pak2 was found to be highly expressed in A2780/Taxol cells compared to parental
A2780 cells, with the most significant down-regulation observed in cells transfected with PAK2-siRNA-3. c. The sensitivity of A2780/Taxol cells to paclitaxel is enhanced through the inhibition of PAK2. a. In terms
of mRNA expression, PAK2 was significantly up-regulated in A2780/Taxol cells compared to parental
A2780 cells. Among the PAK2-siRNA variants, PAK2-siRNA-3 exhibited the highest interference efficiency. b. At the protein level, Pak2 was found to be highly expressed in A2780/Taxol cells compared to parental
A2780 cells, with the most significant down-regulation observed in cells transfected with PAK2-siRNA-3. c. Page 21/24 Page 21/24 A2780/Taxol cells transfected with PAK2-siRNA-3 demonstrated reduced cell proliferation compared to
the NC-siRNA group and un-transfected A2780/Taxol cells(P<0.0001). d. The colony formation assay
confirmed that PAK2 deficiency led to a decrease in the number of colonies(P<0.01). e. The cell apoptosis
assay revealed that PAK2 silencing accelerated apoptosis(P<0.001). f. The wound healing assay
demonstrated a decrease in cell migration after PAK2 knockdown(P<0.0001). GraphPad Prism software
was used for analysis, Student's t-test was used for cell scratching and apoptosis, and Two-way ANOVA
was used for CCK8 and colony formation (at 3 time points). Significant difference: *P<0.05, **P<0.01,
***P<0.001, ****P<0.0001. A2780/Taxol cells transfected with PAK2-siRNA-3 demonstrated reduced cell proliferation compared to
the NC-siRNA group and un-transfected A2780/Taxol cells(P<0.0001). d. The colony formation assay
confirmed that PAK2 deficiency led to a decrease in the number of colonies(P<0.01). e. The cell apoptosis
assay revealed that PAK2 silencing accelerated apoptosis(P<0.001). f. The wound healing assay
demonstrated a decrease in cell migration after PAK2 knockdown(P<0.0001). GraphPad Prism software
was used for analysis, Student's t-test was used for cell scratching and apoptosis, and Two-way ANOVA
was used for CCK8 and colony formation (at 3 time points). Significant difference: *P<0.05, **P<0.01,
***P<0.001, ****P<0.0001. Page 22/24 Figure 4
L
SNHG1 f
i
d
i i
d
RNA (
RNA) b P
23 24
Figure 4
Lnc-SNHG1 functioned as a competitive endogenous RNA (ceRNA) by sequestering miR-216b-5p,
consequently modulating the expression of PAK2. a-d. PAK2 was a direct target of miR-216b-5p. a. Luciferase reporter assay indicated that miR-216b-5p reduced the luciferase activity of PAK2-WT rather
than PAK2-MUT. b. Sequencing was used to identify that the PAK2 and PAK2- MUT plasmid were
successfully constructed for luciferase reporter assay.c-d. Figure 1 A2780/Taxol cells were transfected with MS2 Figure 4 Lnc-SNHG1 functioned as a competitive endogenous RNA (ceRNA) by sequestering miR-216b-5p,
consequently modulating the expression of PAK2. a-d. PAK2 was a direct target of miR-216b-5p. a. Luciferase reporter assay indicated that miR-216b-5p reduced the luciferase activity of PAK2-WT rather
than PAK2-MUT. b. Sequencing was used to identify that the PAK2 and PAK2- MUT plasmid were
successfully constructed for luciferase reporter assay.c-d. A2780/Taxol cells were transfected with MS2- Page 23/24 Page 23/24 tagged PAK2-WT and MS2-tagged PAK2- MUT, and then were assayed by RIP. The RIP-qPCR assay
demonstrated a significant enrichment of miR-216b-5p in the immunoprecipitation (IP) group, suggesting
a binding interaction between AGO2 protein and miR-216b-5p. Additionally, the detection of PAK2 showed
a notable enrichment in the IP group, providing further evidence of a potential correlation between PAK2
and miR-216b-5p. e. The qPCR analysis revealed a decrease in the expression of the PAK2 gene following
treatment with SNHG1-siRNA-2 and miR-216b-5p mimics compared with the normal control group. f-g. The RNA levels of lnc-SNHG1 and miR-216b-5p genes were assessed using qPCR in the seven cell groups
mentioned above. i. Interference of lnc-SNHG1 resulted in a significant decrease in PAK2 protein levels,
while over-expression of miR-216b-5p also led to a notable decrease in PAK2 protein levels. h-j. The RNA
and protein levels of the PAK2 gene were assessed through western blot analysis and qPCR in the above
seven cell groups. Comparison between the second and third groups revealed a significant increase in the
expression level of the target gene PAK2 following knockdown of miR-216b-5p. Comparison between the
second and fourth groups showed a significant decrease in the expression level of the target gene PAK2
following simultaneous knockdown of miR-216b-5p and lnc-SNHG1. Comparison between the second
and sixth groups demonstrated a significant decrease in the expression level of the target gene PAK2
following simultaneous knockdown of miR-216b-5p and PAK2. GraphPad Prism software was used for
analysis, One-way ANOVA was used for qPCR. Significant difference: *P<0.05, **P<0.01, ***P<0.001,
****P<0.0001. tagged PAK2-WT and MS2-tagged PAK2- MUT, and then were assayed by RIP. The RIP-qPCR assay
demonstrated a significant enrichment of miR-216b-5p in the immunoprecipitation (IP) group, suggesting
a binding interaction between AGO2 protein and miR-216b-5p. Additionally, the detection of PAK2 showed
a notable enrichment in the IP group, providing further evidence of a potential correlation between PAK2
and miR-216b-5p. e. Figure 4 The qPCR analysis revealed a decrease in the expression of the PAK2 gene following
treatment with SNHG1-siRNA-2 and miR-216b-5p mimics compared with the normal control group. f-g. The RNA levels of lnc-SNHG1 and miR-216b-5p genes were assessed using qPCR in the seven cell groups
mentioned above. i. Interference of lnc-SNHG1 resulted in a significant decrease in PAK2 protein levels,
while over-expression of miR-216b-5p also led to a notable decrease in PAK2 protein levels. h-j. The RNA
and protein levels of the PAK2 gene were assessed through western blot analysis and qPCR in the above
seven cell groups. Comparison between the second and third groups revealed a significant increase in the
expression level of the target gene PAK2 following knockdown of miR-216b-5p. Comparison between the
second and fourth groups showed a significant decrease in the expression level of the target gene PAK2
following simultaneous knockdown of miR-216b-5p and lnc-SNHG1. Comparison between the second
and sixth groups demonstrated a significant decrease in the expression level of the target gene PAK2
following simultaneous knockdown of miR-216b-5p and PAK2. GraphPad Prism software was used for
analysis, One-way ANOVA was used for qPCR. Significant difference: *P<0.05, **P<0.01, ***P<0.001,
****P<0.0001. tagged PAK2-WT and MS2-tagged PAK2- MUT, and then were assayed by RIP. The RIP-qPCR assay
demonstrated a significant enrichment of miR-216b-5p in the immunoprecipitation (IP) group, suggesting
a binding interaction between AGO2 protein and miR-216b-5p. Additionally, the detection of PAK2 showed Page 24/24
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https://www.nature.com/articles/s41598-022-20846-9.pdf
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An agent-based model of social care provision during the early stages of Covid-19
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Scientific reports
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An agent‑based model of social
care provision during the early
stages of Covid‑19
OPEN Umberto Gostoli1,2 & Eric Silverman1,2* Social care is a frequent topic in UK policy debates, with widespread concern that the country will be
unable to face the challenges posed by the increase in demand for social care. While this is a societal
problem whose dynamics depends on long-term trends, such as the increase of human lifespans and
the drop of birth-rates, a short-term crisis, such as a pandemic, can affect the need and supply of social
care to a considerable, although temporary, extent. Building on previous modelling effort of social
care provision, we present an agent-based computational model to investigate social care provision
in the context of a pandemic (using as an example, the early stages of the Covid-19 pandemic), and
related mitigation policies, on social care demand and supply, using a proof-of-concept agent-based
model (ABM). We show how policy solutions aimed at controlling the pandemic may have substantial
effects on the level of unmet social care need and propose that such models may help policymakers
to compare alternative containment policies, taking into account their side effects on the social care
provision process. Throughout much of the developed world, demographic trends, such as the increase of human lifespans and
birth-rates drop, caused an increase in demand for social care, i.e., the provision of personal and medical care
for people in need of assistance due to age, disability or other factors. In the UK, the social care supply is largely
dependent on informal social care, or care provided free-of-charge by family members and loved ones, in order
to meet the needs of the population. Informal care is enormously widespread in the UK and is much larger than
the formal care infrastructure. The Family Resources Survey 2013/14 showed that there were 5.3 million informal
carers in the UK1, while projections indicate that the number of people receiving informal care will increase by
60% in the period 2015–20352. According to the Health Survey for England 2017, 68% of participants aged 65
and over reported receiving help from unpaid helpers, while 21% said they had received help from both unpaid
helpers and paid helpers3. www.nature.com/scientificreports www.nature.com/scientificreports 1MRC/CSO Social and Public Health Sciences Unit, University of Glasgow, Glasgow G3 7HR, UK. 2These authors
contributed equally: Umberto Gostoli and Eric Silverman. *email: eric.silverman@glasgow.ac.uk Scientific Reports | (2022) 12:16534 www.nature.com/scientificreports/ This model takes into account fertility, mortality and marriage rates for cohorts born between
1908 and 1968 to estimate the amount of care available from partners and children for people aged 50 or over. The DemoCare model uses micro-simulation to generate kinship networks of 10,000 representative agents, based
on the demographic characteristics of each cohort, limiting the network to spouse, children, children-in-law and
grandchildren. Then, through ABM simulations, they estimate the demand for care of these representative agents
and the amount of this need which can be satisfied by spouse and children, taking into account whether they are
working, their state of health and the needs for care of the rest of the family (e.g child care needs). Th
l
d l
d
h
f ll
b
d
l k
d h
h g
y
g
The social care provision model we adopt in this paper is fully agent-based, unlike DemoCare, and this meth-
odology allows us to account for the interactions between kinship networks (which occur when agents belong to
more than one ego network). This interaction which is particularly important when enlarging the group of care
suppliers beyond partners, children, children-in-law and grand-children to include brothers, nephews, aunts
and uncles. Other important differences, such as a more extensive role for social status (which in the DemoCare
model is represented by agents’ educational levels) and the endogenous determination of formal care though the
working agents’ care-work choice, will be discussed in detail in the next section.h g g
Agent-based modelling methodology has only recently been applied widely to the study of pandemics. The
module presented in this paper shares some features with the work of Wilder et al.13, who proposed an agent-
based model of Covid-19 spread taking into account age and comorbidity distributions, age-stratified contact
patterns and household structures. The model follows the standard SEIR agent classification, with four severity
levels for infectious agents (asymptomatic, mild, severe and critical) and levels of infectiousness depending on
the severity level (asymptomatic and symptomatic) and the stage of infection (before and after the onset of symp-
toms). In addition, agents are isolated if they are in the severe and critical severity levels while agents with mild
symptoms become isolated after a number of days determined through a stochastic process. Agents can become
exposed through in-household and out-of-household contacts, with the in-household contacts being more likely
to transmit the virus. www.nature.com/scientificreports/ and disadvantages of alternative containment policies, their overall effect, i.e., including the effect on social care
provision, is taken into account. In this paper, we investigate the interaction between the social care provision process and pandemic dynam-
ics, using the example of the SARS-CoV-2 pandemic, commonly known as Covid-19. As such, this paper fills a
gap between the (sparse) literature focusing on the modelling of social care provision and the growing literature
focusing on agent-based models of pandemic dynamics. In this paper we present an agent-based simulation
model of social care in the United Kingdom, which includes a model of the early stages of the Covid-19 pandemic
and its impact on individuals. The novelty of our framework is represented by the integration of the pandemic
dynamics with the process of informal social care provision, a framework through which we can therefore
account for the interdependence between the two processes.i p
p
To be sure, the model we presented is not meant to provide specific policy recommendations, as they would
require an evaluation of the desirability or value of various outcomes which can be only the result of a political
process. Rather, we propose this framework as a tool allowing the policymakers to better assess the effects of the
pandemic’s containment policies on social care provided. Moreover, while the demographic and institutional
elements of the model presented in this paper have been developed to reflect the specific reality of the UK, the
same framework can be straightforwardly used for other countries, by using the related country-specific demo-
graphic data and institutional settings. Previous works. Our model is composed of two main modules: a social care provision module and a Covid-
19 spread module. The social care provision module adopted in this paper is the latest development of a model-
ling effort spanning the last decade8–11, whose main features will be described in the next section.h f
The adoption of the agent-based methodology to model informal social care provision is quite recent and
previous literature in this field is very sparse. One study related to the social care model used in this paper is a
mixed micro-simulation/agent-based care supply and demand model, called DemoCare, recently proposed by
Spijker et al12. www.nature.com/scientificreports/ The out-of-household contacts are based on a country-specific contact matrix containing
the mean number of daily contacts agents of an age group have with agents from each of the other age groups.hi y
g
g g
p
g
g g
p
The model of Covid-19 spread we present in this paper, it the first attempt, to the best of our knowledge, to
integrate social care provision with the pandemic’s dynamics (although limited to the first stages of it). As such,
it includes significant differences compared to the above mentioned works. While the investigation of social care
provision within the context of a pandemic represents certainly an addition to the literature on social care, our
model contains important novel elements also with regards to Covid-19 modelling effort of Wilder et al. First,
because of the integration with a social care provision model, our pandemic model has an additional exposure
setting, represented by the social care-related interaction between agents. Second, in our model the agents have
additional attributes such as their homes’ geographical location and the social status. These attributes have an
important role in the generation of the contact networks (as we assume that agents close in location and social
status are more likely to be part of the same contact network). Moreover, the agents’ social status affects the prob-
ability they will develop conditions of different severity levels (according to the epidemiological phenomenon of
the social gradient in health). Finally, our model includes a behavioural module representing the decision process
determining the agents’ degree of isolation, as a response to the various kinds of risks associated with Covid-19. An agent‑based model of social
care provision during the early
stages of Covid‑19
OPEN p
p
p
While the dynamics of unmet social care need depends mainly on long-run demographic trends, such as the
increase of human lifespans and the reduction of birth-rates over many decades, relatively short-term crises, such
as global disease outbreaks, can significantly affect social care provision, both directly and indirectly, through
the effect of the policies which health authorities implement to contain the pandemic. The Covid-19 pandemic
has, indeed, increased the pressure on informal carers significantly. Carers UK estimates that before the pan-
demic, the UK had 9.1 million unpaid carers, and that the impact of Covid-19 on vulnerable people generated
an additional 4.5 million unpaid carers, for a total of 13.6 million4. Out of those already providing care before
the arrival of the coronavirus, 81% report spending more time on care than they did previously5. Carers UK
also estimates that carers had provided £135 billion of care between March and November 2020, representing a
substantial increase over previous years. p
y
One important feature of this pandemic, which makes it a good example to investigate the interaction with
social care provision, is its highly unequal effect across demographic groups: the virus has affected vulnerable
populations of older people and adults with disabilities particularly strongly. In the United Kingdom, for exam-
ple, Office for National Statistics data shows that 65% of deaths due to Covid-19 between 12 February 2021 and
6 August 2021 were recorded in adults aged over 756. Amongst adults with disabilities, the risk of death due to
Covid-19 was 3.1 times greater for more-disabled men as compared to non-disabled men, and 3.5 times greater
for more-disabled women7. Given the greatly increased risk of death due to Covid-19 among the groups which
have the highest levels of social care need, it is particularly important for policy makers to understand the
interaction between the pandemic dynamics and social care provision, so that, when assessing the advantages | https://doi.org/10.1038/s41598-022-20846-9 Scientific Reports | (2022) 12:16534 www.nature.com/scientificreports/ www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 1. Share of hospitalized by age group. Figure 1. Share of hospitalized by age group. Figure 1. Share of hospitalized by age group. Figure 2. Policy comparison: outcome dynamics. Figure 2. Policy comparison: outcome dynamics. Figure 2. Policy comparison: outcome dynamics. We can see that, under a lockdown policy which allows freedom of movement for social care purposes (which
is, among the policies we considered in these simulations, the one which most resemble the policy adopted by
the UK government), the empirical shares of hospital admissions by age, as reported in14, are within the 95%
confidence interval for all the age groups, except for the 0-19 age group, which appears to be at the lower limit
of the confidence interval (note, however, that there is a slight discrepancy between the grouping of14, where the
first age group is the 0–17 group, and the grouping of the simulations’ outcomes, where the first age group is the
0–19 group. If we consider that the hospitalization rate increases with age, the empirical shares are higher for
the 0–19 group and lower for the 20-39 group than they appear in Fig. 1). Empirical studies have shown that, among non-pharmaceutical interventions, lockdowns have been the
most effective ones in reducing the spread of Covid-1915,16. In the next set of figures, we compare the effects of
two lockdown policies to the benchmark ‘no-lockdown’ scenario. In these figures, Policy 1 refers to the partial
lockdown policy, i.e. a lockdown which allows movement for social care purposes, while Policy 2 refers to the ‘full
lockdown’ policy. In both cases, the lockdown is imposed 3 days after the first death, and is lifted after 90 days.f titt
Regarding the effects of these policies on the pandemic, we can see from Fig. 2a and b that the two policies
reduce the height of the peak significantly; even a partial lockdown reduces the maximum number of people in
hospital and on the ventilator by half. Figure 3a and b confirm this positive effect, by showing the total number
of days of hospitalization and intensive care. Results n this section, we present the simulations’ results with a particular emphasis on the inequalities characterising
oth the pandemic and the social care outcomes.hi p
The following graphs show, for each output, the mean across 20 repetitions, with 95% confidence intervals. Figure 1 demonstrates the model’s capacity to approximate the distribution of people hospitalized by age group. Scientific Reports | (2022) 12:16534 | https://doi.org/10.1038/s41598-022-20846-9 www.nature.com/scientificreports/
We can see that, under a lockdown policy which allows freedom of movement for social care purposes (which
is, among the policies we considered in these simulations, the one which most resemble the policy adopted by
th UK
t) th
i i
l h
f h
it l d
i i
b
t d i
14
ithi
th 95%
Figure 1. Share of hospitalized by age group. Figure 2. Policy comparison: outcome dynamics. Discussion In this work, we presented a ‘proof of concept’ of an agent-based framework integrating a model of social care
provision with a model of Covid-19 spread, during the early stages of the pandemic. This novel framework,
allowed us to study the effect on the provision of social care of both the Covid-19 spread and the public health
measures taken to contain it. We showed that the model could be used to evaluate the relative advantages and
disadvantages of alternative policies, and can therefore represent an important decision-support tool for policy-
making decisions in this complex and highly uncertain context.hf g
p
g
y
The model results demonstrate that policy-makers have a trade-off to consider when imposing public health
restrictions in these circumstances. When lighter restrictions are imposed, informal social care provision
increases, as informal carers have more time available and are not restricted from providing care; however,
under these conditions the negative outcomes of the pandemic are more modestly reduced. Conversely, under
more severe restrictions informal social care between households becomes impossible, worsening care outcomes
through a significant increase in unmet care need, while the effects of the pandemic are more effectively con-
tained. Models like this one could be used to investigate the nature and extent of these public health trade-offs,
so that policy-makers may make informed decisions based on the current state of the pandemic.hl p
y
y
p
The results also reflect the social gradients of health and care present in these scenarios. Unmet social care
need is distributed highly unequally, with the lowest income quintile having four times as much unmet care need
as the highest quintile. The sensitivity analysis shows that the number of contacts is a significant factor driving
these results, which suggests that the higher income quintiles benefit from having jobs with greater inherent
flexibility, which enables them to work from home in larger numbers and take more time off work to provide
informal care.i Being this work a proof-of-concept, the framework presented in this paper contains many simplifications as
our purpose was not to provide a complete model to make accurate forecasts but rather to develop a minimal
model focusing on the interdependence between the pandemic dynamics and the informal social care provision
process. Therefore, we included a ‘minimal’ model of the Covid-19 spread, which we aim to extend in future
works. www.nature.com/scientificreports/ We can see that Policy 1 and Policy 2 reduce the number of days of
hospitalization by 40% and 60% respectively compared to the benchmark, and the percentage reduction of the
number of days of intensive care is higher still.hf The last four graphs show the effect of these two policies on social care provision. Figure 4a shows that the
two policies have opposite effects on the amount of informal care provided: while total lockdown reduced the
amount of informal care (as potential suppliers cannot provide care to people in need living in other households),
partial lockdown increases the informal care provided, as under partial lockdown people have more time to
allocate to social care (an effect which is consistent with empirical observations). The effect of this variations of
care supply on unmet care need are shown in Fig. 4b, where we can see an increase of unmet care need under
Policy 1 and a decrease of it under Policy 2. https://doi.org/10.1038/s41598-022-20846-9 Scientific Reports | (2022) 12:16534 | www.nature.com/scientificreports/ w.nature.com/scientificreports/
Figure 3. Policy comparison: aggregate outcomes. Figure 4. Policy comparison: care dynamics. Figure 3. Policy comparison: aggregate outcomes. Figure 3. Policy comparison: aggregate outcomes. Figure 4. Policy comparison: care dynamics. Figure 5. Policy comparison: care aggregate outcomes. Figure 3. Policy comparison: aggregate outcomes. Figure 3. Policy comparison: aggregate outcomes. Figure 4. Policy comparison: care dynamics. Figure 4. Policy comparison: care dynamics. g
y
p
y
Figure 5. Policy comparison: care aggregate outcomes. Figure 5. Policy comparison: care aggregate outcomes. The following two figures show the differences between policies in terms of care provision and unmet care. From Fig. 5a and b we can see that the two policies have opposite effects on the amount of informal care and,
therefore, unmet care need. While Policy 1 has a positive effect with regards to social care provision and unmet
care need, the opposite is true for Policy 2, with the level of unmet care need with this latter policy being more
than 20% higher than the level of unmet care need under the former policy. https://doi.org/10.1038/s41598-022-20846-9 Scientific Reports | (2022) 12:16534 | www.nature.com/scientificreports/ Table 1. Sensitivity analysis. www.nature.com/scientificreports/ Parameter
Behaviour affected
Range
Total effect
φ
Domestic/care prudence
[0.25, 1.0]
2.0
ξ
Other-regarding concern
[0.005, 0.02]
33.04
ω
‘Income-effect’
[0.25, 1.0]
2.28
µ
‘Base’ sensitivity to outcomes
[20, 80]
27.06
s
Number of contacts
[0.05, 0.2]
35.54
r
Same-class contacts
[0.1, 0.4]
0.68
h
Same-area contacts
[0.001, 0.004]
1.05
k
Same-friends contacts
[0.05, 0.2]
2.81 To sum up, we can say that our simulations show that while Policy 1 has a positive effect on hospitalisations,
intubations and unmet care need, in the case of Policy 2 the policy maker faces a trade-off posed by the even
greater positive effect on the pandemic outcomes and an increase of about 13% of the level of unmet care need. Sensitivity analysis y
y
Typically, the results of ABM simulations depend on many parameters which are characterized by a high degree
on uncertainty. In our model, in particular, there are parameters related to the agents’ behaviour and the social
networks which are not empirically determined and for which we must perform a sensitivity analysis in order
to determine their importance in driving the simulations’ outcomes. In this sensitivity analysis, we chose the
number of hospitalisations as the main output of interest.f p
p
Table 1 shows the total effect on the outcome’ variance for the eight parameters we included in the sensitivity
analysis, the first four of which determine the agents’ behaviour, while the last four affect the size and structure
of the social networks. To determine the ranges, we halved (lower bound) and doubled (higher bound) the
parameters’ benchmark values.f p
We can see that the ‘base’ sensitivity to risk factors ( µ ) and the effect of income ( ω ) on the overall sensitivity
are two of the most important parameters, with an effect on the overall variance of total hospitalizations of 35%
and 27% respectively. The effect of the knowledge of infection on the behaviour within the household and during
the care provision ( φ ) is the second most important parameter, determining about 33% of the total variance of
the outcome. The other-regarding preferences (affecting the behaviour of knowingly infected people) and the
parameters affecting social network size and structure appear to have a marginal role in driving the outcome
we considered. Scientific Reports | (2022) 12:16534 | Methods
Thi This computational modelling study was carried out following all relevant guidelines; note that we only use pub-
licly available population data, such as the Human Mortality Database17, Eurostat18 and the Office for National
Statistics19. No human experiments were performed, nor was any human data collected during the course of
this study.h y
The model is composed of three main modules: • A demographic module (from year 1860 to year 2019, with yearly steps). g
p
y
y
y
• A social care module (from year 2020, for 180 daily steps). • A Covid-19 spread module (from year 2020, for 180 daily steps). First, the demographic module creates a population on the UK with a realistic demographic structure, starting
in the year 1860 and running until the year 2020 in one-year time steps. Then, from the beginning of the year
2020, a social care module and a Covid-19 spread module generate the social care provision process and the epi-
demiological progression of the pandemic respectively. These process then proceed for 180 one-day time steps
(i.e., we simulate the first 6 months of the pandemic). The demographic module. The initial population of couples is randomly distributed on a 812-cell grid
approximating the geography of the United Kingdom. Agents live in houses which form towns, with the density
of those houses varying in rough proportion to UK population density. The agent population is scaled down
from real UK levels at a factor of roughly 1:10,000. The simulation begins in the year 1860, which allows suffi-
cient time for the population dynamics to stabilise before 1951, at which point UK Census mortality and fertility
data is incorporated into the simulation17,19. Agents form partnerships, have children, start working (and earn an
income), relocate, retire and die, according to sub-modules the details of which have been described in previous
works8–11 and a summary of which is reported in the Supplementary Information. Here we only mention the socioeconomic structure of the population generated by the demographic mod-
ule: agents belong to one of five socioeconomic status groups (SES groups), based on the Approximated Social
Grade from the Office for National Statistics, redistributed as in Gostoli and Silverman10. Discussion For this reason, We did not include, at this stage, important factors affecting the pandemic dynamics, such
as the environmental and the institutional context, and we restricted our analysis to the stage of the pandemic
prior to the introduction of vaccines. In addition, we did not include the effects of common interventions such
as face coverings. In our future work we will continue to refine and extend this model to maximise its utility for
public health policy-makers. Scientific Reports | (2022) 12:16534 | https://doi.org/10.1038/s41598-022-20846-9 www.nature.com/scientificreports/ Table 2. Care need categories/levels and number of hours of care required. Care need category
Care need level
Weekly hours of care required
None
0
0
Low
1
8
Moderate
2
16
Substantial
3
36
Critical
4
84 Table 2. Care need categories/levels and number of hours of care required. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Table 3. Amount of care agents can provide depending on their status and kinship distance from the care
receiver. *Employed agents can provide additional care if they choose to reduce their working hours (i.e. in
case it is more convenient than using income to pay for formal care. See the Formal Care section for details). Agent status
Household (D 0)
D I
D II
D III
Teenager
12
0
0
0
Student
16
8
4
0
Employed*
16
12
8
4
Retired
56
28
16
8 Agent status
Household (D 0)
D I
D II
D III
Teenager
12
0
0
0
Student
16
8
4
0
Employed*
16
12
8
4
Retired
56
28
16
8 Table 3. Amount of care agents can provide depending on their status and kinship distance from the care
receiver. *Employed agents can provide additional care if they choose to reduce their working hours (i.e. in
case it is more convenient than using income to pay for formal care. See the Formal Care section for details Table 3. Amount of care agents can provide depending on their status and kinship distance from the care
receiver. *Employed agents can provide additional care if they choose to reduce their working hours (i.e. in
case it is more convenient than using income to pay for formal care. See the Formal Care section for details). gender, SES, and the cumulative unmet care need experienced by that agent. We therefore assume that large
amounts of unmet care need increase frailty. As for childcare need, we assume that all children, with the exception of newborns, have identical childcare
need, requiring 10 hours of care each day. The net care need for each child agent varies by age, given the presence
of child care and education policies targeted at specific age groups. Newborns have a much higher care need
which must be provided by their mother, who in turn allocates all her care supply for the newborn. Care supply. In this model, the care is provided through three main sources: informal care, privately paid-for
formal care and government care. As for informal care, it is provided by the agents’ network of relatives. Agents
requiring care have kinship networks linking them to households with members having a consanguineous or
affinal relationship with that agent. www.nature.com/scientificreports/ h
p
yi
As outlined in previous work11, agents with care needs may be eligible for publicly funded care, via a govern-
ment-funded care scheme based upon the framework in place in England (see the Supplementary Information
for details). Care provision process. In this model we adopt the care allocation process developed in our previous work11. We simulate care allocation as a negotiation taking place across agent kinship networks. We assume that child-
care needs have priority over social care needs. Therefore, the allocation process unfolds in two stages: 1) the
available care supply (available time/income for care) is allocated to child care need; 2) remaining resources
are provided to social care needs (we refer the reader to the Supplementary Information for details of the care
allocation process). p
)
In both stages the allocation process randomly samples one care-receiving unit (which is an individual in the
case of social care, or a household in the case of child care), with a probability proportional the unmet care need
of that unit. The care receiver is then linked with a care-providing household in the receiver’s kinship network;
potential care-giving households are sampled with a probability proportional to that household’s care supply. After the care supplier has been chosen, a 2-hour unit of care is provided from one member of that household
with available supply to the care receiver. If the supplying household is at distance I, then that household may
choose to provide either time (in the form of informal care) or income (in the form of formal care). p
When choosing between providing assistance in the form of income or time, the choice is made depending
on the hourly wage of the worker in the supplying household with the lowest wage. If the price of formal care is
higher than that wage, then the supplying agent will prefer to take time off work to provide informal care; if the
price of formal care is lower, then the agent will prefer to purchase that care and remain in work. The Covid‑19 spread module. Building on the framework underlying our previous social care models,
we introduce a model of Covid-19 spread which takes into account UK demographic structure, income distribu-
tion, age- and income-specific social mixing patterns, household structure and social care provision networks. The model does not include a range of environmental and institutional factors (e.g. www.nature.com/scientificreports/ Within these networks we define ‘degrees’ of kinship, based on the distances
between the agent and each household in the network. This kinship distance ranges from 0 for agents in the
same household, to III (uncles and aunts, or nieces and nephews). The supply of social care available to an agent
is determined by the size of its kinship network, the kinship distances in that network, and the individual states
of household members contained in that network. The hours of care supply that can be provided by each agent
according to status and network distance are shown in Table 3. In addition to kinship distance, physical distance
also impacts care provision. In this model we assume that care receivers may only receive informal care if the
provider is in the same town. We also assume that kinship distance restricts the provision of formal care; private
paid-for care occurs only between members of the same household, or between parents and children.i p
y
p
Formal care may be provided within the care receiver’s household or by households with first-degree kinship
relationships to the receiver. Income allocated for care may be used to buy private paid-for care, or to reduce
hours spent at work in order to provide informal care (meaning that the income allocated in this case represents
unearned income, rather than spent income). The care receiver may also purchase private care with their own
wealth. The share of their wealth allocated to formal care is positively correlated to their overall financial wealth. As outlined in previous work11, agents with care needs may be eligible for publicly funded care, via a govern-
ment-funded care scheme based upon the framework in place in England (see the Supplementary Information
for details) p
y
p
Formal care may be provided within the care receiver’s household or by households with first-degree kinship
relationships to the receiver. Income allocated for care may be used to buy private paid-for care, or to reduce
hours spent at work in order to provide informal care (meaning that the income allocated in this case represents
unearned income, rather than spent income). The care receiver may also purchase private care with their own
wealth. The share of their wealth allocated to formal care is positively correlated to their overall financial wealth. Methods
Thi Moreover, the model
contains a social mobility process: an agent is assigned the SES group associated with the education level he has
reached, with a probability of moving further up the education ladder depending on the household’ income and
the parents’ level of education. The introduction of SES groups has a number of effects on the various stages of
agent life-courses: a higher SES is associated with lower mortality and fertility rates; higher hourly salaries; and
lower salary growth rate. Socioeconomic status affects the agents’ wealth, which is randomly assigned to agents
according to their accumulated salaries to reproduce the 2016 UK wealth distribution. The socioeconomic posi-
tion of an agent affects social care supply within the household through the agent’s income, as we assume that
the share of income allocated to care supply increases with the household’s per capita income. Moreover, the
inclusion of agents’ SES allows us to take into account the social gradient in health, which affects both the social
care demand, with the probability of transition to higher levels of care need being higher for agents of lower
SESs, and the pandemic dynamics, with the probability of developing more severe infection courses depending
negatively on the agents’ income level. The social care module. The social care module simulates the social and child care provision processes,
starting from the year 2020, for 180 days. This module is based upon previous work on simulating child care
provision11. The social care module can be conveniently described by looking at three key aspects: • The care demand.h • The care supply.h • The care provision process. Care demand. In the model, total care demand is given by two components: childcare and social care demand. As for the latter, agents begin the simulation in a state of good health, and subsequently may develop care needs
according to age-, gender- and SES-specific probabilities. Table 2 shows the five possible categories of care need,
and the hours of care need required at each level. In line with the current understanding of care need progres-
sion, we assume that agents who develop a condition requiring care do not recover, but instead progress to
higher levels of severity and care need over time. This progression through the care need levels depends on age, https://doi.org/10.1038/s41598-022-20846-9 Scientific Reports | (2022) 12:16534 | www.nature.com/scientificreports/ Th
i k f i f
i
h
if k
i
l i f
i • The risk of being infected, if susceptible or if unknowingly infected (e.g. asymptomatic agents). Th
k f
f
h
f k
l
f The risk of being infected, if susceptible or if unknowingly infected (e.g. asymptomatic agents). The risk of infecting others, if knowingly infectious. • The risk of infecting others, if knowingly infectious. • The risk of infecting others, if knowingly infectious. Infected agents become aware of their infection status if they take a test, which is taken with a probability that
depends on the severity of their symptoms. nfected agents become aware of their infection status if they take a test, which is taken with a probability that
epends on the severity of their symptoms. As for susceptible agents, who will be concerned about the risk of being infected, the factor by which they
reduce their social and work activity (the mobility reduction rate (m)) depends on their sensitivity to the virus
spread. The variable measuring the spread ( ρ ) is represented by the weighted moving average of the number of
new cases, relative to the size of population. Formally, the mobility reduction rate is given by: (1)
mb =
1
egρ (1) where g represents the susceptible agent’s overall sensitivity to the risk of being infected, which depends funda-
mentally on two parameters of the behavioural model: where g represents the susceptible agent’s overall sensitivity to the risk of being infected, which depends funda-
mentally on two parameters of the behavioural model: • The infection fatality rate (f). It is a characteristic of the virus, and it is constant as during the simulation the
virus does not evolve. For the agents, it represents the expected ‘cost’ of being infected and it depends on the
agents’ age, income and gender.h • The infection fatality rate (f). It is a characteristic of the virus, and it is constant as during the simulation the
virus does not evolve. For the agents, it represents the expected ‘cost’ of being infected and it depends on the
agents’ age, income and gender.h g
g
g
• The ‘base’ sensitivity to infection’s outcomes ( µ ). It is a behavioural parameter representing the ‘strength’ of
the agents’ reaction to a unit increase of the infection fatality rate. • The ‘base’ sensitivity to infection’s outcomes ( µ ). www.nature.com/scientificreports/ weather, temperature, public
health spending, among others) which have been found to affect the different pandemic dynamics between
countries20–22. By modelling the pandemic dynamics in a single country (the UK), we can consider these con-
textual elements to be fixed, and we can focus on the socio-economic factors determining the inequalities of the
Covid-19 outcomes within the UK. https://doi.org/10.1038/s41598-022-20846-9 Scientific Reports | (2022) 12:16534 | www.nature.com/scientificreports/ www.nature.com/scientificreports/ As in the standard SEIR model, the agents can be in one of four states: susceptible, exposed, infectious or
removed (i.e. recovered or deceased). As for the conditions of the infected agents after the virus incubation period,
we distinguish between: asymptomatic, mild symptomatic (i.e., not hospitalized), severe (i.e., hospitalized but not
in intensive care) and critical (i.e., in intensive care).h An infected agent is given a viral load, which is sampled from a standard uniform distribution. The viral
load determines the agent’s contagiousness and, together with their age and income quintile, the severity of their
symptoms (which we call the agent’s symptoms severity index ( π)). The severity index affects both the agent’s
mobility (which is equal to 0 for those hospitalized) and the probability that it takes a test to determine whether
it is infected.h The main features characterizing our model of the pandemic are: • The inclusion of a behavioural module determining the agents’ behavioural response to the risks of being
infected or infect others. A
l
d f
l
d
d
l • The inclusion of a behavioural module determining the agents’ behavioural response to the risks of being
infected or infect others. A multi-setting exposure process, composed of: a social setting, a domestic setting, and a social care setting. A
i
d
d
ifi
b biliti
f d
l
i
diff
t i
i f ti • A multi-setting exposure process, composed of: a social setting, a domestic setting, and
d
d
fi
b b l
f d
l
d ff
f • Age-, income- and gender-specific probabilities of developing different virus infection courses. , income- and gender-specific probabilities of developing different virus infection courses. Behavioural module. While in standard SEIR models, the behavioural reaction is implicit in the probability of
disease transmission, agent-based modeling allows us to develop an explicit model of the way the agents’ modify
their behaviour in response to the pandemic’s spread. In this model, the agents react to the risks posed by the
pandemic by reducing their social and work activities – by self-isolating. We assume that the agents can be con-
cerned about two kinds of risk: • The risk of being infected, if susceptible or if unknowingly infected (e.g. asymptomatic agents). www.nature.com/scientificreports/ We assume that the probability of being infected by the
care receiver or the care supplier depends on the contagiousness of the infected agent, on the duration of the
interaction and, if the person carrying the virus is the care receiver, on whether the person knows about their
infection status. If the receiver knows they are infectious, we assume that both the care receiver and the care sup-
plier adopt a prudent behaviour which reduces the risk of contagion by a certain factor φ which is a parameter
of the model. When the care supplier is infectious and aware of their status, we assume instead that they do not
provide any care supply. y
y
With regards to domestic interaction, agents can be infected through interactions within their household. The capacity of an infected household member to transmit the virus to a susceptible agent depends on their
contagiousness (their viral load) and whether the infected agent knows they are infected. If the agent knows they
are infected, we assume that households adopt a prudent behaviour reducing the risk of contagion by a certain
factor φ which is a parameter of the model. Finally, agents engage in a series of social interactions through which they come into contact with agents
which are part of their ego network. These ego network of an agent is characterized by a size (i.e. the total num-
ber of contacts) and a distribution of contacts by age group, which depend on the agent’s age group, according
to empirical findings on social mixing patterns (24–26). In line with findings of empirical studies of the effect of
social status on social networks27, we assume that the network size increases with the agents’ social status (rep-
resented by their income quintile), according to a factor s which is a parameter of the model. The probability
that two agents are part of the same network depends negatively on the geographical and social status distances
and positively on the number of common friends, with the ‘strength’ of these three factors being regulated by
three parameters of the model (indicated in the sensitivity analysis below as, respectively, h, r and k). www.nature.com/scientificreports/ words, we should expect agents of the former category to adopt a stricter isolation regime compared to the latter,
for a given increase in case numbers. words, we should expect agents of the former category to adopt a stricter isolation regime compared to the latter,
for a given increase in case numbers. g
As for the knowingly infectious agents, who will be concerned about the risk of infecting others, we assume
hat their mobility reduction depends on their sensitivity to the share of the susceptible population S: (4)
mb = 1
egS (4) For working agents, once again the sensitivity depends on the income and occupation, but differently from the
susceptible agents, we assume that it depends on the general infection fatality rate, F, (as opposed to the agent-
specific infection fatality rate) as it is the variable measuring the virus fatality among the population. Formally,
the infectious agents’ sensitivity to the share of susceptible is given by: (5)
gi
w = µξFωσ (5) gi
w = µξFωσ (6)
gi
u = µξF and for unemployed agents: (6)
gi
u = µξF (6) where ξ is a parameter representing the ‘strength’ of the agents’ social (or other-regarding) preferences (i.e., how
much they care about others’ well-being). where ξ is a parameter representing the ‘strength’ of the agents’ social (or other-regarding) preferences (i.e., how
much they care about others’ well-being). Apart from behavioural reactions to pandemic risks, the mobility of infected agents who are not asymptomatic
may be reduced because of the debilitating effect of the virus. We assume that the extent to which the infected
agent’s mobility is reduced depends on its symptoms severity index, π , according to the formula: (7)
mv = (1 −π)η (7) where η is a parameter determining how the mobility decreases as the severity of the agent’s symptoms increase. Therefore, the overall agent’s mobility reduction rate m, will be given by: (8)
m = min(mb, mv) m = min(mb, mv) m = min(mb, mv) m = min(mb, mv) (8) Exposure settings. In this model, a susceptible agent can become exposed in three settings: • Informal social care setting. • Domestic setting. • Social setting. • Social setting. As for the social care setting, agents with social care needs interact with relatives providing care from other
household, a process through which the virus spreads. www.nature.com/scientificreports/ It is a behavioural parameter representing the ‘strength’ of
the agents’ reaction to a unit increase of the infection fatality rate. or a not-working agent (e.g., a retired agent), the degree of isolation does not affect its income and the occupa-
ional factor is not relevant, so the agent’s sensitivity to risk becomes: (2)
gs
u = f µ (2) In case the agent is working, their sensitivity to the risk of being infected depends also on: he agent is working, their sensitivity to the risk of being infected depends also on: n case the agent is working, their sensitivity to the risk of being infected depends also on: • The agent’s income factor ( ω)h h
• The agent’s occupation factor ( σ) If the agent is working, indeed, reducing its mobility may mean reducing its working hours and therefore its
income. Agents with a relatively high income can afford a higher reduction of their working hours compared
to agents with a low income. Finally, agents with different kinds of occupations differ in their capacity to work
from home. In line with empirical observations23, we assume that people of low social status have jobs with less
inherent flexibility and are less able to work from home, compared to the jobs of people of high social status. The agent’s income quintile is used as a proxy for its social status. Formally, the agent’s sensitivity to the risk of
being infected for a working agent is given by: (3)
gs
w = f µωσ (3) gs
w = f µωσ where g is a proportionality parameter and ω is an increasing function of the agents’ income quintile. h
d b
h
f
h
ld
ld
l
f h h
l
b
h where g is a proportionality parameter and ω is an increasing function of the agents’ income quintile. For the reasons mentioned above, therefore, we should expect old people of high social status to be the most
sensitive to the risk of being infected, and young people of low social status to be the least sensitive; in other For the reasons mentioned above, therefore, we should expect old people of high social status to be the most
ensitive to the risk of being infected, and young people of low social status to be the least sensitive; in other https://doi.org/10.1038/s41598-022-20846-9 Scientific Reports | (2022) 12:16534 | www.nature.com/scientificreports/ www.nature.com/scientificreports/ The risk of domestic exposure, is given by: (12)
d =
h
i=1
viθ d =
h
i=1
viθ (12) where h is the size of the household, vi is the contagiousness of household member i and θ is a prudential behav-
iour, risk-reducing factor, which is equal to 1 if the agents are not infected or are infected but are unaware of
this fact, while θ < 1 otherwise. where h is the size of the household, vi is the contagiousness of household member i and θ is a prudential behav-
iour, risk-reducing factor, which is equal to 1 if the agents are not infected or are infected but are unaware of
this fact, while θ < 1 otherwise. Finally the risk of exposure through social care is given by: Finally the risk of exposure through social care is given by: (13)
c =
k
i=1
(tiθ)φvi (13) where k is the number of social care interactions (the number of people met to receive or provide for care), ti
is the number of hours of care in interaction i, θ is the test-dependent, risk-reducing factor, φ is a parameter
determining how the risk grows with the time of care (in our simulations, φ < 1 , meaning that the risk grows
with time but at a decreasing speed) and vi is the contagiousness of the agent met in interaction i. Virus infection courses. Once an agent has become exposed through one of the three spread settings described
bove, it is assigned one infection course over four possible courses, listed below in order of growing severity: • Asymptomatic • Asymptomatic • Mild conditions (symptomatic not hospitalized) y
• Severe conditions (hospitalized not in intensive care) • Severe conditions (hospitalized not in • Critical conditions (in intensive care) In accordance with empirical studies, we assume that the probabilities through which each exposed agent is
assigned one of these conditions depends on the agent’s age, social status (income quintile) and gender, with
the probability of developing more serious conditions growing with age, decreasing with social status and being
higher for males than for females28–34. www.nature.com/scientificreports/ isolation rates (with the weights being the strength of the network’s links); second, the weights of the links are
reduced by the contacts’ isolation rates (so that the agent is less likely to meet people with higher isolation rates). isolation rates (with the weights being the strength of the network’s links); second, the weights of the links are
reduced by the contacts’ isolation rates (so that the agent is less likely to meet people with higher isolation rates). isolation rates (with the weights being the strength of the network’s links); second, the weights of the links are
reduced by the contacts’ isolation rates (so that the agent is less likely to meet people with higher isolation rates). On any given day, the probability that a given agent becomes exposed, depends on three indexes representing
h i
i
f h i
i
i
h
h
i
i
d b
d h i
i
’
i
hi h On any given day, the probability that a given agent becomes exposed, depends on three indexes representing
the intensity of the interaction in the three settings mentioned above, and the interactions’ contagiousness, which
is 0 for interactions with susceptible or exposed (i.e. infected but not yet contagious) agents. Formally, an agent’s
probability of becoming exposed pe is given by: (9)
pe = er −1
er pe = er −1
er (9) where r is the total risk of infection, which is given by: (10)
r = αs + βd + γ c (10) with s being the risk of exposure by social contact, d the risk of domestic exposure and c the risk of being infected
through the social care process (multiplied by the respective proportionality factors, which are parameters of
the model).h with s being the risk of exposure by social contact, d the risk of domestic exposure and c the risk of being infected
through the social care process (multiplied by the respective proportionality factors, which are parameters of
the model).h The risk of exposure by social contact, in turn, is given by: The risk of exposure by social contact, in turn, is given by: (11)
s = n¯v (11) s = n¯v where n is the number of daily contacts and ¯v is the contacts’ average contagiousness. www.nature.com/scientificreports/ Once a
link is created between two agents, a weight is assigned to the link equal to the probability of its creation, so that
the interaction between pairs of agents is more ‘intense’ the closer they are geographically and socially, and the
higher the number of common friends.f g
Because of the physiological effect of the virus (for infected agents with symptoms) and the agents’ behavioural
reaction to the risks posed by the pandemic (see the previous sub-section), during the pandemic the capacity and
availability of the agents to engage in social interactions may be reduced by a certain factor, which we call the
agent’s isolation rate (i). The values of i can go from 0 to 1 for hospitalized agents. The isolation rate affects the
size and the composition of the list of the agent’s daily contacts. First, the mean number of contacts for an agent
is reduced by a factor equal to the product of that agent’s isolation rate and the weighted average of its contacts’ Scientific Reports | (2022) 12:16534 | https://doi.org/10.1038/s41598-022-20846-9 www.nature.com/scientificreports/ www.nature.com/scientificreports/ The pandemic and the social care provision process affect each
other, and one of our main goals in this paper is to investigate this complex relationship, especially in terms of
unmet social care need inequalities across social classes.f q
In the previous subsection, we have already seen that the social care provision process affects the dynamics
of the pandemic as the care-related interactions represent a channel through which the pandemic spreads. On
the other hand, the pandemic affects both the demand and the supply of social care. f
On the demand side, agents who are hospitalized receive all the care they need in hospitals, and therefore
these parts of social care demand are reduced to 0 for the duration of the hospitalization period. On the other
hand, we assume that infected people may develop symptoms that, although not severe enough to require hos-
pitalization, are sufficiently debilitating that they generate additional social care needs (other than reducing the
capacity to provide for social care to nil, if the agents were normally care suppliers). Further, we assume that in
infected children with symptom severity above a certain threshold, though below hospitalisation level, are not
able to attend school and therefore increase the child care load of their household. On the supply side, symptomatic agents who are not hospitalized have their care supply reduced to nil if the
severity of their symptoms exceeds a certain threshold or if they become aware of their infected status. If the
symptom severity of non-hospitalized agents remains below the threshold, the social care they can normally
supply is reduced by a certain factor which depends on their symptom severity.f pp y
y
p
y
p
y
Besides the pandemic’s direct effects on social care provision, the model we present is a useful tool to inves-
tigate the effects of policies implemented to prevent the spread of Covid-19, such as lockdowns. In the Results
section below, after presenting the results for the ‘No-lockdown’ benchmark scenario, we will show the effects
of two lockdown policies: a ‘full lockdown’ policy, under which the agents are not allowed to visit other house-
holds, and a ‘partial lockdown’ policy, under which people can visit other households for social care purposes. In the latter case, we expect the total care supply to increases as we assume that the normally occupied agent
(i.e. www.nature.com/scientificreports/ g
Upon exposure, the agent is also assigned an incubation period, which, in line with empirical observations35,36,
is drawn from a log-normal distribution with mean of about 5 days, and a recovery period, whose length depends
on the severity level assigned to the agent (in line with the empirical findings, we assume that the more severe the
infection the longer the recovery period), in order to reproduce a log-normal distribution of the recovery period
with mean of about 12 days at the population level. The exposed agent is also assigned a viral load, ǫ , which is
drawn from a standard uniform distribution. After the incubation period, the agent starts to develop symptoms (if not asymptomatic) and, in line with
empirical observations37, we assume that the exposed agent becomes infectious 2 days before the emergence of
symptoms (therefore, 3 days after exposure, on average). We assume that the agent’s contagiousness v is a grow-
ing function of its viral load: (14)
v = εδ v = εδ (14) v = εδ with δ being a parameter regulating the relationship between viral load and contagiousness. W diff
i
h
di i
f
i
b
i
i
h
i with δ being a parameter regulating the relationship between viral load and contagiousness. d ff
h
d
f
b
h
d
b We differentiate the conditions of symptomatic agents by assigning them a symptoms severity index, between
0 and 1 exclusive, with the probability of the agent being assigned a higher value increasing with its viral load
and its age, decreasing with its income quintile and being higher for males than for females. The closer to 1 the https://doi.org/10.1038/s41598-022-20846-9 Scientific Reports | (2022) 12:16534 | www.nature.com/scientificreports/ symptoms severity index, the more severe are the symptoms, the greater is the reduction of the agent’s mobility
and the higher the probability that the agent will take a test.t symptoms severity index, the more severe are the symptoms, the greater is the reduction of the agent’s mobility
and the higher the probability that the agent will take a test.t g
p
y
g
After the recovery period, some agents will die, with a probability that also depends on age, social class and
ender. All other agents recover and we assume that they are immune to Covid-19 thereafter. The pandemic‑social care interaction. References References
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M d 172 577 582 (2020) 4. Verity, R. et al. Author contributions U.G. prepared the simulation and analysed the results, E.S. contributed to simulation development, and both
E.S. and U.G. contributed to the preparation of the manuscript. © The Author(s) 2022 Competing interests h g
The authors declare no competing interests. Additional informationh Additional information
Supplementary Information The online version contains supplementary material available at https://doi.org/
10.1038/s41598-022-20846-9. Supplementary Information The online version contains supplementary material available at https://doi.org/
10.1038/s41598-022-20846-9. Correspondence and requests for materials should be addressed to E.S. Correspondence and requests for materials should be addressed to E.S. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and
institutional affiliations. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and
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